Amato J. Giaccia

All Publications

• Developing high-affinity decoy receptors to treat multiple myeloma and diffuse large B cell lymphoma. The Journal of experimental medicine Miao, Y. R., Thakkar, K., Cenik, C., Jiang, D., Mizuno, K., Jia, C., Li, C. G., Zhao, H., Diep, A., Xu, Y., Zhang, X. E., Yang, T. T., Liedtke, M., Abidi, P., Leung, W., Koong, A. C., Giaccia, A. J. 2022; 219 (9)

  Abstract

  Disease relapse and treatment-induced immunotoxicity pose significant clinical challenges for patients with hematological cancers. Here, we reveal distinctive requirements for neutralizing TNF receptor ligands APRIL and BAFF and their receptor activity in MM and DLBCL, impacting protein translation and production in MM cells and modulating the translation efficiency of the ATM interactor (ATMIN/ACSIZ). Therapeutically, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as an inhibitor of APRIL and BAFF. While wild-type sBCMA-Fc effectively blocked APRIL signaling in MM, it lacked activity in DLBCL due to its weak BAFF binding. To expand the therapeutic utility of sBCMA-Fc, we engineered an affinity-enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) 4- and 500-fold stronger in binding to APRIL and BAFF, respectively. The mutant sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, we also demonstrated an adequate toxicity profile and on-target mechanism of action in nonhuman primate studies.

  View details for DOI 10.1084/jem.20220214

  View details for PubMedID 35881112

• Multi-omics analysis of spatially distinct stromal cells reveals tumor-induced O-glycosylation of the CDK4-pRB axis in fibroblasts at the invasive tumor edge. Cancer research Bouchard, G., Garcia-Marques, F. J., Karacosta, L. G., Zhang, W., Bermudez, A., Riley, N. M., Varma, S., Mehl, L. C., Benson, J. A., Shrager, J. B., Bertozzi, C. R., Pitteri, S. J., Giaccia, A. J., Plevritis, S. K. 2021

  Abstract

  The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that crosstalk between tumor and stromal cells within the tumor microenvironment (TME) results in activation of key biological pathways depending on their position in the tumor (edge vs core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts (TCF) from the tumor core, established from human lung adenocarcinomas. A multi-omics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize crosstalk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential post-translational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 and phosphorylated retinoblastoma protein (CDK4-pRB) axis in the stroma and indirectly modulates pro-invasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma crosstalk and a potential avenue to improve the anti-cancer efficacy of CDK4 inhibitors.

  View details for DOI 10.1158/0008-5472.CAN-21-1705

  View details for PubMedID 34853070

• C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models. Cancer immunology research Nandagopal, S., Li, C. G., Xu, Y., Sodji, Q. H., Graves, E. E., Giaccia, A. J. 2021

  Abstract

  Many solid tumors have low levels of cytotoxic CD56dim NK cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin, LFA-1, orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represents a new strategy to improve the efficiency of immunotherapy for cancer treatment.

  View details for DOI 10.1158/2326-6066.CIR-21-0435

  View details for PubMedID 34819308

• Long-term expression changes of immune-related genes in prostate cancer after radiotherapy. Cancer immunology, immunotherapy : CII Eke, I., Aryankalayil, M. J., Bylicky, M. A., Sandfort, V., Vanpouille-Box, C., Nandagopal, S., Graves, E. E., Giaccia, A. J., Coleman, C. N. 2021

  Abstract

  The expression of immune-related genes in cancer cells can alter the anti-tumor immune response and thereby impact patient outcomes. Radiotherapy has been shown to modulate immune-related genes dependent on the fractionation regimen. To identify long-term changes in gene expression after irradiation, PC3 (p53 deleted) and LNCaP (p53 wildtype) prostate cancer cells were irradiated with either a single dose (SD, 10Gy) or a fractionated regimen (MF) of 10 fractions (1Gy per fraction). Whole human genome arrays were used to determine gene expression at 24h and 2months after irradiation. Immune pathway activation was analyzed with Ingenuity Pathway Analysis software. Additionally, 3D colony formation assays and T-cell cytotoxicity assays were performed. LNCaP had a higher basal expression of immunogenic genes and was more efficiently killed by cytotoxic T-cells compared to PC3. In both cell lines, MF irradiation resulted in an increase in multiple immune-related genes immediately after irradiation, while at 2months, SD irradiation had a more pronounced effect on radiation-induced gene expression. Both immunogenic and immunosuppressive genes were upregulated in the long term in PC3 cells by a 10Gy SD irradiation but not in LNCaP. T-cell-mediated cytotoxicity was significantly increased in 10Gy SD PC3 cells compared to the unirradiated control and could be further enhanced by treatment with immune checkpoint inhibitors. Irradiation impacts the expression of immune-related genes in cancer cells in a fractionation-dependent manner. Understanding and targeting these changes may be a promising strategy for primary prostate cancer and recurrent tumors.

  View details for DOI 10.1007/s00262-021-03036-w

  View details for PubMedID 34435232

• FIBER FINDING ALGORITHM USING STEP-WISE TRACING TO IDENTIFY BIO-POLYMER FIBERS IN NOISY 3D IMAGES. Biophysical journal Rossen, N. S., Kyrsting, A., Giaccia, A. J., Erler, J. T., Oddershede, L. B. 2021

  Abstract

  We present a novel fiber finding algorithm (FFA) that will permit researchers to detect and return traces of individual bio-polymers. Determining the biophysical properties and structural cues of bio-polymers can permit researchers to assess the progression and severity of disease. Confocal microscopy images are a useful method for observing bio-polymer structures in 3D, but their utility for identifying individual bio-polymers is impaired by noise inherent in the acquisition process, including convolution from the point spread function (PSF). The new, iterative FFA we present here (i) measures a microscope's PSF and uses it as a metric for identifying fibers against the background; (ii) traces each fiber within a cone angle; and (iii) blots out the identified trace before identifying another fiber. Blotting out the identified traces in each iteration allows the FFA to detect and return traces of single fibers accurately and efficiently-even within fiber bundles. We used the FFA to trace unlabeled collagen type I fibers-a bio-polymer used to mimic the extra cellular matrix (ECM) in in vitro cancer assays-imaged by confocal reflectance microscopy (CRM) in three dimensions, enabling quantification of fiber contour length, persistence length, and 3D mesh size. Based on 3D CRM images and the PSF, we traced and measured the fibers to confirm that colder gelation temperatures increased fiber contour length, persistence length, and 3D mesh size-thereby demonstrating the FFA's use in quantifying bio-polymers' structural and physical cues from noisy microscope images.

  View details for DOI 10.1016/j.bpj.2021.08.017

  View details for PubMedID 34411578

• Flow radiocytometry using droplet optofluidics. Biosensors & bioelectronics Ha, B., Kim, T. J., Moon, E., Giaccia, A. J., Pratx, G. 2021; 194: 113565

  Abstract

  Flow-based cytometry methods are widely used to analyze heterogeneous cell populations. However, their use for small molecule studies remains limited due to bulky fluorescent labels that often interfere with biochemical activity in cells. In contrast, radiotracers require minimal modification of their target molecules and can track biochemical processes with negligible interference and high specificity. Here, we introduce flow radiocytometry (FRCM) that broadens the scope of current cytometry methods to include beta-emitting radiotracers as probes for single cell studies. FRCM uses droplet microfluidics and radiofluorogenesis to translate the radioactivity of single cells into a fluorescent signal that is then read out using a high-throughput optofluidic device. As a proof of concept, we quantitated [18F]fluorodeoxyglucose radiotracer uptake in single human breast cancer cells and successfully assessed the metabolic flux of glucose and its heterogeneity at the cellular level. We believe FRCM has potential applications ranging from analytical assays for cancer and other diseases to development of small-molecule drugs.

  View details for DOI 10.1016/j.bios.2021.113565

  View details for PubMedID 34492500

• Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models. The Journal of clinical investigation Mehibel, M., Xu, Y., Li, C. G., Moon, E. J., Thakkar, K. N., Diep, A. N., Kim, R. K., Bloomstein, J. D., Xiao, Y., Bacal, J., Saldivar, J. C., Le, Q., Cimprich, K. A., Rankin, E. B., Giaccia, A. J. 2021; 131 (11)

  Abstract

  Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.

  View details for DOI 10.1172/JCI146256

  View details for PubMedID 34060485

• Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination & ndash;deficient cancer models JOURNAL OF CLINICAL INVESTIGATION Mehibel, M., Xu, Y., Li, C. G., Moon, E., Thakkar, K. N., Diep, A. N., Kim, R. K., Bloomstein, J. D., Xiao, Y., Bacal, J., Saldivar, J. C., Le, Q., Cimprich, K. A., Rankin, E. B., Giaccia, A. J. 2021; 131 (11)

  View details for DOI 10.1172/JCI146256.

  View details for Web of Science ID 000656969100004

• Neutralization of PD-L2 is Essential for Overcoming Immune Checkpoint Blockade Resistance in Ovarian Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research Miao, Y. R., Thakkar, K. N., Qian, J., Kariolis, M. S., Huang, W., Nandagopal, S., Yang, T. T., Diep, A. N., Cherf, G. M., Xu, Y., Moon, E. J., Xiao, Y., Alemany, H., Li, T., Yu, W., Wei, B., Rankin, E. B., Giaccia, A. J. 2021

  Abstract

  PURPOSE: Ovarian cancer represents a major clinical hurdle for immune checkpoint blockade (ICB), with reported low patient response rates. We found that the immune checkpoint ligand PD-L2 is robustly expressed in patient samples of ovarian cancers and other malignancies exhibiting suboptimal response to ICB but not in cancers that are ICB sensitive. Therefore we hypothesize that PD-L2 can facilitate immune escape from ICB through incomplete blockade of the PD-1 signaling pathway.EXPERIMENTAL DESIGN: We engineered a soluble form of the PD-1 receptor (sPD-1) capable of binding and neutralizing both PD-L2 and PD-L1 with x200 and x10,000 folds improvement in binding affinity over wild-type PD-1. Leading to superior inhibition of ligand-mediated PD-1 activities.RESULTS: Both In vitro and in vivo analyses performed in this study demonstrated that the high-affinity sPD-1 molecule is superior at blocking both PD-L1 and PD-L2 mediated immune evasion and reducing tumor growth in immune-competent murine models of ovarian cancer.CONCLUSIONS: The data presented in this study provides justification for using a dual targeting, high-affinity sPD-1 receptor as an alternative to PD-1 or PD-L1 therapeutic antibodies for achieving superior therapeutic efficacy in cancers expressing both PD-L2 and PD-L1.

  View details for DOI 10.1158/1078-0432.CCR-20-0482

  View details for PubMedID 34011561

• Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes. Stem cell reports Chang, A. C., Pardon, G., Chang, A. C., Wu, H., Ong, S., Eguchi, A., Ancel, S., Holbrook, C., Ramunas, J., Ribeiro, A. J., LaGory, E. L., Wang, H., Koleckar, K., Giaccia, A., Mack, D. L., Childers, M. K., Denning, C., Day, J. W., Wu, J. C., Pruitt, B. L., Blau, H. M. 2021

  Abstract

  Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.

  View details for DOI 10.1016/j.stemcr.2021.04.018

  View details for PubMedID 34019816

• A NIR fluorescent smart probe for imaging tumor hypoxia. Cancer reports (Hoboken, N.J.) Hettie, K. S., Klockow, J. L., Moon, E. J., Giaccia, A. J., Chin, F. T. 2021: e1384

  Abstract

  BACKGROUND: Tumor hypoxia is a characteristic of paramount importance due to low oxygenation levels in tissue negatively correlating with resistance to traditional therapies. The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia-sensitive smart probe (NO2 -Rosol) for identifying hypoxia via selectively imaging nitroreductase (NTR) activity, which could correlate to oxygen deprivation levels in cells, thereby serving as a proxy. We demonstrated proof of concept by subjecting a glioblastoma (GBM) cell line to extreme stress by evaluating such under radiobiological hypoxic (pO2 ≤~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (pO2 = ~1.7%) which fluctuate little from physiological hypoxic (pO2 = 1.0-3.0%) conditions.AIM: We aimed to evaluate the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (pO2 = 2.0%) within physiological hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain.METHODS: We evaluated multiple GBM cell lines to determine their relative sensitivity to oxygenation levels via measuring carbonic anhydrase IX (CAIX) levels, which is a surrogate marker for indirectly identifying hypoxia by reporting on oxygen deprivation levels and upregulated NTR activity. We evaluated for hypoxia via measuring NTR activity when employing NO2 -Rosol in in vitro and tumor hypoxia imaging studies in vivo.RESULTS: The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. NO2 -Rosol displayed an 8-fold fluorescence enhancement when evaluated in GBM39 cells (pO2 = 2.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant physiological conditions. We demonstrated the feasibility of NO2 -Rosol to afford tumor hypoxia imaging in vivo via it demonstrating a tumor-to-background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue.CONCLUSION: We established the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.

  View details for DOI 10.1002/cnr2.1384

  View details for PubMedID 33811473

• -003 Oxygen dependent resistance to PARP inhibitors Mehibel, M., Xu, J., Li, G., Moon, J., Thakkar, K., Diep, A., Kim, R., Blomstein, J., Xiao, S., Bacal, J., Saldivar, J., Le, Q., Cimprich, K., Rankin, E., Giaccia, A. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000641160600004

• Lipid droplet storage promotes murine pancreatic tumor growth. Oncology reports Grachan, J. J., Kery, M., Giaccia, A. J., Denko, N. C., Papandreou, I. 2021; 45 (4): 1

  Abstract

  Hypoxia Inducible Lipid Droplet Associated (HILPDA) is frequently overexpressed in tumors and promotes neutral lipid storage. The impact of Hilpda on pancreatic ductal adenocarcinoma (PDAC) tumor growth is not known. In order to evaluate Hilpda‑dependent lipid storage mechanisms, expression of Hilpda in murine pancreatic cells(KPC) was genetically manipulated. Lipid droplet(LD) abundance and triglyceride content invitro were measured, and model tumor growth in nu/nu mice was determined. The results showed that excess lipid supply increased triglyceride storage and LD formation in KPC cells in a HILPDA‑dependent manner. Contrary to published results, inhibition of Adipose Triglyceride Lipase (ATGL) did not ameliorate the triglyceride abundance differences between Hilpda WT and KO cells. Hilpda ablation significantly decreased the growth rate of model tumors in immunocompromised mice. In conclusion, Hilpda is a positive regulator of triglyceride storage and lipid droplet formation in murine pancreatic cancer cells invitro and lipid accumulation and tumor growth invivo. Our data suggest that deregulated ATGL is not responsible for the absence of LDs in KO cells in this context.

  View details for DOI 10.3892/or.2021.7972

  View details for PubMedID 33649859

• X-change symposium: status and future of modern radiation oncology-from technology to biology. Radiation oncology (London, England) Corradini, S., Niyazi, M., Verellen, D., Valentini, V., Walsh, S., Grosu, A., Lauber, K., Giaccia, A., Unger, K., Debus, J., Pieters, B. R., Guckenberger, M., Senan, S., Budach, W., Rad, R., Mayerle, J., Belka, C. 2021; 16 (1): 27

  Abstract

  Future radiation oncology encompasses a broad spectrum of topics ranging from modern clinical trial design to treatment and imaging technology and biology. In more detail, the application of hybrid MRI devices in modern image-guided radiotherapy; the emerging field of radiomics; the role of molecular imaging using positron emission tomography and its integration into clinical routine; radiation biology with its future perspectives, the role of molecular signatures in prognostic modelling; as well as special treatment modalities such as brachytherapy or proton beam therapy are areas of rapid development. More clinically, radiation oncology will certainly find an important role in the management of oligometastasis. The treatment spectrum will also be widened by the rational integration of modern systemic targeted or immune therapies into multimodal treatment strategies. All these developments will require a concise rethinking of clinical trial design. This article reviews the current status and the potential developments in the field of radiation oncology as discussed by a panel of European and international experts sharing their vision during the "X-Change" symposium, held in July 2019 in Munich (Germany).

  View details for DOI 10.1186/s13014-021-01758-w

  View details for PubMedID 33541387

• The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling. Nature communications Moon, E. J., Mello, S. S., Li, C. G., Chi, J., Thakkar, K., Kirkland, J. G., Lagory, E. L., Lee, I. J., Diep, A. N., Miao, Y., Rafat, M., Vilalta, M., Castellini, L., Krieg, A. J., Graves, E. E., Attardi, L. D., Giaccia, A. J. 2021; 12 (1): 4308

  Abstract

  Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer.

  View details for DOI 10.1038/s41467-021-24631-6

  View details for PubMedID 34262028

• Wounds Inhibit Tumor Growth In Vivo ANNALS OF SURGERY Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R., Gurtner, G. C., Giaccia, A. J., Lorenz, H., Weissman, I. L., Longaker, M. T. 2021; 273 (1): 173–80

  View details for DOI 10.1097/SLA.0000000000003255

  View details for Web of Science ID 000613348700039

• Intracellular C4BPA Levels Regulate NF-kappaB-Dependent Apoptosis. iScience Olcina, M. M., Kim, R. K., Balanis, N. G., Li, C. G., von Eyben, R., Graeber, T. G., Ricklin, D., Stucki, M., Giaccia, A. J. 2020; 23 (10): 101594

  Abstract

  The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-kappaB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis invitro and invivo. Mechanistically, sensitive C4BPA mutants display increased IkappaBalpha expression and increased inhibitory IkappaBalpha-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-kappaB-dependent apoptosis.

  View details for DOI 10.1016/j.isci.2020.101594

  View details for PubMedID 33205012

• Mitochondrial copper depletion suppresses triple-negative breast cancer in mice. Nature biotechnology Cui, L., Gouw, A. M., LaGory, E. L., Guo, S., Attarwala, N., Tang, Y., Qi, J., Chen, Y., Gao, Z., Casey, K. M., Bazhin, A. A., Chen, M., Hu, L., Xie, J., Fang, M., Zhang, C., Zhu, Q., Wang, Z., Giaccia, A. J., Gambhir, S. S., Zhu, W., Felsher, D. W., Pegram, M. D., Goun, E. A., Le, A., Rao, J. 2020

  Abstract

  Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

  View details for DOI 10.1038/s41587-020-0707-9

  View details for PubMedID 33077961

• Deciphering Warburg effect: hypoxia inhibits tumor cell differentiation through reducing acetyl-CoA generation and chromatin accessibility Ye, J., Li, Y., Gruber, J. J., Litzenburger, U. M., Zhou, Y., Miao, Y. R., LaGory, E. L., Li, A. M., Hu, Z., Hart, L. S., Maris, J. M., Chang, H. Y., Giaccia, A. J. AMER ASSOC CANCER RESEARCH. 2020

  View details for DOI 10.1158/1538-7445.AM2020-5708

  View details for Web of Science ID 000590059302011

• Evaluation of Salmon, Tuna, and Beef Freshness Using a Portable Spectrometer. Sensors (Basel, Switzerland) Moon, E. J., Kim, Y., Xu, Y., Na, Y., Giaccia, A. J., Lee, J. H. 2020; 20 (15)

  Abstract

  There has been strong demand for the development of an accurate but simple method to assess the freshness of food. In this study, we demonstrated a system to determine food freshness by analyzing the spectral response from a portable visible/near-infrared (VIS/NIR) spectrometer using the Convolutional Neural Network (CNN)-based machine learning algorithm. Spectral response data from salmon, tuna, and beef incubated at 25 °C were obtained every minute for 30 h and then categorized into three states of "fresh", "likely spoiled", and "spoiled" based on time and pH. Using the obtained spectral data, a CNN-based machine learning algorithm was built to evaluate the freshness of experimental objects. In addition, a CNN-based machine learning algorithm with a shift-invariant feature can minimize the effect of the variation caused using multiple devices in a real environment. The accuracy of the obtained machine learning model based on the spectral data in predicting the freshness was approximately 85% for salmon, 88% for tuna, and 92% for beef. Therefore, our study demonstrates the practicality of a portable spectrometer in food freshness assessment.

  View details for DOI 10.3390/s20154299

  View details for PubMedID 32752216

• Validated limited gene predictor for cervical cancer lymph node metastases. Oncotarget Bloomstein, J. D., von Eyben, R., Chan, A., Rankin, E. B., Fregoso, D. R., Wang-Chiang, J., Lee, L., Xie, L., David, S. M., Stehr, H., Esfahani, M. S., Giaccia, A. J., Kidd, E. A. 2020; 11 (24): 2302–9

  Abstract

  PURPOSE: Recognizing the prognostic significance of lymph node (LN) involvement for cervical cancer, we aimed to identify genes that are differentially expressed in LN+ versus LN- cervical cancer and to potentially create a validated predictive gene signature for LN involvement.MATERIALS AND METHODS: Primary tumor biopsies were collected from 74 cervical cancer patients. RNA was extracted and RNA sequencing was performed. The samples were divided by institution into a training set (n = 57) and a testing set (n = 17). Differentially expressed genes were identified among the training cohort and used to train a Random Forest classifier.RESULTS: 22 genes showed > 1.5 fold difference in expression between the LN+ and LN- groups. Using forward selection 5 genes were identified and, based on the clinical knowledge of these genes and testing of the different combinations, a 2-gene Random Forest model of BIRC3 and CD300LG was developed. The classification accuracy of lymph node (LN) status on the test set was 88.2%, with an Area under the Receiver Operating Characteristic curve (ROC-AUC) of 98.6%.CONCLUSIONS: We identified a 2 gene Random Forest model of BIRC3 and CD300LG that predicted lymph node involvement in a validation cohort. This validated model, following testing in additional cohorts, could be used to create a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tool that would be useful for helping to identify patients with LN involvement in resource-limited settings.

  View details for DOI 10.18632/oncotarget.27632

  View details for PubMedID 32595829

• An activatable NIR fluorescent rosol for selectively imaging nitroreductase activity SENSORS AND ACTUATORS B-CHEMICAL Klockow, J. L., Hettie, K. S., LaGory, E. L., Moon, E., Giaccia, A. J., Graves, E. E., Chin, F. T. 2020; 306

  View details for DOI 10.1016/j.snb.2019.127446

  View details for Web of Science ID 000507459500002

• An Activatable NIR Fluorescent Rosol for Selectively Imaging Nitroreductase Activity. Sensors and actuators. B, Chemical Klockow, J. L., Hettie, K. S., LaGory, E. L., Moon, E. J., Giaccia, A. J., Graves, E. E., Chin, F. T. 2020; 306

  Abstract

  Hypoxia (pO2 ≤ ~1.5%) is an important characteristic of tumor microenvironments that directly correlates with resistance against first-line therapies and tumor proliferation/infiltration. The ability to accurately identify hypoxic tumor cells/tissue could afford tailored therapeutic regimens for personalized treatment, development of more-effective therapies, and discerning the mechanisms underlying disease progression. Fluorogenic constructs identifying aforesaid cells/tissue operate by targeting the bioreductive activity of primarily nitroreductases (NTRs), but collectively present photophysical and/or physicochemical shortcomings that could limit effectiveness. To overcome these limitations, we present the rational design, development, and evaluation of the first activatable ultracompact xanthene core-based molecular probe (NO 2 -Rosol) for selectively imaging NTR activity that affords an "OFF-ON" near-infrared (NIR) fluorescence response (> 700 nm) alongside a remarkable Stokes shift (> 150 nm) via NTR activity-facilitated modulation to its energetics whose resultant interplay discontinues an intramolecular d-PET fluorescence-quenching mechanism transpiring between directly-linked electronically-uncoupled π-systems comprising its components. DFT calculations guided selection of a suitable fluorogenic scaffold and nitroaromatic moiety candidate that when adjoined could (i) afford such photophysical response upon bioreduction by upregulated NTR activity in hypoxic tumor cells/tissue and (ii) employ a retention mechanism strategy that capitalizes on an inherent physical property of the NIR fluorogenic scaffold for achieving signal amplification. NO 2 -Rosol demonstrated 705 nm NIR fluorescence emission and 157 nm Stokes shift, selectivity for NTR over relevant bioanalytes, and a 28-/12-fold fluorescence enhancement in solution and between cells cultured under different oxic conditions, respectively. In establishing feasibility for NO 2 -Rosol to provide favorable contrast levels in solutio/vitro, we anticipate NO 2 -Rosol doing so in preclinical studies.

  View details for DOI 10.1016/j.snb.2019.127446

  View details for PubMedID 32265579

  View details for PubMedCentralID PMC7138224

• Retraction Note: Lysyl oxidase is essential for hypoxia-induced metastasis. Nature Erler, J. T., Bennewith, K. L., Nicolau, M., Dornhofer, N., Kong, C., Le, Q., Chi, J. A., Jeffrey, S. S., Giaccia, A. J. 2020; 579 (7799): 456

  Abstract

  A Retraction to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41586-020-2112-4

  View details for PubMedID 32188947

• Induced tumor heterogeneity reveals factors informing radiation and immunotherapy combinations. Clinical cancer research : an official journal of the American Association for Cancer Research Aguilera, T. A., Elghonaimy, E., Shehade, H., Rafat, M., Castellini, L., Jiang, D., Kariolis, M., Koong, A., Le, Q., Ellies, L. G., Rankin, E. B., Graves, E. E., Giaccia, A. J. 2020

  Abstract

  PURPOSE: To investigate how induced tumor heterogeneity influences immune responses to radiotherapy (RT) with different proportions of mixed immune responsive and unresponsive tumor cells in a triple negative breast cancer model. It is hypothesized that studying the immune environment of mixed tumors and responses to RT could nominate immune active therapies to enhance immune responses after RT.EXPERIMENTAL DESIGN: Evaluate efficacy and immune responses generated by RT in tumors with different proportions of immunologically responsive and unresponsive tumor cells. Then study the cellular responses and transcriptomic differences between the tumors to nominate immunotherapy combinations with RT and evaluate the combination.RESULTS: The addition of the responsive cells to unresponsive tumors led to a greater than expected therapeutic response to RT with both innate and adaptive immune components. There was a distinct change in myeloid cells, greater inflammatory macrophage activity, and enhanced antigen presentation with responsive cells after RT. Since differences in matrix components, cell adhesion biology, and innate immune signaling correlated with myeloid cell response and phenotype, we hypothesized that RT combined with CD40 agonist antibody would sensitize unresponsive tumors. The combination therapy resulted improved innate and adaptive immune response. Importantly, CD40 treatment increased tumor response to RT and protected against metastatic spread in a metastatic model.CONCLUSIONS: These data combined with transcriptomics from human patients supports RT and myeloid cell targeting for immunologically cold tumors and presents opportunities to investigate the complex overlapping biologic mechanisms that limit immunotherapy and to implement RT with different immunotherapy combinations.

  View details for DOI 10.1158/1078-0432.CCR-19-4220

  View details for PubMedID 32098769

• Acetate supplementation restores chromatin accessibility and promotes tumor cell differentiation under hypoxia. Cell death & disease Li, Y. n., Gruber, J. J., Litzenburger, U. M., Zhou, Y. n., Miao, Y. R., LaGory, E. L., Li, A. M., Hu, Z. n., Yip, M. n., Hart, L. S., Maris, J. M., Chang, H. Y., Giaccia, A. J., Ye, J. n. 2020; 11 (2): 102

  Abstract

  Despite the fact that Otto H. Warburg discovered the Warburg effect almost one hundred years ago, why cancer cells waste most of the glucose carbon as lactate remains an enigma. Warburg proposed a connection between the Warburg effect and cell dedifferentiation. Hypoxia is a common tumor microenvironmental stress that induces the Warburg effect and blocks tumor cell differentiation. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma differentiation model, we discovered that hypoxia repressed cell differentiation through reducing cellular acetyl-CoA levels, leading to reduction of global histone acetylation and chromatin accessibility. The metabolic switch triggering this global histone hypoacetylation was the induction of pyruvate dehydrogenase kinases (PDK1 and PDK3). Inhibition of PDKs using dichloroacetate (DCA) restored acetyl-CoA generation and histone acetylation under hypoxia. Knocking down PDK1 induced neuroblastoma cell differentiation, highlighting the critical role of PDK1 in cell fate control. Importantly, acetate or glycerol triacetate (GTA) supplementation restored differentiation markers expression and neuron differentiation under hypoxia. Moreover, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduced chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. In addition, hypoxia-induced histone hypermethylation by increasing 2-hydroxyglutarate (2HG) and reducing α-ketoglutarate (αKG). αKG supplementation reduced histone hypermethylation upon hypoxia, but did not restore histone acetylation or differentiation markers expression. Together, these findings suggest that diverting pyruvate flux away from acetyl-CoA generation to lactate production is the key mechanism that Warburg effect drives dedifferentiation and tumorigenesis. We propose that combining differentiation therapy with acetate/GTA supplementation might represent an effective therapy against neuroblastoma.

  View details for DOI 10.1038/s41419-020-2303-9

  View details for PubMedID 32029721

• The m6A RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor. Proceedings of the National Academy of Sciences of the United States of America Xiao, Y. n., Thakkar, K. N., Zhao, H. n., Broughton, J. n., Li, Y. n., Seoane, J. A., Diep, A. N., Metzner, T. J., von Eyben, R. n., Dill, D. L., Brooks, J. D., Curtis, C. n., Leppert, J. T., Ye, J. n., Peehl, D. M., Giaccia, A. J., Sinha, S. n., Rankin, E. B. 2020

  Abstract

  Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

  View details for DOI 10.1073/pnas.2000516117

  View details for PubMedID 32817424

• Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation. Communications biology Wu, P. H., Onodera, Y. n., Giaccia, A. J., Le, Q. T., Shimizu, S. n., Shirato, H. n., Nam, J. M. 2020; 3 (1): 620

  Abstract

  Enhanced invasiveness, a critical determinant of metastasis and poor prognosis, has been observed in cancer cells that survive cancer therapy, including radiotherapy. Here, we show that invasiveness in radiation-surviving cancer cells is associated with alterations in lysosomal exocytosis caused by the enhanced activation of Arl8b, a small GTPase that regulates lysosomal trafficking. The binding of Arl8b with its effector, SKIP, is increased after radiation through regulation of BORC-subunits. Knockdown of Arl8b or BORC-subunits decreases lysosomal exocytosis and the invasiveness of radiation-surviving cells. Notably, high expression of ARL8B and BORC-subunit genes is significantly correlated with poor prognosis in breast cancer patients. Sp1, an ATM-regulated transcription factor, is found to increase BORC-subunit genes expression after radiation. In vivo experiments show that ablation of Arl8b decreases IR-induced invasive tumor growth and distant metastasis. These findings suggest that BORC-Arl8b-mediated lysosomal trafficking is a target for improving radiotherapy by inhibiting invasive tumor growth and metastasis.

  View details for DOI 10.1038/s42003-020-01339-9

  View details for PubMedID 33110168

• Rab27b contributes to radioresistance and exerts a paracrine effect via epiregulin in glioblastoma. Neuro-oncology advances Nishioka, S., Wu, P., Yakabe, T., Giaccia, A. J., Le, Q., Aoyama, H., Shimizu, S., Shirato, H., Onodera, Y., Nam, J. 2020; 2 (1): vdaa091

  Abstract

  Background: Radiotherapy is the standard treatment for glioblastoma (GBM). However, radioresistance of GBM cells leads to recurrence and poor patient prognosis. Recent studies suggest that secretion factors have important roles in radioresistance of tumor cells. This study aims to determine whether Rab27b, a small GTPase involved in secretory vesicle trafficking, plays a role in radioresistance of GBM.Methods: Microarray analysis, cell viability analysis, apoptosis assay, immunostaining, and in vivo experiments were performed to assess the effect of Rab27b on radioresistance of GBM. We further investigated paracrine effects mediated by Rab27b after X-ray irradiation using coculture systems of glioma cell lines.Results: Rab27b was specifically upregulated in irradiated U87MG cells. Furthermore, Rab27b knockdown decreased the proliferation of GBM cells after irradiation. Knockdown of Rab27b in U87MG cells combined with radiation treatment suppressed orthotopic tumor growth in the mouse brain and prolonged the survival of recipient mice. Interestingly, the co-upregulation of Rab27b and epiregulin (EREG), a member of the epidermal growth factor (EGF) family, correlated with radioresistance in glioma cell lines. Additionally, EREG, which was secreted from U87MG cells via Rab27b-mediated mechanism, activated EGF receptor and contributed to H4 cell proliferation in a paracrine manner.Conclusions: Our results show that Rab27b mediates the radioresistance of highly malignant GBM cells. Rab27b promotes the proliferation of adjacent cells through EREG-mediated paracrine signaling after irradiation. Thus, the Rab27b-EREG pathway is a novel potential target to improve the efficacy of radiotherapy in GBM.

  View details for DOI 10.1093/noajnl/vdaa091

  View details for PubMedID 33409495

• Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer. British journal of cancer Tailor, D. n., Going, C. C., Resendez, A. n., Kumar, V. n., Nambiar, D. K., Li, Y. n., Dheeraj, A. n., LaGory, E. L., Ghoochani, A. n., Birk, A. M., Stoyanova, T. n., Ye, J. n., Giaccia, A. J., Le, Q. T., Singh, R. P., Sledge, G. W., Pitteri, S. J., Malhotra, S. V. 2020

  Abstract

  To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment.Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models.We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis.SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

  View details for DOI 10.1038/s41416-020-01137-4

  View details for PubMedID 33139797

• Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance. The Journal of clinical investigation Nambiar, D. K., Aguilera, T., Cao, H., Kwok, S., Kong, C., Bloomstein, J., Wang, Z., Rangan, V. S., Jiang, D., von Eyben, R., Liang, R., Agarwal, S., Colevas, A. D., Korman, A., Allen, C. T., Uppaluri, R., Koong, A. C., Giaccia, A., Le, Q. T. 2019

  Abstract

  Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.

  View details for DOI 10.1172/JCI129025

  View details for PubMedID 31710313

• Target-mediated drug disposition pharmacokinetic/pharmacodynamic model-informed dose selection for the first-in-human study of AVB-S6-500. Clinical and translational science Bonifacio, L., Dodds, M., Prohaska, D., Moss, A., Giaccia, A., Tabibiazar, R., McIntyre, G. 2019

  Abstract

  AVB-S6-500 neutralized growth arrest-specific 6 protein (GAS6) and effectively inhibited AXL signaling in preclinical cancer models. A target-mediated drug disposition (TMDD) pharmacokinetic/pharmacodynamic (PK/PD) model was used to select first-in-human (FIH) doses for AVB-S6-500 based on predicted target (GAS6) suppression in the clinic. The effect of TMDD on AVB-S6-500 clearance was incorporated into a standard 2-compartment model, providing parallel linear and nonlinear clearance. Observed AVB-S6-500 and GAS6 concentration data in cynomolgus monkeys and relevant interspecies differences were used to predict the PK (serum concentration)/PD (GAS6 suppression) relationship in humans. Human exposure and GAS6 suppression were simulated for the proposed FIH doses of 1, 2.5, 5, and 10 mg/kg. A dose of 1mg/kg was selected to target GAS6 suppression for 2weeks in the initial healthy volunteer study. The cynomolgus monkey:human ratios for the highest proposed FIH dose were anticipated to yield more than a 10-fold margin to the nonclinical no observed adverse event level while maintaining >90% GAS6 suppression. In human subjects, the first dose (1 mg/kg) model-projected and clinically-observed maximal concentration (Cmax ) was within 10% of predicted; repeat dosing at 5 mg/kg was within 1% (Cmax ) and 45% (AUC0-tau ) of predicted. Predicted GAS6 suppression duration of 14 days was accurate for the 1 mg/kg dose. A PK/PD model expedited clinical development of AVB-S6-500, minimized exposure of cancer patients to subtherapeutic doses, and rationally guided the optimal dosing in patients.

  View details for DOI 10.1111/cts.12706

  View details for PubMedID 31599479

• S100A10 is a critical mediator of GAS6/AXL-induced angiogenesis in renal cell carcinoma. Cancer research Xiao, Y., Zhao, H., Tian, L., Nolley, R., Diep, A. N., Ernst, A., Fuh, K. C., Miao, Y. R., von Eyben, R., Leppert, J. T., Brooks, J. D., Peehl, D. M., Giaccia, A. J., Rankin, E. B. 2019

  Abstract

  Angiogenesis is a hallmark of cancer that promotes tumor progression and metastasis. However, antiangiogenic agents have limited efficacy in cancer therapy due to the development of resistance. In clear cell renal cell carcinoma (ccRCC), AXL expression is associated with antiangiogenic resistance and poor survival. Here, we establish a role for GAS6/AXL signaling in promoting the angiogenic potential of ccRCC cells through the regulation of the plasminogen receptor S100A10. Genetic and therapeutic inhibition of AXL signaling in ccRCC tumor xenografts reduced tumor vessel density and growth under the renal capsule. GAS6/AXL signaling activated the expression of S100A10 through SRC to promote plasmin production, endothelial cell invasion and angiogenesis. Importantly, treatment with the small molecule AXL inhibitor cabozantinib or an ultra-high affinity soluble AXL Fc fusion decoy receptor (sAXL) reduced the growth of a pazopanib-resistant ccRCC patient-derived xenograft. Moreover, the combination of sAXL synergized with pazopanib and axitinib to reduce ccRCC patient-derived xenograft growth and vessel density. These findings highlight a role for AXL/S100A10 signaling in mediating the angiogenic potential of ccRCC cells and support the combination of AXL inhibitors with antiangiogenic agents for advanced ccRCC.

  View details for DOI 10.1158/0008-5472.CAN-19-1366

  View details for PubMedID 31585940

• HILPDA regulates lipid metabolism, lipid droplet abundance and response to microenvironmental stress in solid tumors. Molecular cancer research : MCR VandeKopple, M. J., Wu, J., Auer, E. N., Giaccia, A. J., Denko, N. C., Papandreou, I. 2019

  Abstract

  Accumulation of lipid droplets has been observed in an increasing range of tumors. However, the molecular determinants of this phenotype and the impact of the tumor microenvironment on lipid droplet dynamics are not well defined. The hypoxia-inducible lipid droplet associated protein HILPDA is known to regulate lipid storage and physiological responses to feeding conditions in mice, and was recently shown to promote hypoxic lipid droplet formation through inhibition of the rate-limiting adipose triglyceride lipase (ATGL). Here, we identify fatty acid loading and nutrient deprivation-induced autophagy as stimuli of HILPDA-dependent lipid droplet growth. Using mouse embryonic fibroblasts and human tumor cells, we found that genetic ablation of HILPDA compromised hypoxia- fatty acid- and starvation-induced lipid droplet formation and triglyceride storage. Nutrient deprivation upregulated HILPDA protein post-transcriptionally by a mechanism requiring autophagic flux and lipid droplet turnover, independent of HIF1 transactivation. Mechanistically, loss of HILPDA led to elevated lipolysis which could be corrected by inhibition of ATGL. Lipidomic analysis revealed not only quantitative but also qualitative differences in the glycerolipid and phospholipid profile of HILPDA WT and KO cells, indicating additional HILPDA functions impacting lipid metabolism. Deletion studies of HILPDA mutants identified the N-terminal hydrophobic domain as sufficient for targeting to lipid droplets and restoration of triglyceride storage. In vivo, HILPDA ablated cells showed decreased intratumoral triglyceride levels and impaired xenograft tumor growth associated with elevated levels of apoptosis. Implications: Tumor microenvironmental stresses induce changes in lipid droplet dynamics via HILPDA. Regulation of triglyceride hydrolysis is crucial for cell homeostasis and tumor growth.

  View details for DOI 10.1158/1541-7786.MCR-18-1343

  View details for PubMedID 31308147

• Papaverine and its novel derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism Benej, M., Hong, X., Vibhute, S., Scott, S., Wu, J., Graves, E., Quynh-Thu Le, Koong, A. C., Giaccia, A. J., Chen, C., Yu, B., Papandreou, I., Denko, N. C. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.AM2019-2927

  View details for Web of Science ID 000488279401375

• Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells Li, Y., Zhou, Y., Maris, J. M., Giaccia, A. J., Ye, J. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.SABCS18-3798

  View details for Web of Science ID 000488279403199

• The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment Hacker, B. C., Alves, S. M., Jiang, D., Koong, A. C., Giaccia, A. J., Graves, E. E., Rafat, M. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.AM2019-3931

  View details for Web of Science ID 000488279403328

• Modulating the tumor microenvironment to enhance efficacy of PARP inhibitors Mehibel, M., Xu, J., Diep, A., Thakkar, K., Li, C. G., Xiao, Y., Rankin, E., Giaccia, A. AMER SOC CLINICAL ONCOLOGY. 2019

  View details for DOI 10.1200/JCO.2019.37.15_suppl.e14715

  View details for Web of Science ID 000487345801012

• Targeting the Tumor Microenvironment in Radiation Oncology: Proceedings from the 2018 ASTRO-AACR Research Workshop CLINICAL CANCER RESEARCH McGee, H. M., Jiang, D., Soto-Pantoja, D. R., Nevler, A., Giaccia, A. J., Woodward, W. A. 2019; 25 (10): 2969–74

  View details for DOI 10.1158/1078-0432.CCR-18-3781

  View details for Web of Science ID 000468064200006

• Wounds Inhibit Tumor Growth In Vivo. Annals of surgery Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W. X., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R. E., Gurtner, G. C., Giaccia, A. J., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2019

  Abstract

  OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo.SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo.METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells.RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds.CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

  View details for PubMedID 30829705

• Targeting the Tumor Microenvironment in Radiation Oncology: Proceedings from the 2018 ASTRO-AACR Research Workshop. Clinical cancer research : an official journal of the American Association for Cancer Research McGee, H. M., Jiang, D., Soto-Pantoja, D. R., Nevler, A., Giaccia, A. J., Woodward, W. A. 2019

  Abstract

  The development of cancers and their response to radiation are intricately linked to the tumor microenvironment in which they reside. Tumor cells, immune cells, and stromal cells interact with each other and are influenced by the microbiome and metabolic state of the host, and these interactions are constantly evolving. Stromal cells not only secrete extracellular matrix and participate in wound contraction, but they also secrete fibroblast growth factors (FGFs), which mediate macrophage differentiation. Tumor associated macrophages (TAMs) migrate to hypoxic areas and secrete VEGF to promote angiogenesis. The microbiome and its byproducts alter the metabolic milieu by shifting the balance between glucose utilization and fatty acid oxidation, and these changes subsequently influence the immune response in the tumor microenvironment (TME). Not only does radiation exert cell autonomous effects on tumor cells, but it influences both the tumor-promoting and tumor-suppressive components of the TME. To gain a deeper understanding of how the tumor microenvironment influences the response to radiation, the American Society for Radiation Oncology (ASTRO) and the American Association of Cancer Research (AACR) organized a scientific workshop on July 26-27, 2018, which focused on how the microbiome, the immune response, the metabolome, and the stroma all can shift the balance between radiosensitivity and radioresistance. The proceedings from this workshop are discussed here and highlight recent discoveries in the field as well as the most important areas for future research.

  View details for PubMedID 30723144

• Irradiation or temozolomide chemotherapy enhances anti-CD47 treatment of glioblastoma. Innate immunity Gholamin, S. n., Youssef, O. A., Rafat, M. n., Esparza, R. n., Kahn, S. n., Shahin, M. n., Giaccia, A. J., Graves, E. E., Weissman, I. n., Mitra, S. n., Cheshier, S. H. 2019: 1753425919876690

  View details for DOI 10.1177/1753425919876690

  View details for PubMedID 31547758

• Lambda-Carrageenan Enhances the Effects of Radiation Therapy in Cancer Treatment by Suppressing Cancer Cell Invasion and Metastasis through Racgap1 Inhibition. Cancers Wu, P. H., Onodera, Y. n., Recuenco, F. C., Giaccia, A. J., Le, Q. T., Shimizu, S. n., Shirato, H. n., Nam, J. M. 2019; 11 (8)

  Abstract

  Radiotherapy is used extensively in cancer treatment, but radioresistance and the metastatic potential of cancer cells that survive radiation remain critical issues. There is a need for novel treatments to improve radiotherapy. Here, we evaluated the therapeutic benefit of λ-carrageenan (CGN) to enhance the efficacy of radiation treatment and investigated the underlying molecular mechanism. CGN treatment decreased viability in irradiated cancer cells and enhanced reactive oxygen species accumulation, apoptosis, and polyploid formation. Additionally, CGN suppressed radiation-induced chemoinvasion and invasive growth in 3D lrECM culture. We also screened target molecules using a gene expression microarray analysis and focused on Rac GTPase-activating protein 1 (RacGAP1). Protein expression of RacGAP1 was upregulated in several cancer cell lines after radiation, which was significantly suppressed by CGN treatment. Knockdown of RacGAP1 decreased cell viability and invasiveness after radiation. Overexpression of RacGAP1 partially rescued CGN cytotoxicity. In a mouse xenograft model, local irradiation followed by CGN treatment significantly decreased tumor growth and lung metastasis compared to either treatment alone. Taken together, these results suggest that CGN may enhance the effectiveness of radiation in cancer therapy by decreasing cancer cell viability and suppressing both radiation-induced invasive activity and distal metastasis through downregulating RacGAP1 expression.

  View details for DOI 10.3390/cancers11081192

  View details for PubMedID 31426369

• Suppressing Mitochondrial Respiration Is Critical for Hypoxia Tolerance in the Fetal Growth Plate. Developmental cell Yao, Q. n., Khan, M. P., Merceron, C. n., LaGory, E. L., Tata, Z. n., Mangiavini, L. n., Hu, J. n., Vemulapalli, K. n., Chandel, N. S., Giaccia, A. J., Schipani, E. n. 2019

  Abstract

  Oxygen (O2) is both an indispensable metabolic substrate and a regulatory signal that controls the activity of Hypoxia-Inducible Factor 1α (Hif1a), a mediator of the cellular adaptation to low O2 tension (hypoxia). Hypoxic cells require Hif1a to survive. Additionally, Hif1a is an inhibitor of mitochondrial respiration. Hence, we hypothesized that enhancing mitochondrial respiration is detrimental to the survival of hypoxic cells in vivo. We tested this hypothesis in the fetal growth plate, which is hypoxic. Our findings show that mitochondrial respiration is dispensable for survival of growth plate chondrocytes. Furthermore, its impairment prevents the extreme hypoxia and the massive chondrocyte death observed in growth plates lacking Hif1a. Consequently, augmenting mitochondrial respiration affects the survival of hypoxic chondrocytes by, at least in part, increasing intracellular hypoxia. We thus propose that partial suppression of mitochondrial respiration is crucial during development to protect the tissues that are physiologically hypoxic from lethal intracellular anoxia.

  View details for DOI 10.1016/j.devcel.2019.04.029

  View details for PubMedID 31105007

• Hypoxia-inducible factor 2alpha is a negative regulator of osteoblastogenesis and bone mass accrual. Bone research Merceron, C., Ranganathan, K., Wang, E., Tata, Z., Makkapati, S., Khan, M. P., Mangiavini, L., Yao, A. Q., Castellini, L., Levi, B., Giaccia, A. J., Schipani, E. 2019; 7: 7

  Abstract

  Osteoblasts, which are the bone-forming cells, operate in a hypoxic environment. The transcription factors hypoxia-inducible factor-1alpha (HIF1) and HIF2 are key mediators of the cellular response to hypoxia. Both are expressed in osteoblasts. HIF1 is known to be a positive regulator of bone formation. Conversely, the role of HIF2 in the control osteoblast biology is still poorly understood. In this study, we used mouse genetics to demonstrate that HIF2 is an inhibitor of osteoblastogenesis and bone mass accrual. Moreover, we provided evidence that HIF2 impairs osteoblast differentiation at least in part, by upregulating the transcription factor Sox9. Our findings constitute a paradigm shift, as activation of the hypoxia-signaling pathway has traditionally been associated with increased bone formation through HIF1. Inhibiting HIF2 could thus represent a therapeutic approach for the treatment of the low bone mass observed in chronic diseases, osteoporosis, or aging.

  View details for PubMedID 30792937

• Mutations in an Innate Immunity Pathway Are Associated with Poor Overall Survival Outcomes and Hypoxic Signaling in Cancer. Cell reports Olcina, M. M., Balanis, N. G., Kim, R. K., Aksoy, B. A., Kodysh, J., Thompson, M. J., Hammerbacher, J., Graeber, T. G., Giaccia, A. J. 2018; 25 (13): 3721

  Abstract

  Complement-mediated cytotoxicity may act as a selective pressure for tumor overexpression of complement regulators. We hypothesize that the same selective pressure could lead to complement alterations at the genetic level. We find that, when analyzed as a pathway, mutations in complement genes occur at a relatively high frequency and are associated with changes in overall survival across a number of cancer types. Analysis of pathways expressed in patients with complement mutations that are associated with poor overall survival reveals crosstalk between complement and hypoxia in colorectal cancer. The importance of this crosstalk is highlighted by two key findings: hypoxic signaling is increased in tumors harboring complement mutations, and hypoxic tumor cells are resistant to complement-mediated cytotoxicity due, in part, to hypoxia-induced expression of complement regulator CD55. The range of strategies employed by tumors to dysregulate the complement system testifies to the importance of this pathway in tumor progression.

  View details for PubMedID 30590044

• Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio-protocol Tandon, N., Thakkar, K. N., LaGory, E. L., Liu, Y., Giaccia, A. J. 2018; 8 (21)

  Abstract

  Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.

  View details for PubMedID 30505888

• Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity. Nature communications Chen, X., Litzenburger, U. M., Wei, Y., Schep, A. N., LaGory, E. L., Choudhry, H., Giaccia, A. J., Greenleaf, W. J., Chang, H. Y. 2018; 9 (1): 4590

  Abstract

  Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1alpha protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

  View details for PubMedID 30389926

• Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Benej, M., Hong, X., Vibhute, S., Scott, S., Wu, J., Graves, E., Le, Q., Koong, A. C., Giaccia, A. J., Yu, B., Chen, S., Papandreou, I., Denko, N. C. 2018; 115 (42): 10756–61

  Abstract

  Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.

  View details for PubMedID 30201710

• Endothelial HIF-2alpha is Required for the Maintenance of Airway Microvasculature. Circulation Jiang, X., Tian, W., Tu, A. B., Pasupneti, S., Shuffle, E., Dahms, P., Zhang, P., Cai, H., Dinh, T. T., Liu, B., Cain, C., Giaccia, A. J., Butcher, E. C., Simon, C., Semenza, G. L., Nicolls, M. R. 2018

  Abstract

  BACKGROUND: Hypoxia-inducible factors (HIFs), especially HIF-1alpha and HIF-2alpha, are key mediators of the adaptive response to hypoxic stress and play essential roles in maintaining lung homeostasis. Human and animal genetics studies confirm that abnormal HIF correlates with pulmonary vascular pathology and chronic lung diseases, but it remains unclear whether endothelial cell (EC) HIF production is essential for microvascular health. The large airway has an ideal circulatory bed for evaluating histologic changes and physiology in genetically-modified rodents.METHODS: The tracheal microvasculature of mice, with conditionally-deleted or overexpressed HIF-1alpha or HIF-2alpha, was evaluated for anatomy, perfusion, and permeability. Angiogenic signaling studies assessed vascular changes attributable to dysregulated HIF expression. An orthotopic tracheal transplantation model further evaluated the contribution of individual HIF isoforms in airway ECs.RESULTS: The genetic deletion of Hif-2alpha, but not Hif-1alpha, caused tracheal EC apoptosis, diminished pericyte coverage, reduced vascular perfusion, defective barrier function, overlying epithelial abnormalities and subepithelial fibrotic remodeling. HIF-2alpha promoted microvascular integrity in airways through endothelial angiopoietin-1/TIE2 signaling and Notch activity. In functional tracheal transplants, HIF-2alpha deficiency in airway donors accelerated graft microvascular loss, whereas HIF-2alpha or angiopoietin-1 overexpression prolonged transplant microvascular perfusion. Augmented endothelial HIF-2alpha in transplant donors promoted airway microvascular integrity and diminished alloimmune inflammation.CONCLUSIONS: Our findings reveal that the constitutive expression of endothelial HIF-2alpha is required for airway microvascular health.

  View details for PubMedID 30586708

• Targeting the CLCF1-CNTFR signaling axis using directed evolution for lung cancer therapy Marquez, C., Kim, J., Giaccia, A., Cochran, J., Sweet-Cordero, A. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-3918

  View details for Web of Science ID 000468819502175

• Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis Wu, P., Onodera, Y., Giaccia, A. J., Quynh-Thu Le, Shirato, H., Nam, J. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-3202

  View details for Web of Science ID 000468819501028

• Complement system mutational landscape reveals C4BPA mutations enhance apoptosis in an immune-independent manner Olcina, M. M., Balanis, N. G., Kim, R. K., Thompson, M. J., Graeber, T. G., Giaccia, A. J. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-4382

  View details for Web of Science ID 000468819503046

• The tumour microenvironment links complement system dysregulation and hypoxic signalling. The British journal of radiology Olcina, M. M., Kim, R. K., Melemenidis, S., Graves, E. E., Giaccia, A. J. 2018: 20180069

  Abstract

  The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation.

  View details for PubMedID 29544344

• Selection of external beam radiotherapy approaches for precise and accurate cancer treatment JOURNAL OF RADIATION RESEARCH Shirato, H., Quynh-Thu Le, Kobashi, K., Prayongrat, A., Takao, S., Shimizu, S., Giaccia, A., Xing, L., Umegaki, K. 2018; 59: I2–I10

  Abstract

  Physically precise external-beam radiotherapy (EBRT) technologies may not translate to the best outcome in individual patients. On the other hand, clinical considerations alone are often insufficient to guide the selection of a specific EBRT approach in patients. We examine the ways in which to compare different EBRT approaches based on physical, biological and clinical considerations, and how they can be enhanced with the addition of biophysical models and machine-learning strategies. The process of selecting an EBRT modality is expected to improve in tandem with knowledge-based treatment planning.

  View details for PubMedID 29373709

  View details for PubMedCentralID PMC5868193

• Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence Following Radiation Therapy in Immunosuppressed Patients. Cancer research Rafat, M. n., Aguilera, T. A., Vilalta, M. n., Bronsart, L. L., Soto, L. A., von Eyben, R. n., Golla, M. A., Ahrari, Y. n., Melemenidis, S. n., Afghahi, A. n., Jenkins, M. J., Kurian, A. W., Horst, K. n., Giaccia, A. J., Graves, E. E. 2018

  Abstract

  Although radiation therapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy (BCT). The relationship between RT and local recurrence is unknown. Here we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in TNBC patients, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared to non-recurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8+ T cells. Macrophages (CD11b+F480+) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of TNBC patients.

  View details for PubMedID 29880480

• PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S., Nauta, A. C., Morrison, S. D., Hu, M. S., Zimmermann, A. S., Chung, M. T., Glotzbach, J. P., Wong, V. W., Walmsley, G. G., Lorenz, H., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2018; 141 (1): 55E–67E

  Abstract

  Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing.Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics.PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05).Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

  View details for PubMedID 29280872

  View details for PubMedCentralID PMC5747314

• A Human Genome-wide RNAi Screen Reveals Diverse Modulators that Mediate IRE1α-XBP1 Activation. Molecular cancer research : MCR Yang, Z. n., Zhang, J. n., Jiang, D. n., Khatri, P. n., Solow-Cordero, D. E., Toesca, D. A., Koumenis, C. n., Denko, N. C., Giaccia, A. J., Le, Q. T., Koong, A. C. 2018

  Abstract

  Activation of the unfolded protein response (UPR) signaling pathways is linked to multiple human diseases including cancer. The inositol-requiring kinase 1 (IRE1)-X-box binding protein 1 (XBP1) pathway is the most evolutionarily conserved of the three major signaling branches of the UPR. Here, we performed a genome-wide siRNA screen to obtain a systematic assessment of genes integrated in the IRE1-XBP1 axis. We monitored the expression of an XBP1-luciferase chimeric protein in which luciferase was fused in-frame with the spliced (active) form of XBP1. Using cells expressing this reporter construct, we identified 162 genes for which siRNA inhibition resulted in alteration in XBP1 splicing. These genes express diverse types of proteins modulating a wide range of cellular processes. Pathway analysis identified a set of genes implicated in the pathogenesis of breast cancer. Several genes including BCL10, GCLM, and IGF1R correlated with worse relapse-free survival (RFS) in an analysis of patients with triple negative breast cancer (TNBC). However, in this cohort of 1908 patients, only high GCLM expression correlated with worse RFS in both TNBC and non-TNBC patients. Altogether, our study revealed unidentified roles of novel pathways regulating the UPR and these findings may serve as a paradigm for exploring novel therapeutic opportunities based on modulating the UPR.Genome-wide RNAi screen identifies novel genes/pathways that modulate IRE1-XBP1 signaling in human tumor cells and leads to the development of improved therapeutic approaches targeting the UPR.

  View details for PubMedID 29440447

• Impairment of Mitochondrial Respiration Promotes Survival of Hypoxic Chondrocytes In Vivo. Yao, Q., LaGory, E., Mangiavini, L., Hu, J., Merceron, C., Vemulapalli, K., Khatn, M., Tata, Z., Giaccia, A. J., Schipani, E. WILEY. 2017: S1

  View details for Web of Science ID 000418869200002

• Micro-environmental Features of the Bone and Bone Marrow Associated with Metastatic Colonization. Hartzman, M., Giaccia, A., Wu, C. WILEY. 2017: S313

  View details for Web of Science ID 000418869203196

• Long-range hypoxia signaling in NAFLD NATURE MEDICINE LaGory, E. L., Giaccia, A. J. 2017; 23 (11): 1251–52

  View details for PubMedID 29117167

• Hypoxia and Bone Metastatic Disease. Current osteoporosis reports Johnson, R. W., Sowder, M. E., Giaccia, A. J. 2017; 15 (4): 231-238

  Abstract

  This review highlights our current knowledge of oxygen tensions in the bone marrow, and how low oxygen tensions (hypoxia) regulate tumor metastasis to and colonization of the bone marrow.The bone marrow is a relatively hypoxic microenvironment, but oxygen tensions fluctuate throughout the marrow cavity and across the endosteal and periosteal surfaces. Recent advances in imaging have made it possible to better characterize these fluctuations in bone oxygenation, but technical challenges remain. We have compiled evidence from multiple groups that suggests that hypoxia or hypoxia inducible factor (HIF) signaling may induce spontaneous metastasis to the bone and promote tumor colonization of bone, particularly in the case of breast cancer dissemination to the bone marrow. We are beginning to understand oxygenation patterns within the bone compartment and the role for hypoxia and HIF signaling in tumor cell dissemination to the bone marrow, but further studies are warranted.

  View details for DOI 10.1007/s11914-017-0378-8

  View details for PubMedID 28597139

  View details for PubMedCentralID PMC5667665

• Pre-metastatic niches: organ-specific homes for metastases NATURE REVIEWS CANCER Peinado, H., Zhang, H., Matei, I. R., Costa-Silva, B., Hoshino, A., Rodrigues, G., Psaila, B., Kaplan, R. N., Bromberg, J. F., Kang, Y., Bissell, M. J., Cox, T. R., Giaccia, A. J., Erler, J. T., Hiratsuka, S., Ghajar, C. M., Lyden, D. 2017; 17 (5): 302–17

  Abstract

  It is well established that organs of future metastasis are not passive receivers of circulating tumour cells, but are instead selectively and actively modified by the primary tumour before metastatic spread has even occurred. Sowing the 'seeds' of metastasis requires the action of tumour-secreted factors and tumour-shed extracellular vesicles that enable the 'soil' at distant metastatic sites to encourage the outgrowth of incoming cancer cells. In this Review, we summarize the main processes and new mechanisms involved in the formation of the pre-metastatic niche.

  View details for PubMedID 28303905

• Patterns of Vasculature in Mouse Models of Lung Cancer Are Dependent on Location MOLECULAR IMAGING AND BIOLOGY Vilalta, M., Hughes, N. P., von Eyben, R., Giaccia, A. J., Graves, E. E. 2017; 19 (2): 215-224

  View details for DOI 10.1007/s11307-016-1010-5

  View details for Web of Science ID 000396058200005

• Molecular Pathways: Oncologic Pathways and Their Role in T-cell Exclusion and Immune Evasion-A New Role for the AXL Receptor Tyrosine Kinase. Clinical cancer research : an official journal of the American Association for Cancer Research Aguilera, T. A., Giaccia, A. J. 2017

  Abstract

  With the clinical impact of CTLA-4 and PD-1/PD-L1 immune checkpoint therapies, widespread interest in cancer immunotherapy has been ignited. However, the rate and extent of clinical responses to approved therapies are limited and often nonexistent in many solid tumors. This is partially because immune checkpoint therapies are most effective against T-cell-inflamed tumors, and non-T-cell-inflamed or T-cell-excluded tumors remain a significant barrier. New strategies are needed to overcome immune resistance mechanisms that arise during tumor development, which result in T-cell exclusion. Approaches may need to be combined with conventional therapies such as chemotherapy, radiotherapy, and molecularly targeted therapy, and many clinical trials are ongoing. This review discusses the challenge of T-cell exclusion and innate oncologic pathways that contribute to this problem, including β-catenin, STAT3, NF-κB, PTEN, and AXL tyrosine kinase. The GAS6/AXL pathway is of interest immunologically, as its targeting can lead to greater antitumor immune responses after radiotherapy. In addition, several targeted therapies that are selective and nonselective for AXL are in preclinical and clinical development in acute myelogenous leukemia and renal cell cancer. There remains much to learn, but the future is bright for anti-AXL therapies, though effective combinations and their impact may not be realized for years to come. Clin Cancer Res; 23(12); 2928-33. ©2017 AACR.

  View details for DOI 10.1158/1078-0432.CCR-17-0189

  View details for PubMedID 28289089

• KDM4B/JMJD2B is a p53 target gene that modulates the amplitude of p53 response after DNA damage. Nucleic acids research Castellini, L., Moon, E. J., Razorenova, O. V., Krieg, A. J., von Eyben, R., Giaccia, A. J. 2017

  Abstract

  The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.

  View details for DOI 10.1093/nar/gkw1281

  View details for PubMedID 28073943

• Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies JOURNAL OF CLINICAL INVESTIGATION Kariolis, M. S., Miao, Y. R., Diep, A., Nash, S. E., Olcina, M. M., Jiang, D., Jones, D. S., Kapur, S., Mathews, I. I., Koong, A. C., Rankin, E. B., Cochran, J. R., Giaccia, A. J. 2017; 127 (1): 183-198

  Abstract

  The AXL receptor and its activating ligand, growth arrest-specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. However, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. Here, we have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression in aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Moreover, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.

  View details for DOI 10.1172/JCI85610

  View details for Web of Science ID 000392271300021

  View details for PubMedID 27893463

  View details for PubMedCentralID PMC5199716

• Blimp1 induces transient metastatic heterogeneity in pancreatic cancer. Cancer discovery Chiou, S. H., Risca, V. I., Wang, G. X., Yang, D. n., Grüner, B. M., Kathiria, A. S., Ma, R. K., Vaka, D. n., Chu, P. n., Kozak, M. n., Castellini, L. n., Graves, E. E., Kim, G. E., Mourrain, P. n., Koong, A. C., Giaccia, A. J., Winslow, M. M. 2017

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. Using a novel genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor Blimp1 as a key driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes and hypoxia-mediated induction of Blimp1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which up-regulation of Blimp1 links microenvironmental cues to a metastatic stem cell character.

  View details for PubMedID 28790031

• Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells. International journal of nanomedicine Wu, P. H., Onodera, Y. n., Ichikawa, Y. n., Rankin, E. B., Giaccia, A. J., Watanabe, Y. n., Qian, W. n., Hashimoto, T. n., Shirato, H. n., Nam, J. M. 2017; 12: 5069–85

  Abstract

  Gold nanoparticles (AuNPs) have recently attracted attention as clinical agents for enhancing the effect of radiotherapy in various cancers. Although radiotherapy is a standard treatment for cancers, invasive recurrence and metastasis are significant clinical problems. Several studies have suggested that radiation promotes the invasion of cancer cells by activating molecular mechanisms involving integrin and fibronectin (FN). In this study, polyethylene-glycolylated AuNPs (P-AuNPs) were conjugated with Arg-Gly-Asp (RGD) peptides (RGD/P-AuNPs) to target cancer cells expressing RGD-binding integrins such as α5- and αv-integrins. RGD/P-AuNPs were internalized more efficiently and colocalized with integrins in the late endosomes and lysosomes of MDA-MB-231 cells. A combination of RGD/P-AuNPs and radiation reduced cancer cell viability and increased DNA damage compared to radiation alone in MDA-MB-231 cells. Moreover, the invasive activity of breast cancer cell lines after radiation treatment was significantly inhibited in the presence of RGD/P-AuNPs. Microarray analyses revealed that the expression of FN in irradiated cells was suppressed by combined use of RGD/P-AuNPs. Reduction of FN and downstream signaling may be involved in suppressing radiation-induced invasive activity by RGD/P-AuNPs. Our study suggests that RGD/P-AuNPs can target integrin-overexpressing cancer cells to improve radiation therapy by suppressing invasive activity in addition to sensitization. Thus, these findings provide a possible clinical strategy for using AuNPs to treat invasive breast cancer following radiotherapy.

  View details for PubMedID 28860745

  View details for PubMedCentralID PMC5560413

• Metabolic Alterations in Cancer and Their Potential as Therapeutic Targets. American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Meeting Weyandt, J. D., Thompson, C. B., Giaccia, A. J., Rathmell, W. K. 2017; 37: 825–32

  Abstract

  Otto Warburg's discovery in the 1920s that tumor cells took up more glucose and produced more lactate than normal cells provided the first clues that cancer cells reprogrammed their metabolism. For many years, however, it was unclear as to whether these metabolic alterations were a consequence of tumor growth or an adaptation that provided a survival advantage to these cells. In more recent years, interest in the metabolic differences in cancer cells has surged, as tumor proliferation and survival have been shown to be dependent upon these metabolic changes. In this educational review, we discuss some of the mechanisms that tumor cells use for reprogramming their metabolism to provide the energy and nutrients that they need for quick or sustained proliferation and discuss the potential for therapeutic targeting of these pathways to improve patient outcomes.

  View details for PubMedID 28561705

• Reprogramming the immunological microenvironment through radiation and targeting Axl NATURE COMMUNICATIONS Aguilera, T. A., Rafat, M., Castellini, L., Shehade, H., Kariolis, M. S., Hui, A. B., Stehr, H., von Eyben, R., Jiang, D., Ellies, L. G., Koong, A. C., Diehn, M., Rankin, E. B., Graves, E. E., Giaccia, A. J. 2016; 7

  Abstract

  Increasing evidence suggests that ionizing radiation therapy (RT) in combination with checkpoint immunotherapy is highly effective in treating a subset of cancers. To better understand the limited responses to this combination we analysed the genetic, microenvironmental, and immune factors in tumours derived from a transgenic breast cancer model. We identified two tumours with similar growth characteristics but different RT responses primarily due to an antitumour immune response. The combination of RT and checkpoint immunotherapy resulted in cures in the responsive but not the unresponsive tumours. Profiling the tumours revealed that the Axl receptor tyrosine kinase is overexpressed in the unresponsive tumours, and Axl knockout resulted in slower growth and increased radiosensitivity. These changes were associated with a CD8(+) T-cell response, which was improved in combination with checkpoint immunotherapy. These results suggest a novel role for Axl in suppressing antigen presentation through MHCI, and enhancing cytokine release, which promotes a suppressive myeloid microenvironment.

  View details for DOI 10.1038/ncomms13898

  View details for Web of Science ID 000390399500001

  View details for PubMedID 28008921

  View details for PubMedCentralID PMC5196438

• A New Chromatin-Cytoskeleton Link in Cancer MOLECULAR CANCER RESEARCH Giaccia, A. J. 2016; 14 (12): 1173-1175

  Abstract

  The set domain containing 2 (SETD2) histone methyltransferase, located at 3p2, specifically trimethylates lysine 36 of histone H3 (H3K36me3). H3K36me3 is an active mark involved in transcriptional elongation and RNA processing and a key regulator of DNA repair. In fact, SETD2 is the only methyltransferase that "writes" the H3K36me3 mark. Recent results from Park and colleagues have found a new role for SETD2 in the methylation of K40 of α-tubulin. Loss of SETD2 abolishes methylation of K40 of α-tubulin and results in a dysfunctional mitotic spindle and abnormalities in cytokinesis. Thus, SETD2 links chromatin and cytoskeleton homeostasis through its methyltransferase activity. These studies have important implications on the role of SETD2 mutations in promoting genomic instability and tumor progression. Mol Cancer Res; 14(12); 1173-5. ©2016 AACR.

  View details for DOI 10.1158/1541-7786.MCR-16-0250

  View details for Web of Science ID 000389632700001

  View details for PubMedID 27528705

  View details for PubMedCentralID PMC5136307

• The Receptor Tyrosine Kinase AXL in Cancer Progression. Cancers Rankin, E. B., Giaccia, A. J. 2016; 8 (11)

  Abstract

  The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies have revealed a central role of AXL signaling in tumor proliferation, survival, stem cell phenotype, metastasis, and resistance to cancer therapy. Moreover, AXL is expressed within cellular components of the tumor microenvironment where AXL signaling contributes to the immunosuppressive and protumorigenic phenotypes. A variety of AXL inhibitors have been developed and are efficacious in preclinical studies. These agents offer new opportunities for therapeutic intervention in the prevention and treatment of advanced disease. Here we review the literature that has illuminated the cellular and molecular mechanisms by which AXL signaling promotes tumor progression and we will discuss the therapeutic potential of AXL inhibition for cancer therapy.

  View details for PubMedID 27834845

• Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy. Proceedings of the National Academy of Sciences of the United States of America Chang, A. C., Ong, S., Lagory, E. L., Kraft, P. E., Giaccia, A. J., Wu, J. C., Blau, H. M. 2016

  Abstract

  Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx(4cv)/mTR(G2) mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

  View details for PubMedID 27799523

• Patterns of Vasculature in Mouse Models of Lung Cancer Are Dependent on Location. Molecular imaging and biology Vilalta, M., Hughes, N. P., von Eyben, R., Giaccia, A. J., Graves, E. E. 2016: -?

  Abstract

  Preclinical studies of hypoxia are generally done using ectopic xenograft tumors, which behave differently from human tumors. Our previous findings have shown that subcutaneously implanted lung tumors exhibit more hypoxia than their orthotopic implanted or spontaneous K-ras-induced counterparts. We hypothesize that differences in hypoxia are due to site-specific differences in vascularity and perfusion.To compare the presence and functionality of vessels in these tumor models, we studied vascular perfusion in vivo in real time.Orthotopically implanted and spontaneous K-ras-induced lung tumors showed elevated perfusion, demonstrating vasculature functionality. Little contrast agent uptake was observed within the subcutaneously implanted tumors, indicating vascular dysfunction. These findings were corroborated at the microscopic level with Hoechst 33342 and Meca-32 staining.From these observations, we concluded that differences in hypoxia in experimental models is related to vessel perfusion. Thus, appropriate selection of preclinical lung tumor models is essential for the study of hypoxia, angiogenesis and therapies targeting these phenomena.

  View details for PubMedID 27709411

• Reducing radiation-induced gastrointestinal toxicity - the role of the PHD/HIF axis JOURNAL OF CLINICAL INVESTIGATION Olcina, M. M., Giaccia, A. J. 2016; 126 (10): 3708-3715

  Abstract

  Radiotherapy is an effective treatment strategy for cancer, but a significant proportion of patients experience radiation-induced toxicity due to damage to normal tissue in the irradiation field. The use of chemical or biological approaches aimed at reducing or preventing normal tissue toxicity induced by radiotherapy is a long-held goal. Hypoxia-inducible factors (HIFs) regulate the production of factors that may protect several cellular compartments affected by radiation-induced toxicity. Pharmacological inhibitors of prolyl hydroxylase domain-containing enzymes (PHDs), which result in stabilization of HIFs, have recently been proposed as a new class of radioprotectors. In this review, radiation-induced toxicity in the gastrointestinal (GI) tract and the main cellular compartments studied in this context will be discussed. The effects of PHD inhibition on GI radioprotection will be described in detail.

  View details for DOI 10.1172/JCI84432

  View details for Web of Science ID 000384703300011

  View details for PubMedID 27548524

  View details for PubMedCentralID PMC5096800

• Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the, bone marrow NATURE CELL BIOLOGY Johnson, R. W., Fingers, E. C., Olcina, M. M., Vilalta, M., Aguilera, T., Miao, Y., Merkel, A. R., Johnson, J. R., Sterling, J. A., Wu, J. Y., Giaccia, A. J. 2016; 18 (10): 1078-1089

  Abstract

  Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.

  View details for DOI 10.1038/ncb3408

  View details for Web of Science ID 000384961700007

  View details for PubMedID 27642788

• Acridine Derivatives as Inhibitors of the IRE1a-XBP1 Pathway Are Cytotoxic to Human Multiple Myeloma. Molecular cancer therapeutics Jiang, D., Tam, A. B., Alagappan, M., Hay, M. P., Gupta, A., Kozak, M. M., Solow-Cordero, D. E., Lum, P. Y., Denko, N. C., Giaccia, A. J., Le, Q., Niwa, M., Koong, A. C. 2016; 15 (9): 2055-2065

  Abstract

  Using a luciferase reporter-based high throughput chemical library screen and topological data analysis (TDA), we identified N-acridine-9-yl-N',N'-dimethylpropane-1,3-diamine (DAPA) as a inhibitor of the IRE1α-XBP1 pathway of the unfolded protein response (UPR). We designed a collection of analogues based on the structure of DAPA to explore structure-activity relationships (SAR) and identified N9-(3-(dimethylamino)propyl)-N3,N3,N6,N6-tetramethylacridine-3,6,9-triamine (3,6-DMAD), with 3,6-dimethylamino substitution on the chromophore, as a potent inhibitor. 3,6-DMAD inhibited both IRE1α oligomerization and in vitro endoribonuclease (RNase) activity, while the other analogues only blocked IRE1α oligomerization. Consistent with the inhibition of IRE1α-mediated XBP1 splicing, which is critical for multiple myeloma (MM) cell survival, these analogues were cytotoxic to MM cell lines. Furthermore, 3,6-DMAD inhibited XBP1 splicing and the growth of MM tumor xenografts. Our study not only confirmed the utilization of topological data analysis in drug discovery but also identified a class of compounds with a unique mechanism of action as potent IRE1α-XBP1 inhibitors in the treatment of MM.

  View details for DOI 10.1158/1535-7163.MCT-15-1023

  View details for PubMedID 27307600

• Hypoxia: Signaling the Metastatic Cascade. Trends in cancer Rankin, E. B., Nam, J. M., Giaccia, A. J. 2016; 2 (6): 295-304

  Abstract

  Hypoxia is a potent microenvironmental factor that promotes tumor metastasis. Recent studies have revealed mechanisms by which hypoxia and activation of hypoxia inducible factor (HIF)-dependent signaling promotes metastasis through the regulation of metabolic reprogramming, the stem cell phenotype, invasion, angiogenesis, immune suppression, the premetastatic niche, intravasation and/or extravasation, and resistance to apoptosis. These discoveries suggest novel paradigms in tumor metastasis and identify new opportunities for therapeutic intervention in the prevention and treatment of metastatic disease. Here, we review the impact of hypoxia and hypoxic signaling pathways in tumor and stromal cells on each step of the metastatic cascade.

  View details for DOI 10.1016/j.trecan.2016.05.006

  View details for PubMedID 28741527

  View details for PubMedCentralID PMC5808868

• Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma ONCOGENE Zhou, L., Liu, X., Sun, M., Zhang, X., German, P., Bai, S., Ding, Z., Tannir, N., Wood, C. G., Matin, S. F., Karam, J. A., Tamboli, P., Sircar, K., Rao, P., Rankin, E. B., Laird, D. A., Hoang, A. G., Walker, C. L., Giaccia, A. J., Jonasch, E. 2016; 35 (21): 2687-2697

  Abstract

  Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial-mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and β-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

  View details for DOI 10.1038/onc.2015.343

  View details for Web of Science ID 000377472700003

  View details for PubMedID 26364599

  View details for PubMedCentralID PMC4791213

• Hypoxic control of metastasis SCIENCE Rankin, E. B., Giaccia, A. J. 2016; 352 (6282): 175-180

  Abstract

  Metastatic disease is the leading cause of cancer-related deaths and involves critical interactions between tumor cells and the microenvironment. Hypoxia is a potent microenvironmental factor promoting metastatic progression. Clinically, hypoxia and the expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are associated with increased distant metastasis and poor survival in a variety of tumor types. Moreover, HIF signaling in malignant cells influences multiple steps within the metastatic cascade. Here we review research focused on elucidating the mechanisms by which the hypoxic tumor microenvironment promotes metastatic progression. These studies have identified potential biomarkers and therapeutic targets regulated by hypoxia that could be incorporated into strategies aimed at preventing and treating metastatic disease.

  View details for DOI 10.1126/science.aaf4405

  View details for Web of Science ID 000373681600036

  View details for PubMedID 27124451

  View details for PubMedCentralID PMC4898055

• The ever-expanding role of HIF in tumour and stromal biology. Nature cell biology Lagory, E. L., Giaccia, A. J. 2016; 18 (4): 356-365

  Abstract

  Low oxygen tension (hypoxia) is a hallmark of cancer that influences cancer cell function, but is also an important component of the tumour microenvironment as it alters the extracellular matrix, modulates the tumour immune response and increases angiogenesis. Here we discuss the regulation and role of hypoxia and its key transcriptional mediators, the hypoxia-inducible factor (HIF) family of transcription factors, in the tumour microenvironment and stromal compartments.

  View details for DOI 10.1038/ncb3330

  View details for PubMedID 27027486

• Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1 SCIENTIFIC REPORTS Leszczynska, K. B., Goettgens, E., Biasoli, D., Olcina, M. M., Ient, J., Anbalagan, S., Bernhardt, S., Giaccia, A. J., Hammond, E. M. 2016; 6

  View details for DOI 10.1038/srep21698

  View details for Web of Science ID 000370045100001

  View details for PubMedID 26875667

• Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1. Scientific reports Leszczynska, K. B., Göttgens, E., Biasoli, D., Olcina, M. M., Ient, J., Anbalagan, S., Bernhardt, S., Giaccia, A. J., Hammond, E. M. 2016; 6: 21698-?

  Abstract

  Hypoxia-induced replication stress is one of the most physiologically relevant signals known to activate ATM in tumors. Recently, the ATM interactor (ATMIN) was identified as critical for replication stress-induced activation of ATM in response to aphidicolin and hydroxyurea. This suggests an essential role for ATMIN in ATM regulation during hypoxia, which induces replication stress. However, ATMIN also has a role in base excision repair, a process that has been demonstrated to be repressed and less efficient in hypoxic conditions. Here, we demonstrate that ATMIN is dispensable for ATM activation in hypoxia and in contrast to ATM, does not affect cell survival and radiosensitivity in hypoxia. Instead, we show that in hypoxic conditions ATMIN expression is repressed. Repression of ATMIN in hypoxia is mediated by both p53 and HIF-1α in an oxygen dependent manner. The biological consequence of ATMIN repression in hypoxia is decreased expression of the target gene, DYNLL1. An expression signature associated with p53 activity was negatively correlated with DYNLL1 expression in patient samples further supporting the p53 dependent repression of DYNLL1. Together, these data demonstrate multiple mechanisms of ATMIN repression in hypoxia with consequences including impaired BER and down regulation of the ATMIN transcriptional target, DYNLL1.

  View details for DOI 10.1038/srep21698

  View details for PubMedID 26875667

  View details for PubMedCentralID PMC4753685

• Measuring the Impact of Microenvironmental Conditions on Mitochondrial Dehydrogenase Activity in Cultured Cells. Advances in experimental medicine and biology Sun, R. C., Koong, A., Giaccia, A., Denko, N. C. 2016; 899: 113-120

  Abstract

  Mitochondria are powerhouses of a cell, producing much of the cellular ATP. However, mitochondrial enzymes also participate in many cellular biosynthetic processes. They are responsible for helping to maintain NAD(P)/H and redox balance, supplying metabolic intermediates for cell growth, and regulating several types of programed cell death. Several mitochondrial enzymes have even been shown to participate in the oncogenic process such as isocitrate dehydrogenase, succinate dehydrogenase, and fumarate hydratase. Recent advances have identified significant metabolic changes in the mitochondria that are regulated by malignant transformation and environmental stimuli. Understanding the biological activity and regulation of mitochondrial enzymes can provide insight into how they participate in the process of oncogenic transformation and work to sustain malignant growth. This chapter describes a technique to measure mitochondrial dehydrogenase activities that is faster and more cost effective which can also be scaled up for high throughput.

  View details for DOI 10.1007/978-3-319-26666-4_7

  View details for PubMedID 27325264

  View details for PubMedCentralID PMC5017243

• Identification of Doxorubicin as an Inhibitor of the IRE1a-XBP1 Axis of the Unfolded Protein Response. Scientific reports Jiang, D., Lynch, C., Medeiros, B. C., Liedtke, M., Bam, R., Tam, A. B., Yang, Z., Alagappan, M., Abidi, P., Le, Q., Giaccia, A. J., Denko, N. C., Niwa, M., Koong, A. C. 2016; 6: 33353-?

  Abstract

  Activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR) has been implicated in multiple types of human cancers, including multiple myeloma (MM). Through an in silico drug discovery approach based on protein-compound virtual docking, we identified the anthracycline antibiotic doxorubicin as an in vitro and in vivo inhibitor of XBP1 activation, a previously unknown activity for this widely utilized cancer chemotherapeutic drug. Through a series of mechanistic and phenotypic studies, we showed that this novel activity of doxorubicin was not due to inhibition of topoisomerase II (Topo II). Consistent with its inhibitory activity on the IRE1α-XBP1 branch of the UPR, doxorubicin displayed more potent cytotoxicity against MM cell lines than other cancer cell lines that have lower basal IRE1α-XBP1 activity. In addition, doxorubicin significantly inhibited XBP1 activation in CD138(+) tumor cells isolated from MM patients. Our findings suggest that the UPR-modulating activity of doxorubicin may be utilized clinically to target IRE1α-XBP1-dependent tumors such as MM.

  View details for DOI 10.1038/srep33353

  View details for PubMedID 27634301

  View details for PubMedCentralID PMC5025885

• Isolation of Proteins on Nascent DNA in Hypoxia and Reoxygenation Conditions. Advances in experimental medicine and biology Olcina, M. M., Giaccia, A. J., Hammond, E. M. 2016; 899: 27-40

  Abstract

  During S-phase both DNA replication and histone deposition must be co-ordinated at and around the replication fork. Replication stress can interfere with the fidelity of this process and can result in genomic instability. The study of proteins associated with DNA replication forks is important for a detailed understanding of DNA replication and chromatin assembly both under basal as well as replication stress conditions. iPOND (isolation of Proteins on Nascent DNA) allows the temporal study of proteins and protein modifications associated with replication forks in a variety of conditions, allowing the 'tracing' of protein association and histone deposition and maturation at active, stalled and damaged replication forks. Importantly, low oxygen (hypoxic) conditions, found in tumours, can result in replication stress. Here we describe the adaptation of the iPOND technique allowing the isolation of proteins and protein modifications specifically with replication forks undergoing hypoxia-induced replication stress. Furthermore, we describe the adaptation of this method for the study of factors associated with replication forks recovering from hypoxia-induced replication stress following periods of reoxygenation. These adaptations are important in order to study proteins associated with replication forks undergoing replication stress in physiologically relevant conditions.

  View details for DOI 10.1007/978-3-319-26666-4_3

  View details for PubMedID 27325260

• Fibrosis and Hypoxia-Inducible Factor-1 alpha-Dependent Tumors of the Soft Tissue on Loss of Von Hippel-Lindau in Mesenchymal Progenitors AMERICAN JOURNAL OF PATHOLOGY Mangiavini, L., Merceron, C., Araldi, E., Khatri, R., Gerard-O'Riley, R., Wilson, T. L., Sandusky, G., Abadie, J., Lyons, K. M., Giaccia, A. J., Schipani, E. 2015; 185 (11): 3090-3101

  View details for DOI 10.1016/j.ajpath.2015.07.008

  View details for PubMedID 26348575

• Risk of breast cancer among patients with bioprosthetic or mechanical valve replacement: a population-based study in Sweden. Breast cancer research and treatment Ji, J., Zöller, B., Giaccia, A., Haile, R., Sundquist, J., Sundquist, K. 2015; 154 (2): 369-75

  Abstract

  The association between breast cancer and warfarin is inconclusive as most previous studies examined their association using patients with thromboembolism, whereas thromboembolism itself is a risk factor for cancer. We explored this issue using patients received mechanical heart valves replacement as a proxy for warfarin exposure as these patients need a lifelong warfarin treatment, and compared them with patients received bioprosthesis valves replacement (short-term warfarin treatment) in Sweden between 1987 and 2010. Patients who were operated on for valve replacement were identified from the Swedish Hospital Discharge Registry and linked to the Swedish Cancer Registry to examine the hazard ratios of subsequent breast cancer. A total of 12,242 women were operated on for valve replacement (5481 with mechanical valve and 6401 with bioprosthetic valve). For the entire cohort, the HR of breast cancer was 1.49 (95 % CI 1.09-2.02) among patients with mechanical valve replacement compared to those with bioprosthetic valve replacement. After controlling for a number of confounding factors using propensity score weighting, the HR was 1.69 (95 % CI 1.15-2.47). Our study found that patients with mechanical valve replacement have an increased risk of breast cancer compared to those with bioprosthetic valve replacement. If confirmed, this increased risk should be considered when recommending breast cancer screening for women with mechanical valve replacement. Long-term use of warfarin may explain the observed increase. If so, patients who have used warfarin long-term for other reasons should be studied for a possible increased risk of breast cancer.

  View details for DOI 10.1007/s10549-015-3607-9

  View details for PubMedID 26476725

• Emerging Treatment Paradigms in Radiation Oncology. Clinical cancer research Le, Q., Shirato, H., Giaccia, A. J., Koong, A. C. 2015; 21 (15): 3393-3401

  Abstract

  Rapid advancements in radiotherapy and molecularly targeted therapies have resulted in the development of potential paradigm-shifting use of radiotherapy in the treatment of cancer. In this review, we discuss some of the most promising therapeutic approaches in the field of radiation oncology. These strategies include the use of highly targeted stereotactic radiotherapy and particle therapy as well as combining radiotherapy with agents that modulate the DNA damage response, augment the immune response, or protect normal tissues. Clin Cancer Res; 21(15); 3393-401. ©2015 AACR.

  View details for DOI 10.1158/1078-0432.CCR-14-1191

  View details for PubMedID 25991820

  View details for PubMedCentralID PMC4526434

• Suppression of PGC-1a Is Critical for Reprogramming Oxidative Metabolism in Renal Cell Carcinoma. Cell reports Lagory, E. L., Wu, C., Taniguchi, C. M., Ding, C. C., Chi, J., von Eyben, R., Scott, D. A., Richardson, A. D., Giaccia, A. J. 2015; 12 (1): 116-127

  Abstract

  Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.

  View details for DOI 10.1016/j.celrep.2015.06.006

  View details for PubMedID 26119730

  View details for PubMedCentralID PMC4518559

• Gastrointestinal Toxicities With Combined Antiangiogenic and Stereotactic Body Radiation Therapy INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Pollom, E. L., Deng, L., Pai, R. K., Brown, J. M., Giaccia, A., Loo, B. W., Shultz, D. B., Quynh Thu Le, Q. T., Koong, A. C., Chang, D. T. 2015; 92 (3): 568-576

  Abstract

  Combining the latest targeted biologic agents with the most advanced radiation technologies has been an exciting development in the treatment of cancer patients. Stereotactic body radiation therapy (SBRT) is an ablative radiation approach that has become established for the treatment of a variety of malignancies, and it has been increasingly used in combination with biologic agents, including those targeting angiogenesis-specific pathways. Multiple reports have emerged describing unanticipated toxicities arising from the combination of SBRT and angiogenesis-targeting agents, particularly of late luminal gastrointestinal toxicities. In this review, we summarize the literature describing these toxicities, explore the biological mechanism of action of toxicity with the combined use of antiangiogenic therapies, and discuss areas of future research, so that this combination of treatment modalities can continue to be used in broader clinical contexts.

  View details for DOI 10.1016/j.ijrobp.2015.02.016

  View details for Web of Science ID 000355636800018

• Gastrointestinal Toxicities With Combined Antiangiogenic and Stereotactic Body Radiation Therapy. International journal of radiation oncology, biology, physics Pollom, E. L., Deng, L., Pai, R. K., Brown, J. M., Giaccia, A., Loo, B. W., Shultz, D. B., Le, Q. T., Koong, A. C., Chang, D. T. 2015; 92 (3): 568-576

  Abstract

  Combining the latest targeted biologic agents with the most advanced radiation technologies has been an exciting development in the treatment of cancer patients. Stereotactic body radiation therapy (SBRT) is an ablative radiation approach that has become established for the treatment of a variety of malignancies, and it has been increasingly used in combination with biologic agents, including those targeting angiogenesis-specific pathways. Multiple reports have emerged describing unanticipated toxicities arising from the combination of SBRT and angiogenesis-targeting agents, particularly of late luminal gastrointestinal toxicities. In this review, we summarize the literature describing these toxicities, explore the biological mechanism of action of toxicity with the combined use of antiangiogenic therapies, and discuss areas of future research, so that this combination of treatment modalities can continue to be used in broader clinical contexts.

  View details for DOI 10.1016/j.ijrobp.2015.02.016

  View details for PubMedID 26068491

• HIF targets in bone remodeling and metastatic disease PHARMACOLOGY & THERAPEUTICS Johnson, R. W., Schipani, E., Giaccia, A. J. 2015; 150: 169-177

  Abstract

  The bone marrow is a hypoxic microenvironment that is rich in growth factors and blood vessels and is readily colonized by tumor cells disseminated from numerous cancers including tumors of the breast, prostate, lung, and skin. The origin of metastatic growth promoting factors for tumor cells disseminated to the bone marrow is derived from multiple sources: the bone matrix, which is a reservoir for growth factors, and cells residing in the marrow and along bone surfaces, such as osteoblasts, osteoclasts, macrophages, and T cells, which secrete cytokines and chemokines. Low oxygen levels within the bone marrow induce hypoxia signaling pathways such as hypoxia inducible factor (HIF), which is regulated by oxygen requiring prolyl hydroxylases (PHDs) and von Hippel-Lindau (VHL) tumor suppressor. These hypoxia signaling pathways have profound effects on bone development and homeostasis. Likewise, hypoxic conditions observed in local breast and prostate tumors point to a role for hypoxia-inducible genes in metastasis to and colonization of the bone marrow. This review will explore the role of hypoxia-regulated factors in bone development and remodeling, and how these elements may contribute to solid tumor metastasis to the bone.

  View details for DOI 10.1016/j.pharmthera.2015.02.002

  View details for Web of Science ID 000355028400011

  View details for PubMedID 25681658

  View details for PubMedCentralID PMC4414805

• Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin GENES & DEVELOPMENT Wu, C., Rankin, E. B., Castellini, L., Fernandez-Alcudia, J., Lagory, E. L., Andersen, R., Rhodes, S. D., Wilson, T. L., Mohammad, K. S., Castillo, A. B., Guise, T. A., Schipani, E., Giaccia, A. J. 2015; 29 (8): 817-831

  Abstract

  The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

  View details for DOI 10.1101/gad.255000.114

  View details for Web of Science ID 000353027600004

  View details for PubMedID 25846796

  View details for PubMedCentralID PMC4403258

• Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Finger, E. C., Castellini, L., Rankin, E. B., Vilalta, M., Krieg, A. J., Jiang, D., Banh, A., Zundel, W., Powell, M. B., Giaccia, A. J. 2015; 112 (14): 4441-4446

  Abstract

  Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

  View details for DOI 10.1073/pnas.1418164112

  View details for PubMedID 25792458

• The end of the hypoxic EPOch. International journal of radiation oncology, biology, physics Aguilera, T. A., Giaccia, A. J. 2015; 91 (5): 895-7

  View details for DOI 10.1016/j.ijrobp.2015.01.041

  View details for PubMedID 25832683

• Analysis of p53 Transactivation Domain Mutants Reveals Acad11 as a Metabolic Target Important for p53 Pro-Survival Function. Cell reports Jiang, D., Lagory, E. L., Kenzelmann Brož, D., Bieging, K. T., Brady, C. A., Link, N., Abrams, J. M., Giaccia, A. J., Attardi, L. D. 2015; 10 (7): 1096-1109

  Abstract

  The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

  View details for DOI 10.1016/j.celrep.2015.01.043

  View details for PubMedID 25704813

• Analysis of p53 Transactivation Domain Mutants Reveals Acad11 as a Metabolic Target Important for p53 Pro-Survival Function CELL REPORTS Jiang, D., Lagory, E. L., Broz, D. K., Bieging, K. T., Brady, C. A., Link, N., Abrams, J. M., Giaccia, A. J., Attardi, L. D. 2015; 10 (7): 1096-1109

  Abstract

  The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

  View details for DOI 10.1016/j.celrep.2015.01.043

  View details for Web of Science ID 000349918700007

• Dual roles of NRF2 in tumor prevention and progression: Possible implications in cancer treatment FREE RADICAL BIOLOGY AND MEDICINE Moon, E. J., Giaccia, A. 2015; 79: 292-299

  Abstract

  The cap'n'collar (CNC) family serves as cellular sensors of oxidative and electrophilic stresses and shares structural similarities including basic leucine zipper (bZIP) and CNC domains. They form heterodimers with small MAF proteins to regulate antioxidant and phase II enzymes through antioxidant response element (ARE)-mediated transactivation. Among the CNC family members, NRF2 is required for systemic protection against redox-mediated injury and carcinogenesis. On the other hand, NRF2 is activated by oncogenic pathways, metabolism, and hypoxia. Constitutive NRF2 activation is observed in a variety of human cancers and it is highly correlated with tumor progression and aggressiveness. In this review, we will discuss how NRF2 plays dual roles in cancer prevention and progression depending on the cellular context and environment. Therefore, a better understanding of NRF2 will be necessary to exploit this complex network of balancing antioxidant pathways to inhibit tumor progression.

  View details for DOI 10.1016/j.freeradbiomed.2014.11.009

  View details for Web of Science ID 000350392900028

  View details for PubMedID 25458917

  View details for PubMedCentralID PMC4339613

• Osteoblasts: a novel source of erythropoietin. Current osteoporosis reports Wu, C., Giaccia, A. J., Rankin, E. B. 2014; 12 (4): 428-432

  Abstract

  Osteoblasts are an important cellular component of the bone microenvironment controlling bone formation and hematopoiesis. Understanding the cellular and molecular mechanisms by which osteoblasts regulate these processes is a rapidly growing area of research given the important implications for bone therapy, regenerative medicine, and hematopoietic stem cell transplantation. Here we summarize our current knowledge regarding the cellular and molecular crosstalk driving bone formation and hematopoiesis and will discuss the implications of a recent finding demonstrating that osteoblasts are a cellular source of erythropoietin .

  View details for DOI 10.1007/s11914-014-0236-x

  View details for PubMedID 25204993

• Osteoblasts: a novel source of erythropoietin. Current osteoporosis reports Wu, C., Giaccia, A. J., Rankin, E. B. 2014; 12 (4): 428-432

  View details for DOI 10.1007/s11914-014-0236-x

  View details for PubMedID 25204993

• Galectin-1 Mediates Radiation-Related Lymphopenia and Attenuates NSCLC Radiation Response CLINICAL CANCER RESEARCH Kuo, P., Bratman, S. V., Shultz, D. B., von Eyben, R., Chan, C., Wang, Z., Say, C., Gupta, A., Loo, B. W., Giaccia, A. J., Koong, A. C., Diehn, M., Quynh-Thu Le, Q. T. 2014; 20 (21): 5558-5569

  Abstract

  Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non-small cell lung cancer (NSCLC)] and head and neck cancer patients.LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8(+) T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis. Clin Cancer Res; 20(21); 5558-69. ©2014 AACR.

  View details for DOI 10.1158/1078-0432.CCR-14-1138

  View details for Web of Science ID 000344759100024

  View details for PubMedCentralID PMC4216761

• An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis. Nature chemical biology Kariolis, M. S., Miao, Y. R., Jones, D. S., Kapur, S., Mathews, I. I., Giaccia, A. J., Cochran, J. R. 2014; 10 (11): 977-983

  Abstract

  Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

  View details for DOI 10.1038/nchembio.1636

  View details for PubMedID 25242553

• An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis NATURE CHEMICAL BIOLOGY Kariolis, M. S., Miao, Y. R., Ii, D. S., Kapur, S., Mathews, I. I., Giaccia, A. J., Cochran, J. R. 2014; 10 (11): 977-983

  Abstract

  Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

  View details for DOI 10.1038/NCHEMBIO.1636

  View details for Web of Science ID 000343621500022

  View details for PubMedCentralID PMC4372605

• Galectin-1 mediates radiation-related lymphopenia and attenuates NSCLC radiation response. Clinical cancer research Kuo, P., Bratman, S. V., Shultz, D. B., von Eyben, R., Chan, C., Wang, Z., Say, C., Gupta, A., Loo, B. W., Giaccia, A. J., Koong, A. C., Diehn, M., Le, Q. 2014; 20 (21): 5558-5569

  Abstract

  Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non-small cell lung cancer (NSCLC)] and head and neck cancer patients.LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8(+) T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis. Clin Cancer Res; 20(21); 5558-69. ©2014 AACR.

  View details for DOI 10.1158/1078-0432.CCR-14-1138

  View details for PubMedID 25189484

  View details for PubMedCentralID PMC4216761

• Loss of HIF-1 alpha in the Notochord Results in Cell Death and Complete Disappearance of the Nucleus Pulposus PLOS ONE Merceron, C., Mangiavini, L., Robling, A., Wilson, T. L., Giaccia, A. J., Shapiro, I. M., Schipani, E., Risbud, M. V. 2014; 9 (10)

  Abstract

  The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

  View details for DOI 10.1371/journal.pone.0110768

  View details for Web of Science ID 000343674800073

  View details for PubMedID 25338007

  View details for PubMedCentralID PMC4206488

• Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rankin, E. B., Fuh, K. C., Castellini, L., Viswanathan, K., Finger, E. C., Diep, A. N., Lagory, E. L., Kariolis, M. S., Chan, A., Lindgren, D., Axelson, H., Miao, Y. R., Krieg, A. J., Giaccia, A. J. 2014; 111 (37): 13373-13378

  View details for DOI 10.1073/pnas.1404848111

  View details for Web of Science ID 000341630000041

• Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET. Proceedings of the National Academy of Sciences of the United States of America Rankin, E. B., Fuh, K. C., Castellini, L., Viswanathan, K., Finger, E. C., Diep, A. N., Lagory, E. L., Kariolis, M. S., Chan, A., Lindgren, D., Axelson, H., MIAO, Y. R., Krieg, A. J., Giaccia, A. J. 2014; 111 (37): 13373-13378

  Abstract

  Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

  View details for DOI 10.1073/pnas.1404848111

  View details for PubMedID 25187556

• Molecular Radiobiology: The State of the Art JOURNAL OF CLINICAL ONCOLOGY Giaccia, A. J. 2014; 32 (26): 2871-?

  Abstract

  Traditional cytotoxic agents used in cancer therapy were initially discovered based on their ability to kill rapidly dividing cells. The targets of these early-generation agents were typically one or more aspects of DNA synthesis or mitosis. Thus, dose-limiting toxicities commonly associated with these agents include GI dysfunction, immunosuppression, and other consequences of injury to normal tissues in which cells are replicating under normal physiologic conditions. Although many of these agents still play an important role in cancer therapy when given concurrently with radiation therapy, the major thrust of radiobiology research in the last two decades has focused on discovering tumor-specific traits that might be exploited for more selective targeting that would enhance the efficacy of radiotherapy with less normal tissue toxicity. These newer generation molecular targeted therapies interfere with the growth of tumor cells by inhibiting genes and their protein products that are needed specifically by the tumor for survival and expansion. These agents can be complementary to radiotherapy, a spatially targeted agent. Although there have been extraordinary technical advances in radiotherapy in recent years, we are reaching the limits of improvements that radiotherapy delivery technology can bring and need different approaches. This review will highlight promising new tumor biology-based targets and other novel strategies to reduce normal tissue injury, increase tumor control, and expand the use of radiotherapy to treat widespread metastatic disease.

  View details for DOI 10.1200/JCO.2014.57.2776

  View details for Web of Science ID 000342060700008

  View details for PubMedID 25113768

  View details for PubMedCentralID PMC4152715

• Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development. Developmental biology Mangiavini, L., Merceron, C., Araldi, E., Khatri, R., Gerard-O'Riley, R., LeShan Wilson, T., Rankin, E. B., Giaccia, A. J., Schipani, E. 2014; 393 (1): 124-136

  Abstract

  Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.

  View details for DOI 10.1016/j.ydbio.2014.06.013

  View details for PubMedID 24972088

• Neurotrophic factor GDNF promotes survival of salivary stem cells. journal of clinical investigation Xiao, N., Lin, Y., Cao, H., Sirjani, D., Giaccia, A. J., Koong, A. C., Kong, C. S., Diehn, M., Le, Q. 2014; 124 (8): 3364-3377

  Abstract

  Stem cell-based regenerative therapy is a promising treatment for head and neck cancer patients that suffer from chronic dry mouth (xerostomia) due to salivary gland injury from radiation therapy. Current xerostomia therapies only provide temporary symptom relief, while permanent restoration of salivary function is not currently feasible. Here, we identified and characterized a stem cell population from adult murine submandibular glands. Of the different cells isolated from the submandibular gland, this specific population, Lin-CD24+c-Kit+Sca1+, possessed the highest capacity for proliferation, self renewal, and differentiation during serial passage in vitro. Serial transplantations of this stem cell population into the submandibular gland of irradiated mice successfully restored saliva secretion and increased the number of functional acini. Gene-expression analysis revealed that glial cell line-derived neurotrophic factor (Gdnf) is highly expressed in Lin-CD24+c-Kit+Sca1+ stem cells. Furthermore, GDNF expression was upregulated upon radiation therapy in submandibular glands of both mice and humans. Administration of GDNF improved saliva production and enriched the number of functional acini in submandibular glands of irradiated animals and enhanced salisphere formation in cultured salivary stem cells, but did not accelerate growth of head and neck cancer cells. These data indicate that modulation of the GDNF pathway may have potential therapeutic benefit for management of radiation-induced xerostomia.

  View details for DOI 10.1172/JCI74096

  View details for PubMedID 25036711

  View details for PubMedCentralID PMC4109543

• Recruitment of Circulating Breast Cancer Cells Is Stimulated by Radiotherapy CELL REPORTS Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. 2014; 8 (2): 401-408

  View details for DOI 10.1016/j.celrep.2014.06.011

  View details for Web of Science ID 000341569800010

• Recruitment of circulating breast cancer cells is stimulated by radiotherapy. Cell reports Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. 2014; 8 (2): 402-409

  Abstract

  Radiotherapy (RT) is a localized therapy that is highly effective in killing primary tumor cells located within the field of the radiation beam. We present evidence that irradiation of breast tumors can attract migrating breast cancer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) produced by tumor cells in response to radiation stimulates the recruitment of migrating tumor cells to irradiated tumors, suggesting a mechanism of tumor recurrence after radiation facilitated by transit of unirradiated, viable circulating tumor cells to irradiated tumors. Data supporting this hypothesis are presented through in vitro invasion assays and in vivo orthotopic models of breast cancer. Our work provides a mechanism for tumor recurrence in which RT attracts cells outside the radiation field to migrate to the site of treatment.

  View details for DOI 10.1016/j.celrep.2014.06.011

  View details for PubMedID 25017065

  View details for PubMedCentralID PMC4121080

• PHD Inhibition Mitigates and Protects Against Radiation-Induced Gastrointestinal Toxicity via HIF2. Science translational medicine Taniguchi, C. M., Miao, Y. R., Diep, A. N., Wu, C., Rankin, E. B., Atwood, T. F., Xing, L., Giaccia, A. J. 2014; 6 (236): 236ra64-?

  Abstract

  Radiation-induced gastrointestinal (GI) toxicity can be a major source of morbidity and mortality after radiation exposure. There is an unmet need for effective preventative or mitigative treatments against the potentially fatal diarrhea and water loss induced by radiation damage to the GI tract. We report that prolyl hydroxylase inhibition by genetic knockout or pharmacologic inhibition of all PHD (prolyl hydroxylase domain) isoforms by the small-molecule dimethyloxallyl glycine (DMOG) increases hypoxia-inducible factor (HIF) expression, improves epithelial integrity, reduces apoptosis, and increases intestinal angiogenesis, all of which are essential for radioprotection. HIF2, but not HIF1, is both necessary and sufficient to prevent radiation-induced GI toxicity and death. Increased vascular endothelial growth factor (VEGF) expression contributes to the protective effects of HIF2, because inhibition of VEGF function reversed the radioprotection and radiomitigation afforded by DMOG. Additionally, mortality from abdominal or total body irradiation was reduced even when DMOG was given 24 hours after exposure. Thus, prolyl hydroxylase inhibition represents a treatment strategy to protect against and mitigate GI toxicity from both therapeutic radiation and potentially lethal radiation exposures.

  View details for DOI 10.1126/scitranslmed.3008523

  View details for PubMedID 24828078

  View details for PubMedCentralID PMC4136475

• PHD inhibition mitigates and protects against radiation-induced gastrointestinal toxicity via HIF2. Science translational medicine Taniguchi, C. M., Miao, Y. R., Diep, A. N., Wu, C., Rankin, E. B., Atwood, T. F., Xing, L., Giaccia, A. J. 2014; 6 (236): 236ra64-?

  Abstract

  Radiation-induced gastrointestinal (GI) toxicity can be a major source of morbidity and mortality after radiation exposure. There is an unmet need for effective preventative or mitigative treatments against the potentially fatal diarrhea and water loss induced by radiation damage to the GI tract. We report that prolyl hydroxylase inhibition by genetic knockout or pharmacologic inhibition of all PHD (prolyl hydroxylase domain) isoforms by the small-molecule dimethyloxallyl glycine (DMOG) increases hypoxia-inducible factor (HIF) expression, improves epithelial integrity, reduces apoptosis, and increases intestinal angiogenesis, all of which are essential for radioprotection. HIF2, but not HIF1, is both necessary and sufficient to prevent radiation-induced GI toxicity and death. Increased vascular endothelial growth factor (VEGF) expression contributes to the protective effects of HIF2, because inhibition of VEGF function reversed the radioprotection and radiomitigation afforded by DMOG. Additionally, mortality from abdominal or total body irradiation was reduced even when DMOG was given 24 hours after exposure. Thus, prolyl hydroxylase inhibition represents a treatment strategy to protect against and mitigate GI toxicity from both therapeutic radiation and potentially lethal radiation exposures.

  View details for DOI 10.1126/scitranslmed.3008523

  View details for PubMedID 24828078

  View details for PubMedCentralID PMC4136475

• CTGF is a therapeutic target for metastatic melanoma. Oncogene Finger, E. C., Cheng, C., Williams, T. R., Rankin, E. B., Bedogni, B., Tachiki, L., Spong, S., Giaccia, A. J., Powell, M. B. 2014; 33 (9): 1093-1100

  Abstract

  Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.47.

  View details for DOI 10.1038/onc.2013.47

  View details for PubMedID 23435419

• The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells. Molecular and cellular biology Razorenova, O. V., Castellini, L., Colavitti, R., Edgington, L. E., Nicolau, M., Huang, X., Bedogni, B., Mills, E. M., Bogyo, M., Giaccia, A. J. 2014; 34 (4): 739-751

  Abstract

  The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp -190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

  View details for DOI 10.1128/MCB.00644-12

  View details for PubMedID 24344197

  View details for PubMedCentralID PMC3911479

• Identifying novel targets in renal cell carcinoma: Design and synthesis of affinity chromatography reagents BIOORGANIC & MEDICINAL CHEMISTRY Bonnet, M., Flanagan, J. U., Chan, D. A., Giaccia, A. J., Hay, M. P. 2014; 22 (2): 711-720

  Abstract

  Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel-Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatography reagents to aid the identification of the molecular targets of these two classes. Structure-activity data and computational models were used to predict possible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with 2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of 4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized as an affinity reagent.

  View details for DOI 10.1016/j.bmc.2013.12.028

  View details for Web of Science ID 000329576400005

  View details for PubMedID 24387866

• Epidermal or Dermal Specific Knockout of PHD-2 Enhances Wound Healing and Minimizes Ischemic Injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

• Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  Abstract

  Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).PHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

  View details for PubMedCentralID PMC3973687

• HIF-2: the missing link between obesity and cardiomyopathy. Journal of the American Heart Association Giaccia, A. J. 2013; 2 (6): e000710

  View details for DOI 10.1161/JAHA.113.000710

  View details for PubMedID 24342999

  View details for PubMedCentralID PMC3886772

• A liver Hif-2 alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition NATURE MEDICINE Wei, K., Piecewicz, S. M., McGinnis, L. M., Taniguchi, C. M., Wiegand, S. J., Anderson, K., Chan, C. W., Mulligan, K. X., Kuo, D., Yuan, J., Vallon, M., Morton, L. C., Lefai, E., Simon, M. C., Maher, J. J., Mithieux, G., Rajas, F., Annes, J. P., McGuinness, O. P., Thurston, G., Giaccia, A. J., Kuo, C. J. 2013; 19 (10): 1331-?

  Abstract

  Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

  View details for DOI 10.1038/nm.3295

  View details for Web of Science ID 000325531700034

  View details for PubMedID 24037094

  View details for PubMedCentralID PMC3795838

• A liver Hif-2a-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nature medicine Wei, K., Piecewicz, S. M., McGinnis, L. M., Taniguchi, C. M., Wiegand, S. J., Anderson, K., Chan, C. W., Mulligan, K. X., Kuo, D., Yuan, J., Vallon, M., Morton, L. C., Lefai, E., Simon, M. C., Maher, J. J., Mithieux, G., Rajas, F., Annes, J. P., McGuinness, O. P., Thurston, G., Giaccia, A. J., Kuo, C. J. 2013; 19 (10): 1331-1337

  Abstract

  Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

  View details for DOI 10.1038/nm.3295

  View details for PubMedID 24037094

• Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes NATURE MEDICINE Taniguchi, C. M., Finger, E. C., Krieg, A. J., Wu, C., Diep, A. N., Lagory, E. L., Wei, K., McGinnis, L. M., Yuan, J., Kuo, C. J., Giaccia, A. J. 2013; 19 (10): 1325-?

  Abstract

  Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.

  View details for DOI 10.1038/nm.3294

  View details for Web of Science ID 000325531700033

  View details for PubMedID 24037093

  View details for PubMedCentralID PMC4089950

• A Novel Aldehyde Dehydrogenase-3 Activator (Alda-89) Protects Submandibular Gland Function from Irradiation without Accelerating Tumor Growth. Clinical cancer research Xiao, N., Cao, H., Chen, C., Kong, C. S., Ali, R., Chan, C., Sirjani, D., Graves, E., Koong, A., Giaccia, A., Mochly-Rosen, D., Le, Q. 2013; 19 (16): 4455-4464

  Abstract

  To determine the effect of Alda-89 (an ALDH3 activitor) on (i) the function of irradiated (radiotherapy) submandibular gland (SMG) in mice, (ii) its toxicity profile, and (iii) its effect on the growth of head and neck cancer (HNC) in vitro and in vivo.Adult mice were infused with Alda-89 or vehicle before, during, and after radiotherapy. Saliva secretion was monitored weekly. Hematology, metabolic profile, and postmortem evaluation for toxicity were examined at the time of sacrifice. Alda-89 or vehicle was applied to HNC cell lines in vitro, and severe combined immunodeficient (SCID) mice transplanted with HNC in vivo with or without radiation; HNC growth was monitored. The ALDH3A1 and ALDH3A2 protein expression was evaluated in 89 patients with HNC and correlated to freedom from relapse (FFR) and overall survival (OS).Alda-89 infusion significantly resulted in more whole saliva production and a higher percentage of preserved acini after radiotherapy compared with vehicle control. There was no difference in the complete blood count, metabolic profile, and major organ morphology between the Alda-89 and vehicle groups. Compared with vehicle control, Alda-89 treatment neither accelerated HNC cell proliferation in vitro, nor did it affect tumor growth in vivo with or without radiotherapy. Higher expression of ALDH3A1 or ALDH3A2 was not significantly associated with worse FFR or OS in either human papillomavirus (HPV)-positive or HPV-negative group.Alda-89 preserves salivary function after radiotherapy without affecting HNC growth or causing measurable toxicity in mice. It is a promising candidate to mitigate radiotherapy-related xerostomia.

  View details for DOI 10.1158/1078-0432.CCR-13-0127

  View details for PubMedID 23812668

• A Novel Aldehyde Dehydrogenase-3 Activator (Alda-89) Protects Submandibular Gland Function from Irradiation without Accelerating Tumor Growth CLINICAL CANCER RESEARCH Xiao, N., Cao, H., Chen, C., Kong, C. S., Ali, R., Chan, C., Sirjani, D., Graves, E., Koong, A., Giaccia, A., Mochly-Rosen, D., Quynh-Thu Le, Q. T. 2013; 19 (16): 4455-4464

  Abstract

  To determine the effect of Alda-89 (an ALDH3 activitor) on (i) the function of irradiated (radiotherapy) submandibular gland (SMG) in mice, (ii) its toxicity profile, and (iii) its effect on the growth of head and neck cancer (HNC) in vitro and in vivo.Adult mice were infused with Alda-89 or vehicle before, during, and after radiotherapy. Saliva secretion was monitored weekly. Hematology, metabolic profile, and postmortem evaluation for toxicity were examined at the time of sacrifice. Alda-89 or vehicle was applied to HNC cell lines in vitro, and severe combined immunodeficient (SCID) mice transplanted with HNC in vivo with or without radiation; HNC growth was monitored. The ALDH3A1 and ALDH3A2 protein expression was evaluated in 89 patients with HNC and correlated to freedom from relapse (FFR) and overall survival (OS).Alda-89 infusion significantly resulted in more whole saliva production and a higher percentage of preserved acini after radiotherapy compared with vehicle control. There was no difference in the complete blood count, metabolic profile, and major organ morphology between the Alda-89 and vehicle groups. Compared with vehicle control, Alda-89 treatment neither accelerated HNC cell proliferation in vitro, nor did it affect tumor growth in vivo with or without radiotherapy. Higher expression of ALDH3A1 or ALDH3A2 was not significantly associated with worse FFR or OS in either human papillomavirus (HPV)-positive or HPV-negative group.Alda-89 preserves salivary function after radiotherapy without affecting HNC growth or causing measurable toxicity in mice. It is a promising candidate to mitigate radiotherapy-related xerostomia.

  View details for DOI 10.1158/1078-0432.CCR-13-0127

  View details for Web of Science ID 000323147700018

  View details for PubMedCentralID PMC3745542

• A low-carb diet kills tumor cells with a mutant p53 tumor suppressor gene: the Atkins diet suppresses tumor growth. Cell cycle Lagory, E. L., Giaccia, A. J. 2013; 12 (5): 718-719

  View details for DOI 10.4161/cc.23948

  View details for PubMedID 23422857

  View details for PubMedCentralID PMC3610718

• Regulation of Bone Marrow Angiogenesis by Osteoblasts during Bone Development and Homeostasis. Frontiers in endocrinology Schipani, E., Wu, C., Rankin, E. B., Giaccia, A. J. 2013; 4: 85-?

  Abstract

  Bone marrow is a highly heterogeneous and vascularized tissue. The various cell types populating the bone marrow extensively communicate with each other, and cell-to-cell cross talk is likely to be essential for proper bone development and homeostasis. In particular, the existence of osteogenesis and angiogenesis coupling has been recently proposed. Despite its high degree of vascularization, a gradient of oxygenation is present in the bone marrow, and the endosteal surface of cortical bone appears to be among the most hypoxic areas in the body. Oxygen (O2) is both an essential metabolic substrate and a regulatory signal that is in charge of a specific genetic program. An important component of this program is the family of transcription factors known as hypoxia-inducible factors (HIFs). In this Perspective, we will summarize our current knowledge about the role of the HIF signaling pathway in controlling bone development and homeostasis, and especially in regulating the crosstalk between osteoblasts, progenitor cells, and bone marrow blood vessels.

  View details for DOI 10.3389/fendo.2013.00085

  View details for PubMedID 23847596

• Prolyl Hydroxylase Inhibition Protects Against and Mitigates Radiation-induced Gastrointestinal Toxicity Through Improved Integrity and Function of the GI Epithelium 54th Annual Meeting of the American-Society-for-Radiation-Oncology (ASTRO) Taniguchi, C. M., Atwood, T., Giaccia, A. J. ELSEVIER SCIENCE INC. 2012: S106–S107

  View details for Web of Science ID 000310542900265

• The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis ONCOTARGET Catalina-Rodriguez, O., Kolukula, V. K., Tomita, Y., Preet, A., Palmieri, F., Wellstein, A., Byers, S., Giaccia, A. J., Glasgow, E., Albanese, C., Avantaggiati, M. L. 2012; 3 (10): 1220-1235

  Abstract

  Dysregulation of the pathways that preserve mitochondrial integrity hallmarks many human diseases including diabetes, neurodegeration, aging and cancer. The mitochondrial citrate transporter gene, SLC25A1 or CIC, maps on chromosome 22q11.21, a region amplified in some tumors and deleted in developmental disorders known as velo-cardio-facial- and DiGeorge syndromes. We report here that in tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence promoting proliferation. CIC levels are increased in human cancers and its inhibition has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial homeostasis in metabolically active, high proliferating tissues and imply that this protein is a therapeutic target in cancer and likely, in other human diseases.

  View details for Web of Science ID 000312410800019

  View details for PubMedID 23100451

• Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss MOLECULAR HUMAN REPRODUCTION Krieg, S. A., Fan, X., Hong, Y., Sang, Q., Giaccia, A., Westphal, L. M., Lathi, R. B., Krieg, A. J., Nayak, N. R. 2012; 18 (9): 442-450

  Abstract

  Recurrent pregnancy loss (RPL) occurs in ∼5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.

  View details for DOI 10.1093/molehr/gas017

  View details for Web of Science ID 000308243000003

  View details for PubMedID 22505054

  View details for PubMedCentralID PMC3431184

• The Apoptosis Repressor With a CARD Domain (ARC) is Essential for the Survival of VHL Deficient Renal Cancer Cells 22nd Biennial Congress of the European-Association-for-Cancer-Research Razorenova, O. V., Colavitti, R., Castellini, L., Edgington, L. E., Huang, X., Nicolau, M., Bedogni, B., Mills, E. M., Bogyo, M., Giaccia, A. J. ELSEVIER SCI LTD. 2012: S120–S120

  View details for Web of Science ID 000313036501011

• Identification of Novel p53-regulated Genes Involved in Chromatin Remodeling in Response to DNA Damage 22nd Biennial Congress of the European-Association-for-Cancer-Research Castellini, L., Krieg, A. J., Mello, S. S., Giaccia, A. J. ELSEVIER SCI LTD. 2012: S147–S147

  View details for Web of Science ID 000313036501114

• State of the Science: An Update on Renal Cell Carcinoma MOLECULAR CANCER RESEARCH Jonasch, E., Futreal, P. A., Davis, I. J., Bailey, S. T., Kim, W. Y., Brugarolas, J., Giaccia, A. J., Kurban, G., Pause, A., Frydman, J., Zurita, A. J., Rini, B. I., Sharma, P., Atkins, M. B., Walker, C. L., Rathmell, W. K. 2012; 10 (7): 859-880

  Abstract

  Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.

  View details for DOI 10.1158/1541-7786.MCR-12-0117

  View details for Web of Science ID 000308027300001

  View details for PubMedID 22638109

• Blood and bones Osteoblastic HIF signaling regulates erythropoiesis CELL CYCLE Wu, C., Rankin, E. B., Giaccia, A. J. 2012; 11 (12): 2221-2222

  View details for DOI 10.4161/cc.20635

  View details for Web of Science ID 000305353000003

  View details for PubMedID 22627672

  View details for PubMedCentralID PMC3383578

• The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO CELL Rankin, E. B., Wu, C., Khatri, R., Wilson, T. L., Andersen, R., Araldi, E., Rankin, A. L., Yuan, J., Kuo, C. J., Schipani, E., Giaccia, A. J. 2012; 149 (1): 63-74

  Abstract

  Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.

  View details for DOI 10.1016/j.cell.2012.01.051

  View details for Web of Science ID 000302235400010

  View details for PubMedID 22464323

  View details for PubMedCentralID PMC3408231

• Prognostic and Predictive Significance of Plasma HGF and IL-8 in a Phase III Trial of Chemoradiation with or without Tirapazamine in Locoregionally Advanced Head and Neck Cancer CLINICAL CANCER RESEARCH Quynh-Thu Le, Q. T., Fisher, R., Oliner, K. S., Young, R. J., Cao, H., Kong, C., Graves, E., Hicks, R. J., McArthur, G. A., Peters, L., O'Sullivan, B., Giaccia, A., Rischin, D. 2012; 18 (6): 1798-1807

  Abstract

  Hepatocyte growth factor (HGF) is a hypoxia-induced secreted protein that binds to cMet and regulates interleukin (IL)-8 expression. We evaluated the role of circulating HGF and IL-8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin.Patients with stages III to IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or CIS plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL-8 assay by ELISA and no major radiation deviations (N = 498). Analyses included adjustment for major prognostic factors. p16(INK4A) staining (human papillomavirus surrogate) was carried out on available tumors. Thirty-nine patients had hypoxia imaging with (18)F-fluoroazomycin arabinoside ((18)FAZA)-positron emission tomography.Elevated IL-8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (P = 0.053); elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16(INK4A)-negative patients. Four subgroups defined by high and low HGF/IL-8 levels were examined for TPZ effect; the test for interaction with arm was P = 0.099. TPZ/CIS seemed to be beneficial for patients with high HGF and IL-8 but adverse for low HGF and high IL-8. Only HGF correlated with (18)FAZA tumor standard uptake value.IL-8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL-8/HGF levels seemed to do better with TPZ/CIS while others did worse, highlighting the complexity of hypoxia targeting in unselected patients.

  View details for DOI 10.1158/1078-0432.CCR-11-2094

  View details for PubMedID 22383739

• VEGF-independent cell-autonomous functions of HIF-1a regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival JOURNAL OF BONE AND MINERAL RESEARCH Maes, C., Araldi, E., Haigh, K., Khatri, R., Van Looveren, R., Giaccia, A. J., Haigh, J. J., Carmeliet, G., Schipani, E. 2012; 27 (3): 596-609

  Abstract

  Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1α and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.

  View details for DOI 10.1002/jbmr.1487

  View details for Web of Science ID 000300682500010

  View details for PubMedID 22162090

• Prognostic Significance of Plasma Osteopontin in Patients with Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Treated on TROG 02.02 Phase III Trial CLINICAL CANCER RESEARCH Lim, A. M., Rischin, D., Fisher, R., Cao, H., Kwok, K., Truong, D., McArthur, G. A., Young, R. J., Giaccia, A., Peters, L., Le, Q. 2012; 18 (1): 301-307

  Abstract

  High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in head and neck squamous cell carcinomas (HNSCC), correlate with tumor hypoxia, and be predictive of benefit from hypoxia-targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial.Patients with stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N = 578). OPN concentrations were analyzed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was carried out in patients with available tumor p16(INK4A) staining status.The median OPN level was 544 ng/mL (range: 7-2,640). High OPN levels were not associated with worse OS (relative HR, 1.03 for highest tertile) or TTLRF (relative HR 0.91 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (P = 0.93 for OS; P = 0.87 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR = 1.11, P = 0.67). Similarly for p16(INK4A) negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR = 1.05, P = 0.86).We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy.

  View details for DOI 10.1158/1078-0432.CCR-11-2295

  View details for Web of Science ID 000298758900032

  View details for PubMedID 22096023

  View details for PubMedCentralID PMC3251655

• Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction Annual Meeting of the American-Heart-Association Huang, M., Nguyen, P., Jia, F., Hu, S., Gong, Y., de Almeida, P. E., Wang, L., Nag, D., Kay, M. A., Giaccia, A. J., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2011: S46–S54

  Abstract

  Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively.We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

  View details for DOI 10.1161/CIRCULATIONAHA.110.014019

  View details for Web of Science ID 000294782800006

  View details for PubMedID 21911818

  View details for PubMedCentralID PMC3181087

• Targeting Galectin-1, a Hypoxia Induced Protein, in Non-Small Cell Lung Cancers European Multidisciplinary Cancer Congress on Integrating Basic and Translational Science, Surgery, Radiotherapy, Medical oncology, Advocacy and Care Banh, A., Zhang, J., Cao, H., Bouley, D., Kwok, S., Kong, C., Giaccia, A., Koong, A., Le, Q. ELSEVIER SCI LTD. 2011: S60–S60

  View details for Web of Science ID 000295752800212

• Targeting GLUT1 and the Warburg Effect in Renal Cell Carcinoma by Chemical Synthetic Lethality SCIENCE TRANSLATIONAL MEDICINE Chan, D. A., Sutphin, P. D., Nguyen, P., Turcotte, S., Lai, E. W., Banh, A., Reynolds, G. E., Chi, J., Wu, J., Solow-Cordero, D. E., Bonnet, M., Flanagan, J. U., Bouley, D. M., Graves, E. E., Denny, W. A., Hay, M. P., Giaccia, A. J. 2011; 3 (94)

  Abstract

  Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.

  View details for DOI 10.1126/scitranslmed.3002394

  View details for PubMedID 21813754

• Tumor Galectin-1 Mediates Tumor Growth and Metastasis through Regulation of T-Cell Apoptosis CANCER RESEARCH Banh, A., Zhang, J., Cao, H., Bouley, D. M., Kwok, S., Kong, C., Giaccia, A. J., Koong, A. C., Le, Q. 2011; 71 (13): 4423-4431

  Abstract

  Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.

  View details for DOI 10.1158/0008-5472.CAN-10-4157

  View details for PubMedID 21546572

• SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells BIOORGANIC & MEDICINAL CHEMISTRY Bonnet, M., Flanagan, J. U., Chan, D. A., Lai, E. W., Phuong Nguyen, P., Giaccia, A. J., Hay, M. P. 2011; 19 (11): 3347-3356

  Abstract

  We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.

  View details for DOI 10.1016/j.bmc.2011.04.042

  View details for Web of Science ID 000290955400005

  View details for PubMedID 21561782

  View details for PubMedCentralID PMC3115526

• A central role for hypoxic signaling in cartilage, bone, and hematopoiesis. Current osteoporosis reports Rankin, E. B., Giaccia, A. J., Schipani, E. 2011; 9 (2): 46-52

  Abstract

  Hypoxic signaling plays an essential role in maintaining oxygen homeostasis and cell survival. Hypoxia-inducible transcription factors HIF-1 and HIF-2 are central mediators of the cellular response to hypoxia by regulating the expression of genes controlling metabolic adaptation, oxygen delivery, and survival in response to oxygen deprivation. Recent studies have identified an important role for HIF-1 and HIF-2 in the regulation of skeletal development, bone formation, and regeneration, as well as joint formation and homeostasis. In addition, overexpression of HIF-1 and HIF-2 is clinically associated with osteosarcoma and osteoarthritis. Together, these findings implicate hypoxic signaling as a central regulator of bone biology and disease.

  View details for DOI 10.1007/s11914-011-0047-2

  View details for PubMedID 21360287

• Harnessing synthetic lethal interactions in anticancer drug discovery NATURE REVIEWS DRUG DISCOVERY Chan, D. A., Giaccia, A. J. 2011; 10 (5): 351-364

  Abstract

  Unique features of tumours that can be exploited by targeted therapies are a key focus of current cancer research. One such approach is known as synthetic lethality screening, which involves searching for genetic interactions of two mutations whereby the presence of either mutation alone has no effect on cell viability but the combination of the two mutations results in cell death. The presence of one of these mutations in cancer cells but not in normal cells can therefore create opportunities to selectively kill cancer cells by mimicking the effect of the second genetic mutation with targeted therapy. Here, we summarize strategies that can be used to identify synthetic lethal interactions for anticancer drug discovery, describe examples of such interactions that are currently being investigated in preclinical and clinical studies of targeted anticancer therapies, and discuss the challenges of realizing the full potential of such therapies.

  View details for DOI 10.1038/nrd3374

  View details for Web of Science ID 000290017300017

  View details for PubMedID 21532565

• Hypoxia and Senescence: The Impact of Oxygenation on Tumor Suppression MOLECULAR CANCER RESEARCH Welford, S. M., Giaccia, A. J. 2011; 9 (5): 538-544

  Abstract

  Cellular senescence has emerged as a biological response to two major pathophysiological states of our being: cancer and aging. In the course of the transformation of a normal cell to a cancerous cell, senescence is frequently induced to suppress tumor development. In aged individuals, senescence is found in cells that have exhausted their replication potential. The similarity in these responses suggests that understanding how senescence is mediated can provide insight into both cancer and aging. One environmental factor that is implicated in both of these states is tissue hypoxia, which increases with aging and can inhibit senescence. Hypoxia is particularly important in normal physiology to maintain the stem cell niche; but at the same time, hypoxic inhibition of an essential tumor suppressor response can theoretically contribute to cancer initiation.

  View details for DOI 10.1158/1541-7786.MCR-11-0065

  View details for Web of Science ID 000290610600002

  View details for PubMedID 21385881

• Combinatorial methods for engineering antagonists of the Axl receptor tyrosine kinase 241st National Meeting and Exposition of the American-Chemical-Society (ACS) Kariolis, M. S., Jones, D. S., Giaccia, A. J., Cochran, J. R. AMER CHEMICAL SOC. 2011

  View details for Web of Science ID 000291982802267

• VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC delta-driven migration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Razorenova, O. V., Finger, E. C., Colavitti, R., Chernikova, S. B., Boiko, A. D., Chan, C. K., Krieg, A., Bedogni, B., LaGory, E., Weissman, I. L., Broome-Powell, M., Giaccia, A. J. 2011; 108 (5): 1931-1936

  Abstract

  A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

  View details for DOI 10.1073/pnas.1011777108

  View details for PubMedID 21233420

• Lack of HIF-2 alpha in limb bud mesenchyme causes a modest and transient delay of endochondral bone development NATURE MEDICINE Araldi, E., Khatri, R., Giaccia, A. J., Simon, M. C., Schipani, E. 2011; 17 (1): 25-26

  View details for DOI 10.1038/nm0111-25

  View details for Web of Science ID 000285994000018

  View details for PubMedID 21217667

• Control of lysyl oxidase activity through site-specific deuteration of lysine BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Pestov, N. B., Okkelman, I. A., Shmanai, V. V., Hurski, A. L., Giaccia, A. J., Shchepinov, M. S. 2011; 21 (1): 255-258

  Abstract

  Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.

  View details for DOI 10.1016/j.bmcl.2010.11.018

  View details for Web of Science ID 000285544400049

  View details for PubMedID 21106372

• Basic Principles of Radiobiology Breast Surgical Techniques and Interdisciplinary Management Horst KC, Giaccia AJ 2011: 799-812
• Hypoxia in Models of Lung Cancer: Implications for Targeted Therapeutics CLINICAL CANCER RESEARCH Graves, E. E., Vilalta, M., Cecic, I. K., Erler, J. T., Tran, P. T., Felsher, D., Sayles, L., Sweet-Cordero, A., Le, Q., Giaccia, A. J. 2010; 16 (19): 4843-4852

  Abstract

  To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response.Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ or subcutaneously were studied using fluorodeoxyglucose and fluoroazomycin arabinoside positron emission tomography, and postmortem by immunohistochemical observation of the hypoxia marker pimonidazole. The response of these models to the hypoxia-activated cytotoxin PR-104 was also quantified by the formation of γH2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers.Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping of both hypoxia probes. These observations correlated with the response of these tumors to PR-104, and with the reduced incidence of hypoxia in human lung cancers relative to other solid tumor types.These findings suggest that in situ models of lung cancer in mice may be more reflective of the human disease, and encourage judicious selection of preclinical tumor models for the study of hypoxia imaging and antihypoxic cell therapies.

  View details for DOI 10.1158/1078-0432.CCR-10-1206

  View details for Web of Science ID 000282647900017

  View details for PubMedID 20858837

  View details for PubMedCentralID PMC2948600

• Renal Oxygenation Suppresses VHL Loss-Induced Senescence That Is Caused by Increased Sensitivity to Oxidative Stress MOLECULAR AND CELLULAR BIOLOGY Welford, S. M., Dorie, M. J., Li, X., Haase, V. H., Giaccia, A. J. 2010; 30 (19): 4595-4603

  Abstract

  Loss of the VHL tumor suppressor is regarded as an initiating event in the development of clear-cell renal carcinoma. Surprisingly, loss of VHL induces senescence in mouse fibroblasts in vitro, a response that would restrict development of renal carcinoma in vivo. Typical in vitro cell culture levels of oxygen, however, are significantly higher than physiological levels of oxygen, which have been shown to abrogate senescence induced by many stimuli. Therefore, we investigated the oxygen dependence of VHL loss-induced senescence. Using mouse fibroblasts and primary renal epithelial cells in vitro, we found that VHL loss leads to senescence under atmospheric conditions (21% O(2)), partly through increasing p27 levels, but not under physiological oxygenation (2% to 5% O(2)), despite maintaining increased p27 expression. This suggests that VHL inactivation sensitizes cells to oxidative stress. In support of this concept, senescence following VHL loss depends on p53 activity, which decreases under the less stressful conditions of mild hypoxia. We confirmed these observations in vivo by treating kidney-specific VHL knockout animals with the potent oxidizer paraquat and observed a robust induction of cellular senescence. Together, these data demonstrate that in vivo oxygenation promotes tolerance of VHL loss in renal epithelia, which may promote the development of renal carcinoma.

  View details for DOI 10.1128/MCB.01618-09

  View details for Web of Science ID 000281721400004

  View details for PubMedID 20679489

  View details for PubMedCentralID PMC2950534

• AXL Is an Essential Factor and Therapeutic Target for Metastatic Ovarian Cancer CANCER RESEARCH Rankin, E. B., Fuh, K. C., Taylor, T. E., Krieg, A. J., Musser, M., Yuan, J., Wei, K., Kuo, C. J., Longacre, T. A., Giaccia, A. J. 2010; 70 (19): 7570-7579

  Abstract

  The receptor tyrosine kinase AXL is thought to play a role in metastasis; however, the therapeutic efficacy of an AXL-targeting agent remains largely untested in metastatic disease. In this study, we defined AXL as a therapeutic target for metastatic ovarian cancer. AXL is primarily expressed in metastases and advanced-stage human ovarian tumors but not in normal ovarian epithelium. Genetic inhibition of AXL in human metastatic ovarian tumor cells is sufficient to prevent the initiation of metastatic disease in vivo. Mechanistically, inhibition of AXL signaling in animals with metastatic disease results in decreased invasion and matrix metalloproteinase activity. Most importantly, soluble human AXL receptors that imposed a specific blockade of the GAS6/AXL pathway had a profound inhibitory effect on progression of established metastatic ovarian cancer without normal tissue toxicity. These results offer the first genetic validation of GAS6/AXL targeting as an effective strategy for inhibition of metastatic tumor progression in vivo. Furthermore, this study defines the soluble AXL receptor as a therapeutic candidate agent for treatment of metastatic ovarian cancer, for which current therapies are ineffective.

  View details for DOI 10.1158/0008-5472.CAN-10-1267

  View details for Web of Science ID 000282647700021

  View details for PubMedID 20858715

  View details for PubMedCentralID PMC3408227

• Hypoxia-inducible Factor-1 Activation in Nonhypoxic Conditions: The Essential Role of Mitochondrial-derived Reactive Oxygen Species MOLECULAR BIOLOGY OF THE CELL Patten, D. A., Lafleur, V. N., Robitaille, G. A., Chan, D. A., Giaccia, A. J., Richard, D. E. 2010; 21 (18): 3247-3257

  Abstract

  Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.

  View details for Web of Science ID 000281761300014

  View details for PubMedID 20660157

• Toxoplasma gondii Activates Hypoxia-inducible Factor (HIF) by Stabilizing the HIF-1 alpha Subunit via Type I Activin-like Receptor Kinase Receptor Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Wiley, M., Sweeney, K. R., Chan, D. A., Brown, K. M., McMurtrey, C., Howard, E. W., Giaccia, A. J., Blader, I. J. 2010; 285 (35): 26852-26860

  Abstract

  Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the alpha subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1alpha is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are alpha-ketoglutarate-dependent dioxygenases that have low K(m) values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1alpha is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1alpha stability by preventing HIF-1alpha prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1alpha. The effects of Toxoplasma on HIF-1alpha abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.

  View details for DOI 10.1074/jbc.M110.147041

  View details for Web of Science ID 000281172700016

  View details for PubMedID 20581113

• PHD2 in tumour angiogenesis BRITISH JOURNAL OF CANCER Chan, D. A., Giaccia, A. J. 2010; 103 (1): 1-5

  Abstract

  Originally identified as the enzymes responsible for catalysing the oxidation of specific, conserved proline residues within hypoxia-inducible factor-1alpha (HIF-1alpha), the additional roles for the prolyl hydroxylase domain (PHD) proteins have remained elusive. Of the four identified PHD enzymes, PHD2 is considered to be the key oxygen sensor, as knockdown of PHD2 results in elevated HIF protein. Several recent studies have highlighted the importance of PHD2 in tumourigenesis. However, there is conflicting evidence as to the exact role of PHD2 in tumour angiogenesis. The divergence seems to be because of the contribution of stromal-derived PHD2, and in particular the involvement of endothelial cells, vs tumour-derived PHD2. This review summarises our current understanding of PHD2 and tumour angiogenesis, focusing on the influences of PHD2 on vascular normalisation and neovascularisation.

  View details for DOI 10.1038/sj.bjc.6605682

  View details for Web of Science ID 000279374800001

  View details for PubMedID 20461086

• Hypoxia, inflammation, and the tumor microenvironment in metastatic disease CANCER AND METASTASIS REVIEWS Finger, E. C., Giaccia, A. J. 2010; 29 (2): 285-293

  Abstract

  Metastasis, the leading cause of cancer deaths, is an intricate process involving many important tumor and stromal proteins that have yet to be fully defined. This review discusses critical components necessary for the metastatic cascade, including hypoxia, inflammation, and the tumor microenvironment. More specifically, this review focuses on tumor cell and stroma interactions, which allow cell detachment from a primary tumor, intravasation to the blood stream, and extravasation at a distant site where cells can seed and tumor metastases can form. Central players involved in this process and discussed in this review include integrins, matrix metalloproteinases, and soluble growth factors/matrix proteins, including the connective tissue growth factor and lysyl oxidase.

  View details for DOI 10.1007/s10555-010-9224-5

  View details for Web of Science ID 000277411300006

  View details for PubMedID 20393783

• MiR-210-micromanager of the hypoxia pathway TRENDS IN MOLECULAR MEDICINE Huang, X., Le, Q., Giaccia, A. J. 2010; 16 (5): 230-237

  Abstract

  Hypoxia inducible factors (HIFs) regulate a variety of genes to prepare cells to adapt and survive under a hypoxic environment. Recently, microRNAs (miRNAs) have emerged as a new class of genes regulated by HIFs in response to hypoxia, of which miR-210 is the most consistently and predominantly upregulated miRNA. Functional studies have demonstrated that miR-210 is a versatile gene that regulates many aspects of hypoxia pathways, both in physiological and malignant conditions. Here, we summarize recent findings on the mechanism of hypoxia regulation of miR-210 expression and its multifaceted biological functions in normal physiological and malignant conditions, and discuss the challenges we face in elucidating the biological functions of miR-210 and exploring its potential use for therapeutics.

  View details for DOI 10.1016/j.molmed.2010.03.004

  View details for Web of Science ID 000278669500004

  View details for PubMedID 20434954

  View details for PubMedCentralID PMC3408219

• Targeting cancer cells through autophagy for anticancer therapy CURRENT OPINION IN CELL BIOLOGY Turcotte, S., Giaccia, A. J. 2010; 22 (2): 246-251

  Abstract

  Autophagy is a cellular degradation process in which portions of the cell's cytoplasm and organelles are sequestered in a double-membrane bound vesicle called an autophagosome. Fusion of autophagosomes with lysosomes results in the formation of autolysosomes, where the proteins and organelles are degraded. This degradation pathway is induced under nutrient deprivation, metabolic stress or microenvironmental conditions to ensure energy balance, clearance of damaged proteins and adaptation to stress. Disruption of autophagy is involved in diverse human diseases including cancer. In particular, the regulation of autophagy in cancer cells is complex since it can enhance tumor cell survival in response to certain stresses, yet it can also act to suppress the initiation of tumor growth. Understanding the signaling pathways involved in the regulation of autophagy as well as the autophagy process itself represents new directions in the development of anticancer therapies. In this review, we discuss recent advances in our understanding the complexity of the autophagy process and the development of targeted therapies that modulate autophagy in cancer cells in the clinic.

  View details for DOI 10.1016/j.ceb.2009.12.007

  View details for Web of Science ID 000277845300019

  View details for PubMedID 20056398

• The RGD Domain of Human Osteopontin Promotes Tumor Growth and Metastasis through Activation of Survival Pathways PLOS ONE Courter, D., Cao, H., Kwok, S., Kong, C., Banh, A., Kuo, P., Bouley, D. M., Vice, C., Brustugun, O. T., Denko, N. C., Koong, A. C., Giaccia, A., Le, Q. 2010; 5 (3)

  Abstract

  Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models.Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-kappaB activation and FAK phosphorylation. Inhibition of NF-kappaB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells.Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-kappaB activation.

  View details for DOI 10.1371/journal.pone.0009633

  View details for PubMedID 20224789

• Differential Gene Expression in the Decidua of Recurrent Pregnancy Loss Patients. 57th Annual Meeting of the Society-for-Gynecologic-Investigation Krieg, S. A., Krieg, A. J., Fan, X., Dahl, S., Giaccia, A., Westphal, L. M., Lathi, R., Nayak, N. R. SAGE PUBLICATIONS INC. 2010: 361A–361A

  View details for Web of Science ID 000275558601500

• 4-Pyridylanilinothiazoles That Selectively Target von Hippel-Lindau Deficient Renal Cell Carcinoma Cells by Inducing Autophagic Cell Death JOURNAL OF MEDICINAL CHEMISTRY Hay, M. P., Turcotte, S., Flanagan, J. U., Bonnet, M., Chan, D. A., Sutphin, P. D., Nguyen, P., Giaccia, A. J., Denny, W. A. 2010; 53 (2): 787-797

  Abstract

  Renal cell carcinomas (RCC) are refractory to standard therapy with advanced RCC having a poor prognosis; consequently treatment of advanced RCC represents an unmet clinical need. The von Hippel-Lindau (VHL) tumor suppressor gene is mutated or inactivated in a majority of RCCs. We recently identified a 4-pyridyl-2-anilinothiazole (PAT) with selective cytotoxicity against VHL-deficient renal cells mediated by induction of autophagy and increased acidification of autolysosomes. We report exploration of structure-activity relationships (SAR) around this PAT lead. Analogues with substituents on each of the three rings, and various linkers between rings, were synthesized and tested in vitro using paired RCC4 cell lines. A contour map describing the relative spatial contributions of different chemical features to potency illustrates a region, adjacent to the pyridyl ring, with potential for further development. Examples probing this domain validated this approach and may provide the opportunity to develop this novel chemotype as a targeted approach to the treatment of RCC.

  View details for DOI 10.1021/jm901457w

  View details for Web of Science ID 000273672100025

  View details for PubMedID 19994864

  View details for PubMedCentralID PMC2838715

• DNA Damage and Repair Textbook of Radiation Oncology, 3rd Edition Hammond EM, Pires I, Giaccia AJ 2010: 31-39
• Role of Carcinoma-Associated Fibroblasts and Hypoxia in Tumor Progression DIVERSE EFFECTS OF HYPOXIA ON TUMOR PROGRESSION Giaccia, A. J., Schipani, E. 2010; 345: 31-45

  Abstract

  In recent years, a variety of experimental evidence has convincingly shown that progression of malignant tumors does not depend exclusively on cell-autonomous properties of the cancer cells, but can also be influenced by the tumor stroma. The concept that cancer cells are subjected to microenvironmental control has thus emerged as an important chapter in cancer biology. Recent findings have suggested an important role, in particular, for macrophages, endothelial cells, and cancer-associated fibroblasts (CAFs) in tumor growth and progression. Numerous lines of evidence indicate that the bone marrow is the source, at least in part, of these cells. This chapter summarizes our current knowledge of how bone marrow contributes to the tumor stroma, with particular emphasis on CAFs. The potential role of hypoxia in modulating the differentiation and activity of CAFs, and the therapeutical implications of targeting CAFs for anticancer therapy are discussed.

  View details for DOI 10.1007/82_2010_73

  View details for Web of Science ID 000282105100003

  View details for PubMedID 20517716

• Prematastatic Niches Tumor Microenvironment Bennewith KL, Erler JT, Giaccia AJ 2010: 161-182
• Regulation of the Histone Demethylase JMJD1A by Hypoxia-Inducible Factor 1 alpha Enhances Hypoxic Gene Expression and Tumor Growth MOLECULAR AND CELLULAR BIOLOGY Krieg, A. J., Rankin, E. B., Chan, D., Razorenova, O., Fernandez, S., Giaccia, A. J. 2010; 30 (1): 344-353

  Abstract

  The hypoxia-inducible transcription factors (HIFs) directly and indirectly mediate cellular adaptation to reduced oxygen tensions. Recent studies have shown that the histone demethylase genes JMJD1A, JMJD2B, and JARID1B are HIF targets, suggesting that HIFs indirectly influence gene expression at the level of histone methylation under hypoxia. In this study, we identify a subset of hypoxia-inducible genes that are dependent on JMJD1A in both renal cell and colon carcinoma cell lines. JMJD1A regulates the expression of adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) under hypoxia by decreasing promoter histone methylation. In addition, we demonstrate that loss of JMJD1A is sufficient to reduce tumor growth in vivo, demonstrating that histone demethylation plays a significant role in modulating growth within the tumor microenvironment. Thus, hypoxic regulation of JMJD1A acts as a signal amplifier to facilitate hypoxic gene expression, ultimately enhancing tumor growth.

  View details for DOI 10.1128/MCB.00444-09

  View details for Web of Science ID 000272569200028

  View details for PubMedID 19858293

  View details for PubMedCentralID PMC2798291

• Role of carcinoma-associated fibroblasts and hypoxia in tumor progression Diverse Effects of Hypoxia on Tumor Progression Giaccia AJ, Schipani E 2010: 31-45
• Hypoxia, Gene Expression and Metastasis The Tumor Microenvironment Razorenova OV, Giaccia AJ 2010: 43-58
• Pharmacologically Increased Tumor Hypoxia Can Be Measured by F-18-Fluoroazomycin Arabinoside Positron Emission Tomography and Enhances Tumor Response to Hypoxic Cytotoxin PR-104 CLINICAL CANCER RESEARCH Cairns, R. A., Bennewith, K. L., Graves, E. E., Giaccia, A. J., Chang, D. T., Denko, N. C. 2009; 15 (23): 7170-7174

  Abstract

  Solid tumors contain microenvironmental regions of hypoxia that present a barrier to traditional radiotherapy and chemotherapy, and this work describes a novel approach to circumvent hypoxia. We propose to overcome hypoxia by augmenting the effectiveness of drugs that are designed to specifically kill hypoxic tumor cells.We have constructed RKO colorectal tumor cells that express a small RNA hairpin that specifically knocks down the hypoxia-inducible factor 1a (HIF1a) transcription factor. We have used these cells in vitro to determine the effect of HIF1 on cellular sensitivity to the hypoxic cytotoxin PR-104, and its role in cellular oxygen consumption in response to the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA). We have further used these cells in vivo in xenografted tumors to determine the role of HIF1 in regulating tumor hypoxia in response to DCA using (18)F-fluoroazomycin arabinoside positron emission tomography, and its role in regulating tumor sensitivity to the combination of DCA and PR-104.HIF1 does not affect cellular sensitivity to PR-104 in vitro. DCA transiently increases cellular oxygen consumption in vitro and increases the extent of tumor hypoxia in vivo as measured with (18)F-fluoroazomycin arabinoside positron emission tomography. Furthermore, we show that DCA-dependent alterations in hypoxia increase the antitumor activity of the next-generation hypoxic cytotoxin PR-104.DCA interferes with the HIF-dependent "adaptive response," which limits mitochondrial oxygen consumption. This approach transiently increases tumor hypoxia and represents an important method to improve antitumor efficacy of hypoxia-targeted agents, without increasing toxicity to oxygenated normal tissue.

  View details for DOI 10.1158/1078-0432.CCR-09-1676

  View details for Web of Science ID 000272363700011

  View details for PubMedID 19920111

  View details for PubMedCentralID PMC2810128

• Inhibition of Histone Deacetylase Activity in Reduced Oxygen Environment Enhances the Osteogenesis of Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Xu, Y., Hammerick, K. E., James, A. W., Carre, A. L., Leucht, P., Giaccia, A. J., Longaker, M. T. 2009; 15 (12): 3697-3707

  Abstract

  Recent studies suggest that oxygen tension has a great impact on the osteogenic differentiation capacity of mesenchymal cells derived from adipose tissue: reduced oxygen impedes osteogenesis. We have found that expansion of mouse adipose-derived stromal cells (mASCs) in reduced oxygen tension (10%) results in increased cell proliferation along with induction of histone deacetylase (HDAC) activity. In this study, we utilized two HDAC inhibitors (HDACi), sodium butyrate (NaB) and valproic acid (VPA), and studied their effects on mASCs expanded in various oxygen tensions (21%, 10%, and 1% O(2)). Significant growth inhibition was observed with NaB or VPA treatment in each oxygen tension. Osteogenesis was enhanced by treatment with NaB or VPA, particularly in reduced oxygen tensions (10% and 1% O(2)). Conversely, adipogenesis was decreased with treatments of NaB or VPA at all oxygen tensions. Finally, NaB- or VPA-treated, reduced oxygen tension-exposed (1% O(2)) ASCs were grafted into surgically created mouse tibial defects and resulted in significantly increased bone regeneration. In conclusion, HDACi significantly promote the osteogenic differentiation of mASCs exposed to reduced oxygen tension; HDACi may hold promise for future clinical applications of ASCs for skeletal regeneration.

  View details for DOI 10.1089/ten.tea.2009.0213

  View details for Web of Science ID 000272528400003

  View details for PubMedID 19505250

  View details for PubMedCentralID PMC2792078

• Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling CELL Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, S. F., Csiszar, K., Giaccia, A., Weninger, W., Yamauchi, M., Gasser, D. L., Weaver, V. M. 2009; 139 (5): 891-906

  Abstract

  Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

  View details for DOI 10.1016/j.cell.2009.10.027

  View details for Web of Science ID 000272169400014

  View details for PubMedID 19931152

• Bioreductively Activated Lysyl Oxidase Inhibitors against Hypoxic Tumours CHEMMEDCHEM Granchi, C., Funaioli, T., Erler, J. T., Giaccia, A. J., Macchia, M., Minutolo, F. 2009; 4 (10): 1590-1594

  View details for DOI 10.1002/cmdc.200900247

  View details for Web of Science ID 000270767900003

  View details for PubMedID 19685543

• Hypoxia-Inducible mir-210 Regulates Normoxic Gene Expression Involved in Tumor Initiation MOLECULAR CELL Huang, X., Ding, L., Bennewith, K. L., Tong, R. T., Welford, S. M., Ang, K. K., Story, M., Le, Q., Giaccia, A. J. 2009; 35 (6): 856-867

  Abstract

  Previous studies have suggested that the HIF transcription factors can both activate and inhibit gene expression. Here we show that HIF1 regulates the expression of mir-210 in a variety of tumor types through a hypoxia-responsive element. Expression analysis in primary head and neck tumor samples indicates that mir-210 may serve as an in vivo marker for tumor hypoxia. By Argonaute protein immunoprecipitation, we identified 50 potential mir-210 targets and validated randomly selected ones. The majority of these 50 genes are not classical hypoxia-inducible genes, suggesting mir-210 represses genes expressed under normoxia that are no longer necessary to adapt and survive in a hypoxic environment. When human head and neck or pancreatic tumor cells ectopically expressing mir-210 were implanted into immunodeficient mice, mir-210 repressed initiation of tumor growth. Taken together, these data implicate an important role for mir-210 in regulating the hypoxic response of tumor cells and tumor growth.

  View details for DOI 10.1016/j.molcel.2009.09.006

  View details for Web of Science ID 000270559100018

  View details for PubMedID 19782034

  View details for PubMedCentralID PMC2782615

• Validation of Lysyl Oxidase As a Prognostic Marker for Metastasis and Survival in Head and Neck Squamous Cell Carcinoma: Radiation Therapy Oncology Group Trial 90-03 JOURNAL OF CLINICAL ONCOLOGY Le, Q., Harris, J., Magliocco, A. M., Kong, C. S., Diaz, R., Shin, B., Cao, H., Trotti, A., Erler, J. T., Chung, C. H., Dicker, A., Pajak, T. F., Giaccia, A. J., Ang, K. K. 2009; 27 (26): 4281-4286

  Abstract

  To validate lysyl oxidase (LOX), a hypoxia-related protein, as a marker for metastasis in an independent head and neck cancer (HNC) patient group enrolled onto a prospective trial.We performed traditional immunohistochemical (IHC) staining and automated quantitative analysis (AQUA) for LOX expression in 66 HNC patients from one institution. We also performed AQUA staining for LOX in 306 of 1,113 patients treated on a phase III trial comparing four radiation fractionation schedules in locally advanced HNC (RTOG 90-03). Pretreatment characteristics and outcome were similar between patients with and without LOX assessment. We correlated AQUA LOX expression with time to metastasis (TTM), time to progression (TTP), and overall survival (OS).LOX expression from both staining methods predicted for TTM in the first 66 patients. Multivariate analysis, controlling for significant parameters including nodal stage and performance status, revealed tumor LOX expression, as a continuous variable, was an independent predictor for TTM (hazard ratio [HR], 1.21; 95% CI, 1.10 to 1.33; P = .0001), TTP (HR, 1.06; 95% CI, 1.02 to 1.10; P = .0069), and OS (HR, 1.04; 95% CI, 1.00 to 1.07; P = .0311) in RTOG 90-03 patients. This translates into a 259% increase in metastatic risk for a patient at the 75th percentile of LOX compared with one at the 25th percentile.AQUA LOX expression was strongly associated with increased metastasis, progression, and death in RTOG 90-03 patients. This study validates that LOX is a marker for metastasis and survival in HNC.

  View details for DOI 10.1200/JCO.2008.20.6003

  View details for PubMedID 19667273

• Stabilization of hypoxia-inducible factor-1 enhances proangiogenic potential of bone marrow-derived mesenchymal stem cells 95th Annual Clinical Congress of the American-College-of-Surgeons/64th Annual Sessions of the Owen H Wangensteen Forum on Fundamental Surgical Problems Ko, S. H., Chan, D. A., Glotzbach, J. P., Giaccia, A. J., Gurtner, G. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2009: S88–S88

  View details for Web of Science ID 000269755300195

• Bringing H2AX into the Angiogenesis Family CANCER CELL Rankin, E. B., Giaccia, A. J., Hammond, E. M. 2009; 15 (6): 459-461

  Abstract

  The cell's ability to sense and respond to DNA damage is critical to maintain homeostasis and prevent the development of cancer. Paradoxically, Economopoulou et al. recently reported that a DNA damage response protein, H2AX, promotes tumor growth and angiogenesis.

  View details for DOI 10.1016/j.ccr.2009.05.004

  View details for Web of Science ID 000266686500003

  View details for PubMedID 19477424

  View details for PubMedCentralID PMC3408225

• Tumor Vasculature Is Regulated by PHD2-Mediated Angiogenesis and Bone Marrow-Derived Cell Recruitment CANCER CELL Chan, D. A., Kawahara, T. L., Sutphin, P. D., Chang, H. Y., Chi, J., Giaccia, A. J. 2009; 15 (6): 527-538

  Abstract

  Sustained angiogenesis, through either local sprouting (angiogenesis) or the recruitment of bone marrow-derived cells (BMDCs) (vasculogenesis), is essential to the development of a tumor. How BMDCs are recruited to the tumor and their contribution to the tumor vasculature is poorly understood. Here, we demonstrate that both IL-8 and angiogenin contribute to the complementary pathways of angiogenesis and BMDC mobilization to increase tumor growth. These two factors are regulated by PHD2 in a HIF-independent but NF-kappaB-dependent manner. PHD2 levels are decreased in human cancers, compared with corresponding normal tissue, and correlate with an increase in mature blood vessels. Thus, PHD2 plays a critical role in regulating tumor angiogenesis.

  View details for DOI 10.1016/j.ccr.2009.04.010

  View details for PubMedID 19477431

• Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells BLOOD Ghosh, A. K., Shanafelt, T. D., Cimmino, A., Taccioli, C., Volinia, S., Liu, C., Calin, G. A., Croce, C. M., Chan, D. A., Giaccia, A. J., Secreto, C., Wellik, L. E., Lee, Y. K., Mukhopadhyay, D., Kay, N. E. 2009; 113 (22): 5568-5574

  Abstract

  The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1alpha under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1alpha degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1alpha can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.

  View details for DOI 10.1182/blood-2008-10-185686

  View details for Web of Science ID 000266634700026

  View details for PubMedID 19336759

  View details for PubMedCentralID PMC2689054

• The Role of Tumor Cell-Derived Connective Tissue Growth Factor (CTGF/CCN2) in Pancreatic Tumor Growth CANCER RESEARCH Bennewith, K. L., Huang, X., Ham, C. M., Graves, E. E., Erler, J. T., Kambham, N., Feazell, J., Yang, G. P., Koong, A., Giaccia, A. J. 2009; 69 (3): 775-784

  Abstract

  Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.

  View details for DOI 10.1158/0008-5472.CAN-08-0987

  View details for Web of Science ID 000263048700010

  View details for PubMedID 19179545

  View details for PubMedCentralID PMC2747032

• ATM Activation and Signaling under Hypoxic Conditions MOLECULAR AND CELLULAR BIOLOGY Bencokova, Z., Kaufmann, M. R., Pires, I. M., Lecane, P. S., Giaccia, A. J., Hammond, E. M. 2009; 29 (2): 526-537

  Abstract

  The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.

  View details for DOI 10.1128/MCB.01301-08

  View details for Web of Science ID 000262045800020

  View details for PubMedID 18981219

• Hypoxia-Induced Lysyl Oxidase Is a Critical Mediator of Bone Marrow Cell Recruitment to Form the Premetastatic Niche CANCER CELL Erler, J. T., Bennewith, K. L., Cox, T. R., Lang, G., Bird, D., Koong, A., Le, Q., Giaccia, A. J. 2009; 15 (1): 35-44

  Abstract

  Tumor cell metastasis is facilitated by "premetastatic niches" formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment and metastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease.

  View details for DOI 10.1016/j.ccr.2008.11.012

  View details for Web of Science ID 000262334800007

  View details for PubMedID 19111879

  View details for PubMedCentralID PMC3050620

• Hypoxia-Mediated Signaling Pathways Handbook of Cell Signaling, 2nd Edition Chan DA, Koong AC, Giaccia AJ 2009; 3: 2241-2245
• Hypoxia, angiogenesis and oral cancer metastasis Oral Cancer Metastasis Le Q, Courter D, Giaccia AJ 2009: 299-321
• Ph.D. Training in Cancer Biology CANCER RESEARCH Torti, F. M., Altieri, D., Broach, J., Fan, H., Lotze, M., Manfredi, J., Matrisian, L., Yu, D., Giaccia, A. 2008; 68 (22): 9122-9124

  View details for DOI 10.1158/0008-5472.CAN-08-2626

  View details for Web of Science ID 000261136600004

  View details for PubMedID 19010881

• Notch1 is an effector of Akt and hypoxia in melanoma development JOURNAL OF CLINICAL INVESTIGATION Bedogni, B., Warneke, J. A., Nickoloff, B. J., Giaccia, A. J., Powell, M. B. 2008; 118 (11): 3660-3670

  Abstract

  Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

  View details for DOI 10.1172/JCI36157

  View details for Web of Science ID 000260622600020

  View details for PubMedID 18924608

  View details for PubMedCentralID PMC2567838

• Targeted therapy for the loss of von Hippel-Lindau in renal cell carcinoma - A novel molecule that induces autophagic cell death AUTOPHAGY Turcotte, S., Sutphin, P. D., Giaccia, A. J. 2008; 4 (7): 944-946

  Abstract

  Radiation and conventional cytotoxic chemotherapies are ineffective in treating renal cancer. Approximately 75 percent of renal cell carcinoma (RCC) is associated with an inactivation of the tumor suppressor gene von Hippel-Lindau (VHL). We exploited the possibility of targeting VHL-deficient RCC through synthetic lethality using a high-throughput screening approach. In this screen, STF-62247 was identified to be selectively toxic and growth inhibitory to renal cells lacking VHL. We recently demonstrated that the cytotoxicity of STF-62247 is due to dysregulated autophagy. Furthermore, the reduction of protein levels of essential autophagy pathway components such as Atg5, Atg7 and Atg9 reduces sensitivity of VHL-deficient cells to killing by STF-62247. Loss of proteins involved in Golgi trafficking sensitized RCC with wild-type VHL to killing by STF-62247, indicating a potential role for these proteins as a target of the compound. Our study supports the concept of using synthetic lethality to selectively kill VHL-deficient cells that represents a new type of targeted therapy for the treatment of RCC.

  View details for Web of Science ID 000259829700018

  View details for PubMedID 18769110

• Targeting cancer cells by synthetic lethality CELL CYCLE Chan, D. A., Giaccia, A. J. 2008; 7 (19): 2987-2990

  Abstract

  Standard cytotoxic agents for treating cancer were developed based on their effectiveness to kill rapidly dividing cells, not on their ability to selectively kill cancer cells and spare normal tissue. Much of contemporary cancer research is aimed at identifying specific molecular features of cancers to directly target tumor cells with the hope of reducing or eliminating unwanted side effects. Targeted therapy for the treatment of cancer can be divided into two main categories: monoclonal antibodies and small molecules. In this Perspective, we review the approach of synthetic lethality to target cancer, specifically renal cell carcinoma. The concept of synthetic lethality is used to describe a genetic interaction of two non-allelic and non-lethal genes that when mutated simultaneously results in cell death. Recently, we identified a compound, STF-62247, that functions in a synthetic lethal manner to the loss of VHL, a mutation found in the majority of renal cell carcinomas.

  View details for Web of Science ID 000260263000010

  View details for PubMedID 18818511

• Short hairpin RNA interference therapy for ischemic heart disease 80th Annual Scientific Session of the American-Heart-Association (AHA) Huang, M., Chan, D. A., Jia, F., Xie, X., Li, Z., Hoyt, G., Robbins, R. C., Chen, X., Giaccia, A. J., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2008: S226–U289

  Abstract

  During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.

  View details for DOI 10.1161/CIRCULATIONAHA.107.760785

  View details for Web of Science ID 000259648600033

  View details for PubMedID 18824759

  View details for PubMedCentralID PMC3657507

• (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a matrix metalloprotease inhibitor, is a novel and potent activator of hypoxia-inducible factors MOLECULAR PHARMACOLOGY Lauzier, M., Robitaille, G. A., Chan, D. A., Giaccia, A. J., Richard, D. E. 2008; 74 (1): 282-288

  Abstract

  Hypoxia-inducible factors (HIFs) are unstable heterodimeric transcription factors and decisive elements for the transcriptional regulation of genes important in the adaptation to low-oxygen conditions. Hypoxia is the ubiquitous inducer of HIFs, stabilizing the alpha-subunit and permitting the formation of a functional HIF complex. Here, we identify (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a commercially available metalloprotease-2 and -9 inhibitor, as a rapid and potent inducer of HIFs. We show that in different cell lines, BiPS induces the HIF-alpha subunit by inhibiting its degradation through stabilization of its labile oxygen-dependent degradation domain. This is achieved through the inhibition of HIF-1alpha hydroxylation. The HIF-1 complex, formed after BiPS treatment, is capable of DNA binding and activation of HIF target genes, including the expression of vascular endothelial growth factor. Because novel HIF activators have generated considerable interest in the possible treatment of different ischemic diseases, we believe that BiPS and derivative molecules could have strong therapeutic potential.

  View details for DOI 10.1124/mol.108.045690

  View details for Web of Science ID 000256889600029

  View details for PubMedID 18424552

• A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy CANCER CELL Turcotte, S., Chan, D. A., Sutphin, P. D., Hay, M. P., Denny, W. A., Giaccia, A. J. 2008; 14 (1): 90-102

  Abstract

  Renal cell carcinomas (RCCs) are refractory to standard therapies. The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in 75% of RCCs. By screening for small molecules selectively targeting VHL-deficient RCC cells, we identified STF-62247. STF-62247 induces cytotoxicity and reduces tumor growth of VHL-deficient RCC cells compared to genetically matched cells with wild-type VHL. STF-62247-stimulated toxicity occurs in a HIF-independent manner through autophagy. Reduction of protein levels of essential autophagy pathway components reduces sensitivity of VHL-deficient cells to STF-62247. Using a yeast deletion pool, we show that loss of proteins involved in Golgi trafficking increases killing by STF-62247. Thus, we have found a small molecule that selectively induces cell death in VHL-deficient cells, representing a paradigm shift for targeted therapy.

  View details for DOI 10.1016/j.ccr.2008.06.004

  View details for Web of Science ID 000257655100012

  View details for PubMedID 18598947

  View details for PubMedCentralID PMC2819422

• The metastasis-associated gene Prl-3 is a p53 target involved in cell-cycle regulation MOLECULAR CELL Basak, S., Jacobs, S. B., Krieg, A. J., Pathak, N., Zeng, Q., Kaldis, P., Giaccia, A. J., Attardi, L. D. 2008; 30 (3): 303-314

  Abstract

  The p53 tumor suppressor restricts tumorigenesis through the transcriptional activation of target genes involved in cell-cycle arrest and apoptosis. Here, we identify Prl-3 (phosphatase of regenerating liver-3) as a p53-inducible gene. Whereas previous studies implicated Prl-3 in metastasis because of its overexpression in metastatic human colorectal cancer and its ability to promote invasiveness and motility, we demonstrate here that Prl-3 is an important cell-cycle regulator. Consistent with a role in DNA damage-induced cell-cycle arrest, Prl-3 overexpression induces G(1) arrest downstream of p53 by triggering a PI3K-Akt-activated negative feedback loop. Surprisingly, attenuation of Prl-3 expression also elicits an arrest response, suggesting that basal level Prl-3 expression is pivotal for normal cell-cycle progression. Our findings highlight key dose-dependent functions of Prl-3 in both positive and negative regulation of cell-cycle progression and provide insight into Prl-3's role in cancer progression.

  View details for DOI 10.1016/j.molcel.2008.04.002

  View details for Web of Science ID 000255761200008

  View details for PubMedID 18471976

• Molecular Imaging of Hypoxia-Inducible Factor 1 alpha and von Hippel-Lindau Interaction in Mice MOLECULAR IMAGING Choi, C. Y., Chau, D. A., Paulmurugan, R., Sutphin, P. D., Le, Q., Koong, A. C., Zundel, W., Gambhir, S. S., Giaccia, A. J. 2008; 7 (3): 139-146

  Abstract

  Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.

  View details for DOI 10.2310/7290.2008.00017

  View details for Web of Science ID 000260954700004

  View details for PubMedID 19123984

• The role of hypoxia-inducible factors in tumorigenesis CELL DEATH AND DIFFERENTIATION Rankin, E. B., Giaccia, A. J. 2008; 15 (4): 678-685

  Abstract

  Hypoxia-inducible factors (HIFs) are essential mediators of the cellular oxygen-signaling pathway. They are heterodimeric transcription factors consisting of an oxygen-sensitive alpha subunit (HIF-alpha) and a constitutive beta subunit (HIF-beta) that facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating the expression of genes that control glucose uptake, metabolism, angiogenesis, erythropoiesis, cell proliferation, and apoptosis. In most experimental models, the HIF pathway is a positive regulator of tumor growth as its inhibition often results in tumor suppression. In clinical samples, HIF is found elevated and correlates with poor patient prognosis in a variety of cancers. In summary, HIF regulates multiple aspects of tumorigenesis, including angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy, making it a critical regulator of the malignant phenotype.

  View details for DOI 10.1038/cdd.2008.21

  View details for Web of Science ID 000254041800010

  View details for PubMedID 18259193

  View details for PubMedCentralID PMC3050610

• Principles of Radiation Oncology Principles & Practice of Oncology, 8th Edition Lawrence TS, Ten Haken RK, Giaccia AJ 2008: 307-327
• Hypoxia-inducible factor-1 alpha stabilization in nonhypoxic conditions: Role of oxidation and intracellular ascorbate depletion MOLECULAR BIOLOGY OF THE CELL Page, E. L., Chan, D. A., Giaccia, A. J., Levine, M., Richard, D. E. 2008; 19 (1): 86-94

  Abstract

  Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1alpha, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1alpha accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein-coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1alpha protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1alpha and allowing HIF-1alpha protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1alpha stability is mediated through the Ang II-mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1alpha stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II-related cell responses.

  View details for DOI 10.1091/mbc.E07-06-0612

  View details for Web of Science ID 000258847500009

  View details for PubMedID 17942596

• Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1 alpha stabilization during ischemia CIRCULATION Chang, E. I., Loh, S. A., Ceradini, D. J., Chang, E. I., Lin, S., Bastidas, N., Aarabi, S., Chan, D. A., Freedman, M. L., Giaccia, A. J., Gurtner, G. C. 2007; 116 (24): 2818-2829

  Abstract

  Advanced age is known to impair neovascularization. Because endothelial progenitor cells (EPCs) participate in this process, we examined the effects of aging on EPC recruitment and vascular incorporation.Murine neovascularization was examined by use of an ischemic flap model, which demonstrated aged mice (19 to 24 months) had decreased EPC mobilization (percent mobilized 1.4+/-0.2% versus 0.4+/-0.1%, P<0.005) that resulted in impaired gross tissue survival compared with young mice (2 to 6 months). This decrease correlated with diminished tissue perfusion (P<0.005) and decreased CD31+ vascular density (P<0.005). Gender-mismatched bone marrow transplantation demonstrated significantly fewer chimeric vessels in aged mice (P<0.05), which confirmed a deficit in bone marrow-mediated vasculogenesis. Age had no effect on total EPC number in mice or humans. Reciprocal bone marrow transplantations confirmed that impaired neovascularization resulted from defects in the response of aged tissue to hypoxia and not from intrinsic defects in EPC function. We demonstrate that aging decreased hypoxia-inducible factor 1alpha stabilization in ischemic tissues because of increased prolyl hydroxylase-mediated hydroxylation (P<0.05) and proteasomal degradation. This resulted in a diminished hypoxia response, including decreased stromal cell-derived factor 1 (P<0.005) and vascular endothelial growth factor (P<0.0004). This effect can be reversed with the iron chelator deferoxamine, which results in hypoxia-inducible factor 1alpha stabilization and increased tissue survival.Aging impairs EPC trafficking to sites of ischemia through a failure of aged tissues to normally activate the hypoxia-inducible factor 1alpha-mediated hypoxia response.

  View details for DOI 10.1161/CIRCULATIONAHA.107.715847

  View details for Web of Science ID 000251542200009

  View details for PubMedID 18040029

• Oxygen sensing and the DNA-damage response CURRENT OPINION IN CELL BIOLOGY Hammond, E. M., Kaufmann, M. R., Giaccia, A. J. 2007; 19 (6): 680-684

  View details for DOI 10.1016/j.ceb.2007.10.002

  View details for Web of Science ID 000252143000012

  View details for PubMedID 18023567

• In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis TISSUE ENGINEERING Xu, Y., Malladi, P., Chiou, M., Bekerman, E., Giaccia, A. J., Longaker, M. T. 2007; 13 (12): 2981-2993

  Abstract

  Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

  View details for DOI 10.1089/ten.2007.0050

  View details for Web of Science ID 000251788400018

  View details for PubMedID 17916040

• Short hairpin RNA interference therapy in murine myocardial infarction 80th Annual Scientific Session of the American-Heart-Association (AHA) Huang, M., Chan, D., Jia, F., Xie, X., Li, Z., Giaccia, A., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2007: 590–90

  View details for Web of Science ID 000250394302665

• Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas 48th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Le, Q., Kong, C., Lavori, P. W., O'Byrne, K., Erler, J. T., Huang, X., Chen, Y., Cao, H., Tibshiran, R., Denko, N., Giaccia, A. J., Koong, A. C. ELSEVIER SCIENCE INC. 2007: 167–75

  Abstract

  To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO(2) and prognosis.We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IkappaB kinase beta, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO(2) measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO(2), and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis.Osteopontin expression correlated with tumor pO(2) (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age).We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy.

  View details for DOI 10.1016/j.ijrobp.2007.01.071

  View details for PubMedID 17707270

• Oxygen sensitivity of reporter genes: Implications for preclinical imaging of tumor hypoxia MOLECULAR IMAGING Cecic, I., Chan, D. A., Sutphin, P. D., Ray, P., Gambhir, S. S., Giaccia, A. J., Gravcs, E. E. 2007; 6 (4): 219-228

  Abstract

  Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

  View details for DOI 10.2310/7290.2007.00017

  View details for Web of Science ID 000249349100001

  View details for PubMedID 17711777

• Targeting the loss of the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells CANCER RESEARCH Sutphin, P. D., Chan, D. A., Li, J. M., Turcotte, S., Krieg, A. J., Giaccia, A. J. 2007; 67 (12): 5896-5905

  Abstract

  Late-stage clear cell renal carcinoma poses a formidable clinical challenge due to the high mortality rate associated with this disease. Molecular and genetic studies have identified functional loss of the von Hippel-Lindau (VHL) gene as a frequent and crucial event in the development of the malignant phenotype of clear cell renal carcinomas. Loss of VHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. The objective of this study was to evaluate the possibility of targeting VHL loss through pharmacologic means. Chromomycin A3 (ChA3) was identified through in silico analysis of existing publicly available drug profiles from the National Cancer Institute as an agent that seemed to selectively target VHL-deficient clear cell renal carcinoma cells. Genotype-selective toxicity was first determined through short-term viability assays and then confirmed with clonogenic studies. Coculture of fluorescently labeled VHL-deficient and VHL-positive cells showed discriminate killing of the VHL-deficient cells with ChA3. Mechanistically, overexpression of hypoxia-inducible factor (HIF)-2alpha in VHL-positive clear cell renal carcinoma cells phenocopied loss of VHL with respect to ChA3 toxicity, establishing ChA3 as a HIF-dependent cytotoxin. This study shows the feasibility of selectively targeting the loss of the VHL tumor suppressor gene in clear cell renal carcinoma for potential clinical benefit and may have greater ramifications in the development of new targeted therapies for the treatment of cancer and other genetic diseases.

  View details for DOI 10.1158/0008-5472.CAN-07-0604

  View details for Web of Science ID 000247360100042

  View details for PubMedID 17575159

• Energy regulation: HIF MXles it up with the C-MYC powerhouse DEVELOPMENTAL CELL Sutphin, P. D., Giaccia, A. J., Chan, D. A. 2007; 12 (6): 845-846

  Abstract

  The phenomenon of aerobic glycolysis observed in cancer cells has remained unexplained. In the May 2007 issue of Cancer Cell, Zhang and colleagues determine that the hypoxia-inducible factor family of transcriptional factors regulate mitochondrial biogenesis through inhibition of C-MYC.

  View details for DOI 10.1016/j.devcel.2007.05.006

  View details for Web of Science ID 000247119700004

  View details for PubMedID 17543856

• Hypoxia inducible factor-1 alpha deficiency affects chondrogenesis of adipose-derived adult stromal cells TISSUE ENGINEERING Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2007; 13 (6): 1159-1171

  Abstract

  Increased cartilage-related disease, poor regeneration of cartilage tissue, and limited treatment options have led to intense research in tissue engineering of cartilage. Adipose-derived adult stromal cells (ADAS) are a promising cell source for skeletal tissue engineering; understanding ADAS cellular signaling and chondrogenesis will advance cell-based therapies in cartilage repair. Chondrocytes are unique-they are continuously challenged by a hypoxic microenvironment. Hypoxia inducible factor-1-alpha (HIF-1alpha), a critical mediator of a cell's response to hypoxia, plays a significant role in chondrocyte survival, growth arrest, and differentiation. By using an established in vitro 3-dimensional micromass system, we investigated the role of HIF-1alpha in chondrogenesis. Targeted deletion of HIF-1alpha in ADAS substantially inhibited the chondrogenic pathway specifically. In marked contrast, deletion of HIF-1alpha did not affect osteogenic differentiation but enhanced adipogenic differentiation. This study demonstrates the critical and specific interplay between HIF-1alpha and chondrogenesis in vitro.

  View details for DOI 10.1089/ten.2006.0265

  View details for Web of Science ID 000247237600003

  View details for PubMedID 17518738

• Hypoxia, gene expression, and metastasis CANCER AND METASTASIS REVIEWS Chan, D. A., Giaccia, A. J. 2007; 26 (2): 333-339

  Abstract

  Hypoxia poses many problems to the treatment of cancer. Hypoxic tumors are more resistant to chemotherapy and radiation. In addition, hypoxia induces a number of genes responsible for increased invasion, aggressiveness, and metastasis of tumors. The augmented metastatic potential due to hypoxia-mediated gene expression is discussed in this section. Particular attention is given to recent studies of specific genes involved in the key steps of metastasis, including extracellular matrix interactions, migration, and proliferation.

  View details for DOI 10.1007/s10555-007-9063-1

  View details for Web of Science ID 000248300100012

  View details for PubMedID 17458506

• Hif-1 alpha regulates differentiation of limb bud mesenchyme and joint development JOURNAL OF CELL BIOLOGY Provot, S., Zinyk, D., Gunes, Y., Kathri, R., Le, Q., Kronenberg, H. M., Johnson, R. S., Longaker, M. T., Giaccia, A. J., Schipani, E. 2007; 177 (3): 451-464

  Abstract

  Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1alpha. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1alpha is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1alpha in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1alpha in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1alpha in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1alpha in early chondrogenesis and joint formation.

  View details for DOI 10.1083/jcb.200612023

  View details for Web of Science ID 000246580000009

  View details for PubMedID 17470636

  View details for PubMedCentralID PMC2064828

• Imaging tumoral hypoxia: Oxygen concentrations and beyond ONCOLOGY-NEW YORK Graves, E. E., Giaccia, A. J. 2007; 21 (3): 368-376

  Abstract

  The role of hypoxia as a key determinant of outcome for human cancers has encouraged efforts to noninvasively detect and localize regions of poor oxygenation in tumors. In this review, we will summarize existing and developing techniques for imaging tumoral hypoxia. A brief review of the biology of tumor oxygenation and its effect on tumor cells will be provided initially. We will then describe existing methods for measurement of tissue oxygenation status. An overview of emerging molecular imaging techniques based on radiolabeled hypoxic markers such as misonidazole or hypoxia-related genes and proteins will then be given, and the usefulness of these approaches toward targeting hypoxia directly will be assessed. Finally, we will evaluate the clinical potential of oxygen- and molecular-specific techniques for imaging hypoxia, and discuss how these methods will individually and collectively advance oncology.

  View details for Web of Science ID 000253822400010

  View details for PubMedID 17447439

• Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro MOLECULAR AND CELLULAR BIOLOGY Pan, Y., Mansfield, K. D., Bertozzi, C. C., Rudenko, V., Chan, D. A., Giaccia, A. J., Simon, M. C. 2007; 27 (3): 912-925

  Abstract

  Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.

  View details for DOI 10.1128/MCB.01223-06

  View details for Web of Science ID 000243946000011

  View details for PubMedID 17101781

  View details for PubMedCentralID PMC1800695

• Noninvasive monitoring of ligand-dependent VEGF receptor-2 dimerization with split firefly luciferase 49th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Lee, P., Chan, C., Hua, A., Paulmurugan, R., Chan, D., Gambhir, S., Le, Q., Giaccia, A. ELSEVIER SCIENCE INC. 2007: S96–S97

  View details for Web of Science ID 000249950200173

• HIF gene expression in cancer therapy OXYGEN BIOLOGY AND HYPOXIA Chan, D. A., Krieg, A. J., Turcotte, S., Giaccia, A. J. 2007; 435: 323-?

  Abstract

  Tumor hypoxia is a feature common to almost all solid tumors due to malformed vasculature and inadequate perfusion. Tumor cells have evolved mechanisms that allow them to respond and adapt to a hypoxic microenvironment. The hypoxia-inducible transcription factor (HIF) family is comprised of oxygen-sensitive alpha (alpha) subunits that respond rapidly to decreased oxygen levels and oxygen-insensitive beta (beta) subunits. HIF binds to specific recognition sequences in the genome and increases the transcription of genes involved in a variety of metabolic and enzymatic pathways that are necessary for cells to respond to an oxygen-poor environment. The critical role of this family of transcriptional regulators in maintaining oxygen homeostasis is supported by multiple regulatory mechanisms that allow the cell to control the levels of HIF as well as its transcriptional activity. This review will focus on how the transcriptional activity of HIF is studied and how it can be exploited for cancer therapy.

  View details for DOI 10.1016/S0076-6879(07)35016-7

  View details for Web of Science ID 000251162300016

  View details for PubMedID 17998061

• HIF1 alpha delays premature senescence through the activation of MIF GENES & DEVELOPMENT Welford, S. M., Bedogni, B., Gradin, K., Poellinger, L., Powell, M. B., Giaccia, A. J. 2006; 20 (24): 3366-3371

  Abstract

  Premature senescence in vitro has been attributed to oxidative stress leading to a DNA damage response. In the absence of oxidative damage that occurs at atmospheric oxygen levels, proliferation of untransformed cells continues for extended periods of time. We have investigated the role of the hypoxia-inducible factor 1alpha (HIF1alpha) transcription factor in preventing senescence in aerobic and hypoxic conditions. Using embryonic fibroblasts from a conditional HIF1alpha knockout mouse, we found that loss of HIF1alpha under aerobic conditions significantly accelerated the onset of cellular senescence, and decreased proliferation under hypoxia. Furthermore, we identify the macrophage migration inhibitory factor (MIF) as a crucial effector of HIF1alpha that delays senescence. Inhibition of MIF phenocopies loss of HIF1alpha. Our findings highlight a novel role for HIF1alpha under aerobic conditions, and identify MIF as a target responsible for this function.

  View details for DOI 10.1101/gad.1471106

  View details for Web of Science ID 000242996100004

  View details for PubMedID 17142669

  View details for PubMedCentralID PMC1698444

• Plasma osteopontin is an independent prognostic marker for head and neck cancers 41st Annual Meeting of the American-Society-of-Clinical-Oncology Petrik, D., Lavori, P. W., Cao, H., Zhu, Y., Wong, P., Christofferson, E., Kaplan, M. J., Pinto, H. A., Sutphin, P., Koong, A. C., Giaccia, A. J., Le, Q. AMER SOC CLINICAL ONCOLOGY. 2006: 5291–97

  Abstract

  To confirm the relationship between plasma osteopontin (OPN) levels and treatment outcomes in head and neck squamous cell carcinoma (HNSCC) patients in an expanded study.One hundred forty patients with newly diagnosed HNSCC were enrolled onto this study, 54 previously reported and 86 new patients. Pretreatment plasma OPN levels were assessed in all patients by an enzyme-linked immunosorbent assay method. OPN levels were correlated to treatment outcomes in the new group of patients. Detailed analyses were also performed on the relationship between OPN and tumor control rate, event-free survival (EFS), and postrelapse survival for the entire group.Using a previously defined cut off point of 450 ng/mL, there was a significant correlation between OPN and freedom-from-relapse (P = .047), overall survival (P = .019), and EFS (P = .023) in the new, independent patient cohort (n = 86). Sequence of event analyses using the entire group (N = 140) revealed that OPN was an independent prognostic factor for initial tumor control, EFS in those who have achieved tumor control, and postrelapse survival.In this expanded study, we were able to replicate the prognostic significance of OPN using a predefined cut off point in an independent patient group and demonstrated that plasma OPN is an independent prognostic marker for HNSCC.

  View details for DOI 10.1200/JCO.2006.06.8627

  View details for Web of Science ID 000242342800017

  View details for PubMedID 17114663

• Elevated HIF-1 alpha levels in CLLB cells may explain their autocrine VEGF secretion. 48th Annual Meeting of the American-Society-of-Hematology Lee, Y. K., Shanafelt, T., Chan, D., Giaccia, A., Mukhopadhyay, D., Taccioli, C., Calin, G., Croce, C., Kay, N. E. AMER SOC HEMATOLOGY. 2006: 176A–176A

  View details for Web of Science ID 000242440000584

• Lysyl oxidase mediates hypoxic control of metastasis CANCER RESEARCH Erler, J. T., Giaccia, A. J. 2006; 66 (21): 10238-10241

  Abstract

  Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.

  View details for DOI 10.1158/0008-5472.CAN-06-3197

  View details for Web of Science ID 000241781600003

  View details for PubMedID 17079439

• Hypoxia: Importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy INTERNATIONAL JOURNAL OF RADIATION BIOLOGY Tatum, J. L., Kelloff, G. J., Gillies, R. J., Arbeit, J. M., Brown, J. M., Chao, K. S., Chapman, J. D., Eckelman, W. C., Fyles, A. W., Giaccia, A. J., Hill, R. P., Koch, C. J., Krishna, M. C., Krohn, K. A., Lewis, J. S., Mason, R. P., Melillo, G., Padhani, A. R., Powis, G., Rajendran, J. G., Reba, R., Robinson, S. P., Semenza, G. L., Swartz, H. M., Vaupel, P., Yang, D., Croft, B., Hoffman, J., Liu, G., Stone, H., Sullivan, D. 2006; 82 (10): 699-757

  Abstract

  The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques.This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

  View details for DOI 10.1080/09553000601002324

  View details for Web of Science ID 000242762100001

  View details for PubMedID 17118889

• Functional analysis of p53 binding under differential stresses MOLECULAR AND CELLULAR BIOLOGY Krieg, A. J., Hammond, E. M., Giaccia, A. J. 2006; 26 (19): 7030-7045

  Abstract

  Hypoxia and DNA damage stabilize the p53 protein, but the subsequent effect that each stress has on transcriptional regulation of known p53 target genes is variable. We have used chromatin immunoprecipitation followed by CpG island (CGI) microarray hybridization to identify promoters bound by p53 under both DNA-damaging and non-DNA-damaging conditions in HCT116 cells. Using gene-specific PCR analysis, we have verified an association with CGIs of the highest enrichment (> 2.5-fold) (REV3L, XPMC2H, HNRPUL1, TOR1AIP1, glutathione peroxidase 1, and SCFD2), with CGIs of intermediate enrichment (> 2.2-fold) (COX7A2L, SYVN1, and JAG2), and with CGIs of low enrichment (> 2.0-fold) (MYC and PCNA). We found little difference in promoter binding when p53 is stabilized by these two distinctly different stresses. However, expression of these genes varies a great deal: while a few genes exhibit classical induction with adriamycin, the majority of the genes are unchanged or are mildly repressed by either hypoxia or adriamycin. Further analysis using p53 mutated in the core DNA binding domain revealed that the interaction of p53 with CGIs may be occurring through both sequence-dependent and -independent mechanisms. Taken together, these experiments describe the identification of novel p53 target genes and the subsequent discovery of distinctly different expression phenomena for p53 target genes under different stress scenarios.

  View details for DOI 10.1128/MCB.00322-06

  View details for Web of Science ID 000240740200002

  View details for PubMedID 16980608

  View details for PubMedCentralID PMC1592883

• Hypoxia-inducible factor-1 and p53: Friends, acquaintances, or strangers? CLINICAL CANCER RESEARCH Hammond, E. M., Giaccia, A. J. 2006; 12 (17): 5007-5009

  View details for DOI 10.1158/1078-0432.CCR-06-0613

  View details for Web of Science ID 000240392000003

  View details for PubMedID 16951213

• The roles of Chk 1 and Chk 2 in hypoxia and reoxygenation CANCER LETTERS Hammond, E. M., Freiberg, R. A., Giaccia, A. J. 2006; 238 (2): 161-167

  Abstract

  Both Chk 1 and Chk 2 are critically important checkpoint kinases. Chk 1 is an essential gene that is required for normal cell division and Chk 2 has been found to be mutated in an ever-growing list of human malignancies. Our recent studies indicate that both Chk 1 and Chk 2 have roles to play in the physiological stress of hypoxia/reoxygenation. Loss or inhibition of either kinase sensitizes cells to hypoxia/reoxygenation indicating that either or both could represent significant therapeutic targets.

  View details for DOI 10.1016/j.canlet.2005.06.029

  View details for Web of Science ID 000238904400001

  View details for PubMedID 16085356

• Checking in on hypoxia/reoxygenation CELL CYCLE Freiberg, R. A., Krieg, A. J., Giaccia, A. J., Hammond, E. M. 2006; 5 (12): 1304-1307

  Abstract

  Hypoxia/reoxygenation is a physiological stress that activates the DNA damage pathway. Significantly, this pathway is initiated during hypoxia, in the absence of detectable DNA damage. Our most recent study determined that during hypoxia, Chk 2 is phosphorylated in an ATM-dependent manner. In addition to this finding, we found that components of the MRN complex were not required for Chk 2 phosphorylation during hypoxia/reoxygenation. Once activated, Chk 2 initiates a signaling cascade, which induces a cell cycle arrest in the G2 phase. Loss of the Chk 2-mediated arrest correlated with an increase in sensitivity to hypoxia/reoxygenation. In contrast, loss of a p53-mediated reoxygenation-induced G1 arrest does not correlate with increased sensitivity to hypoxia/reoxygenation.

  View details for Web of Science ID 000238581200013

  View details for PubMedID 16760660

• Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis CANCER RESEARCH Dornhoefer, N., Spong, S., Bennewith, K., Salim, A., Klaus, S., Kambham, N., Wong, C., Kaper, F., Sutphin, P., Nacalumi, R., Hoeckel, M., Le, Q., Longaker, M., Yang, G., Koong, A., Giaccia, A. 2006; 66 (11): 5816-5827

  Abstract

  Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.

  View details for DOI 10.1158/0008-5472.CAN-06-0081

  View details for Web of Science ID 000238003100038

  View details for PubMedID 16740721

• Genome-wide analysis of p53 under hypoxic conditions MOLECULAR AND CELLULAR BIOLOGY Hammond, E. M., Mandell, D. J., Salim, A., Krieg, A. J., Johnson, T. M., Shirazi, H. A., Attardi, L. D., Giaccia, A. J. 2006; 26 (9): 3492-3504

  Abstract

  Hypoxia is an important nongenotoxic stress that modulates the tumor suppressor activity of p53 during malignant progression. In this study, we investigated how genotoxic and nongenotoxic stresses regulate p53 association with chromatin, p53 transcriptional activity, and p53-dependent apoptosis. We found that genotoxic and nongenotoxic stresses result in the accumulation and binding of the p53 tumor suppressor protein to the same cognate binding sites in chromatin. However, it is the stress that determines whether downstream signaling is mediated by association with transcriptional coactivators. In contrast to p53 induced by DNA-damaging agents, hypoxia-induced p53 has primarily transrepression activity. Using extensive microarray analysis, we identified families of repressed targets of p53 that are involved in cell signaling, DNA repair, cell cycle control, and differentiation. Following our previous study on the contribution of residues 25 and 26 to p53-dependent hypoxia-induced apoptosis, we found that residues 25-26 and 53-54 and the polyproline- and DNA-binding regions are also required for both gene repression and the induction of apoptosis by p53 during hypoxia. This study defines a new role for residues 53 and 54 of p53 in regulating transrepression and demonstrates that 25-26 and 53-54 work in the same pathway to induce apoptosis through gene repression.

  View details for DOI 10.1128/MCB.26.9.3492-3504.2006

  View details for Web of Science ID 000236993300013

  View details for PubMedID 16611991

  View details for PubMedCentralID PMC1447427

• Lysyl oxidase is essential for hypoxia-induced metastasis NATURE Erler, J. T., Bennewith, K. L., Nicolau, M., Dornhofer, N., Kong, C., Le, Q. T., Chi, J. T., Jeffrey, S. S., Giaccia, A. J. 2006; 440 (7088): 1222-1226

  Abstract

  Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.

  View details for DOI 10.1038/nature04695

  View details for PubMedID 16642001

• Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2006; 290 (4): C1139-C1145

  Abstract

  Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.

  View details for DOI 10.1152/ajpcell.00415.2005

  View details for PubMedID 16291817

• Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers PLOS MEDICINE Chi, J. T., Wang, Z., Nuyten, D. S., Rodriguez, E. H., Schaner, M. E., Salim, A., Wang, Y., Kristensen, G. B., Helland, A., Borresen-Dale, A. L., Giaccia, A., Longaker, M. T., Hastie, T., Yang, G. P., Van de Vijver, M. J., Brown, P. O. 2006; 3 (3): 395-409

  Abstract

  Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

  View details for DOI 10.1371/journal.pmed.0030047

  View details for Web of Science ID 000236897500020

  View details for PubMedID 16417408

  View details for PubMedCentralID PMC1334226

• An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers CLINICAL CANCER RESEARCH Le, Q. T., Chen, E., Salim, A., Cao, H. B., Kong, C. S., Whyte, R., Donington, J., Cannon, W., Wakelee, H., Tibshirani, R., Mitchell, J. D., Richardson, D., O'Byrne, K. J., Koong, A. C., Giaccia, A. J. 2006; 12 (5): 1507-1514

  Abstract

  To directly assess tumor oxygenation in resectable non-small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes.Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes.The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001).Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors.

  View details for DOI 10.1158/1078-0432.CCR-05-2049

  View details for PubMedID 16533775

• LOX is essential for hypoxia-induced metastasis 3rd International Conference on Translational Research and Pre-Clinical Strategies in Radiation Oncology Erler, J. T., Dornhoefer, N., Le, Q. T., Kong, C., Chi, J. T., Nicolau, M., Jeffrey, S. S., Giaccia, A. J. ELSEVIER IRELAND LTD. 2006: S5–S5

  View details for Web of Science ID 000237599000013

• DNA damage during reoxygenation elicits a Chk2-dependent checkpoint response MOLECULAR AND CELLULAR BIOLOGY Freiberg, R. A., Hammond, E. M., Dorie, M. J., Welford, S. M., Giaccia, A. J. 2006; 26 (5): 1598-1609

  Abstract

  Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2-/- cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.

  View details for DOI 10.1128/MCB.26.5.1598-1609.2006

  View details for Web of Science ID 000235602700003

  View details for PubMedID 16478982

  View details for PubMedCentralID PMC1430245

• The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels CELLULAR MICROBIOLOGY Spear, W., Chan, D., Coppens, I., Johnson, R. S., Giaccia, A., Blader, I. J. 2006; 8 (2): 339-352

  Abstract

  Toxoplasma gondii is an obligate intracellular protozoan pathogen. We previously found that genes mediating cellular responses to hypoxia were upregulated in Toxoplasma -infected cells but not in cells infected with another intracellular pathogen, Trypanosoma cruzi. The inducible expression of these genes is controlled by the hypoxia-inducible factor 1 (HIF1) transcription factor, which is the master regulator of cells exposed to low oxygen. Because this response may be important for parasites to grow at physiological oxygen levels, we tested the hypothesis that HIF1 is important for Toxoplasma growth. Here, we demonstrate that Toxoplasma infection rapidly increased the abundance of the HIF1alpha subunit and activated HIF1 reporter gene expression. In addition, we found that Toxoplasma growth and survival was severely reduced in HIF1alpha knockout cells at 3% oxygen. While HIF1alpha was not required for parasite invasion, we determined that HIF1 was required for parasite cell division and organelle maintenance at 3% oxygen. These data indicate that Toxoplasma activates HIF1 and requires HIF1 for growth and survival at physiologically relevant oxygen levels.

  View details for DOI 10.1111/j.1462-5822.2005.00628.x

  View details for Web of Science ID 000234667200015

  View details for PubMedID 16441443

• Mutations in the PI3K/PTEN/TSC2 pathway contribute to mammalian target of rapamycin activity and increased translation under hypoxic conditions CANCER RESEARCH Kaper, F., Dornhoefer, N., Giaccia, A. J. 2006; 66 (3): 1561-1569

  Abstract

  Decreased oxygen causes a rapid inhibition of mRNA translation. An important regulatory mechanism of translational repression under hypoxic conditions involves inhibition of the mammalian target of rapamycin (mTOR). mTOR is a target of the phosphatase and tensin homologue detected on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase/AKT/TSC2 pathway, a pathway that is frequently mutated in human cancers. Although hypoxia has been shown to inhibit mTOR activity, we show here that the hypoxia-induced inhibition of mTOR activity is attenuated in cells lacking TSC2 or PTEN, resulting in a higher translation rate even under hypoxic conditions. Comparison of mTOR inhibition by hypoxia alone or in combination with rapamycin showed that prolonged exposure to hypoxia was required to fully inhibit mTOR activity even in wild-type cells. Increased mTOR activity and protein synthesis did not translate into enhanced cell proliferation rates. However, lack of TSC2 resulted in a survival advantage when cells were exposed to hypoxia. Protection against hypoxia-induced cell death due to TSC2 deficiency is rapamycin-resistant, suggesting that TSC2 affects an apoptotic pathway. Tumors derived from TSC2 wild-type cells exhibited a growth delay compared with TSC2-deficient tumors, indicating that enhanced mTOR activity is advantageous in the initial phase of tumor growth. Therefore, failure to inhibit mTOR under oxygen-limiting conditions can be affected by upstream activating mutations and increases the survival and growth of hypoxic tumor cells.

  View details for DOI 10.1158/0008-5472.CAN-05-3375

  View details for Web of Science ID 000235095900040

  View details for PubMedID 16452213

• Comment on: Osteopontin as toxic marker RADIOTHERAPY AND ONCOLOGY Le, Q. T., Cao, H. B., Giaccia, A. 2006; 78 (2): 230-230

  View details for DOI 10.1016/j.radonc.2005.12.011

  View details for Web of Science ID 000236490300018

  View details for PubMedID 16442647

• Radiobiology for the Radiologist, Sixth Edition Hall EJ, Giaccia AJ 2006
• Hypoxia in Cancer Apoptosis and Cancer Chemotherapy: From Cutting-edge Science to Novel Therapeutic Concepts Hammond EM, Papandreou I, Giaccia AJ 2006; 2: 777-798
• Expression and prognostic significance of a panel of tissue hypoxia markers in head and neck squamous cell carcinomas (HNSCC) 48th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Le, Q., Kong, C., Lavori, P. W., Erler, J., Huang, X., Chen, Y., Cao, H., Denko, N., Giaccia, A., Koong, A. ELSEVIER SCIENCE INC. 2006: S186–S187

  View details for Web of Science ID 000241221600312

• Molecular imaging of HIF-1 alpha and pVHL interaction in living subjects using firefly luciferase complementation - Improvement over the Renilla luciferase system 48th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Choi, C. Y., Paulmurugan, R., Chan, D. A., Sutphin, P. D., Le, Q., Koong, A., Gambhir, S. S., Giaccia, A. J. ELSEVIER SCIENCE INC. 2006: S48–S49

  View details for Web of Science ID 000241221600083

• Galectin-1: A link between tumor hypoxia and tumor immune privilege 46th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology Le, Q. T., Shi, G. Y., Cao, H. B., Nelson, D. W., Wang, Y. Y., CHEN, E. Y., Zhao, S. C., Kong, C., Richardson, D., O'Byrne, K. J., Giaccia, A. J., Koong, A. C. AMER SOC CLINICAL ONCOLOGY. 2005: 8932–41

  Abstract

  To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression.We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CD3 (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes.SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CD3 staining (P = .01). Expression of galectin-1 and CD3 were significant predictors for overall survival on multivariate analysis.Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege.

  View details for DOI 10.1200/JCO.2005.02.0206

  View details for PubMedID 16219933

• The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation CANCER CELL Bedogni, B., Welford, S. M., Cassarino, D. S., Nickoloff, B. J., Giaccia, A. J., Powell, M. B. 2005; 8 (6): 443-454

  Abstract

  Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.

  View details for DOI 10.1016/j.ccr.2005.11.005

  View details for Web of Science ID 000234164200004

  View details for PubMedID 16338658

• Hypoxia inducible factor-1 alpha is over expressed in CLL B cells because of an impaired proteasome pathway associated with defective interaction with von Hippel-Landau protein. 47th Annual Meeting of the American-Society-of-Hematology Lee, Y. K., Strege, A. K., Bone, N. D., Wellik, L. E., Chan, D. A., Giaccia, A. J., Mukhopadhyay, D., Kay, N. E. AMER SOC HEMATOLOGY. 2005: 598A–598A

  View details for Web of Science ID 000233426003477

• Hypoxia upregulates osteopontin expression in NIH-3T3 cells via a ras-activated enhancer ONCOGENE Zhu, Y. H., Denhardt, D. T., Cao, H. B., Sutphin, P. D., Koong, A. C., Giaccia, A. J., Le, Q. T. 2005; 24 (43): 6555-6563

  Abstract

  Osteopontin (OPN) is a secreted phosphoglycoprotein that has been linked to tumor progression and survival in several solid tumors, including head and neck cancers. Previous studies showed that OPN expression is induced by tumor hypoxia, and its plasma levels can serve as a surrogate marker for tumor hypoxia and treatment outcome in head and neck cancer patients. In this study, we investigate the transcriptional mechanism by which hypoxia enhances OPN expression. We found that OPN is induced in head and neck squamous cell carcinoma (HNSCC) cell lines and in NIH3T3 cells by hypoxia at both mRNA and protein levels in a time-dependent manner. Actinomycin D chase experiments showed that hypoxic induction of OPN was not due to increased mRNA stability. Deletion analyses of the mouse OPN promoter regions indicated that a ras-activated enhancer (RAE) located at -731 to -712 relative to the transcription start site was essential for hypoxia-enhanced OPN transcription. Using electrophoretic mobility shift assays with the RAE DNA sequence, we found that hypoxia induced sequence-specific DNA-binding complexes. Furthermore, hypoxia and ras exposure resulted in an additive induction of OPN protein and mRNA levels that appeared to be mediated by the RAE. Induction of OPN through the RAE element by hypoxia is mediated by an Akt-kinase signaled pathway as decreasing Akt levels with dominant negative constructs resulted in inhibition of OPN induction by hypoxia. Taken together, these results have identified a new hypoxia responsive transcriptional enhancer that is regulated by Akt signaling.

  View details for DOI 10.1038/sj.onc.1208800

  View details for Web of Science ID 000232204100009

  View details for PubMedID 16007184

• The effect of pre-exposure to hypoxia on in vitro chondrogenesis of adipose-derived mesenchymal cells (AMCs) 91st Annual Clinical Congress of the American-College-of-Surgeons Malladi, P., Xu, Y., Giaccia, A., Longaker, M. T. ELSEVIER SCIENCE INC. 2005: S49–S49

  View details for Web of Science ID 000231745800097

• Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha MOLECULAR AND CELLULAR BIOLOGY Chan, D. A., Sutphin, P. D., Yen, S. E., Giaccia, A. J. 2005; 25 (15): 6415-6426

  Abstract

  Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1alpha to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1alpha under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1alpha are unknown. Here we show that the two ODDs of HIF-1alpha have independent yet interactive roles in the regulation of HIF-1alpha protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1alpha protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1alpha regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1alpha has a distinct activity in controlling HIF-1alpha stability in response to different levels of oxygenation.

  View details for DOI 10.1128/MCB.25.15.6415-6426.2005

  View details for Web of Science ID 000230684400011

  View details for PubMedID 16024780

  View details for PubMedCentralID PMC1190339

• A noninvasive approach for assessing tumor hypoxia in xenografts: Developing a urinary marker for hypoxia CANCER RESEARCH Nelson, D. W., Cao, H. B., Zhu, Y. H., Sunar-Reeder, B., Choi, C. Y., Faix, J. D., Brown, J. M., Koong, A. C., Giaccia, A. J., Le, Q. T. 2005; 65 (14): 6151-6158

  Abstract

  Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary beta-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the beta-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete beta-hCG in response to hypoxia or hypoxia-inducible factor 1alpha (HIF-1alpha) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1alpha pathways, including topotecan, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between beta-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and beta-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of beta-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary beta-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE beta-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.

  View details for Web of Science ID 000230633400024

  View details for PubMedID 16024616

• Angiogenesis is required for successful bone induction during distraction osteogenesis JOURNAL OF BONE AND MINERAL RESEARCH Fang, T. D., Salim, A., Xia, W., Nacamuli, R. P., Guccione, S., Song, H. M., Carano, R. A., Filvaroff, E. H., Bednarski, M. D., Giaccia, A. J., Longaker, M. T. 2005; 20 (7): 1114-1124

  Abstract

  The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis.Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis.Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41.Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.

  View details for DOI 10.1359/JBMR.050301

  View details for Web of Science ID 000230134100005

  View details for PubMedID 15940364

• A comprehensive evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers (NSCLC) 11th World Conference on Lung Cancer Le, Q., Chen, E., Salim, A., Cao, H., Kong, C., O'Byrne, K., Mitchell, J., Donington, J., Whyte, R., Giaccia, A. ELSEVIER IRELAND LTD. 2005: S134–S134

  View details for Web of Science ID 000231307800444

• The role of p53 in hypoxia-induced apoptosis BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hammond, E. M., Giaccia, A. J. 2005; 331 (3): 718-725

  Abstract

  Hypoxia represents one of the most physiologically relevant stresses, having significant roles in both normal development and malignant progression. Exposure to severe hypoxia leads to the accumulation of p53 which can in turn lead to rapid apoptosis. In contrast to the response to DNA-damaging agents, hypoxia-induced p53 has little or no transcriptional transactivation capabilities and instead seems to function primarily as a transrepressor in order to induce apoptosis.

  View details for Web of Science ID 000229135900007

  View details for PubMedID 15865928

• An expanded study of the relationship between osteopontin, tumor pO(2) and prognosis in head and neck squamous cell carcinomas. 41st Annual Meeting of the American-Society-of-Clinical-Oncology Petrik, D. W., Cao, H., Zhu, Y., Wong, P., Sutphin, P., Giaccia, A. J., Koong, A. A., Le, Q. T. AMER SOC CLINICAL ONCOLOGY. 2005: 505S–505S

  View details for Web of Science ID 000230326603245

• Inhibition of pancreatic tumor growth and progression by a human monoclonal antibody specific for connective tissue growth factor. 41st Annual Meeting of the American-Society-of-Clinical-Oncology Spong, S. M., Dornhofer, N., Wong, C., Lomongsod, E., Salim, A., Le, Q., Koong, A., Klaus, S., Giaccia, A. AMER SOC CLINICAL ONCOLOGY. 2005: 345S–345S

  View details for Web of Science ID 000230326602201

• Anoxia is necessary for tumor cell toxicity caused by a low-oxygen environment CANCER RESEARCH Papandreou, I., Krishna, C., Kaper, F., Cai, D. L., Giaccia, A. J., Denko, N. C. 2005; 65 (8): 3171-3178

  Abstract

  Cells exposed to oxygen deprivation in vitro have been shown to reduce proliferation and/or engage in programmed cell death. There is considerable controversy in the literature as to the role of hypoxia-inducible factor-1 (HIF-1) and HIF-1 target genes in initiating these responses. We therefore examined the oxygen dependence and the role of the hypoxia-responsive transcription factor HIF-1 in making the cellular death decision. Oxygen concentrations as low as 0.5% did not alter the growth of HIF-1-proficient or HIF-1-deficient murine fibroblasts, or human tumor cells, despite the appropriate induction of HIF-1 target genes. Severe hypoxia (<0.01% oxygen) did induced apoptosis, resulting in decreased colony formation, chromatin condensation, DNA fragmentation, and caspase activation but also independent of HIF1alpha status. Transcriptional induction of HIF-1-dependent genes putatively involved in cell death like BNip3 and BNip3L was therefore disassociated from hypoxia-dependent toxicity. Likewise, forced overexpression of a nondegradable form of HIF-1alpha in several human tumor cell lines was not sufficient to induce apoptosis under normoxic conditions. Taken together, these findings indicate that additional molecular events are triggered by anoxia in a HIF-1-independent manner, and these changes are necessary for cell death observed in low-oxygen environments.

  View details for Web of Science ID 000228424800029

  View details for PubMedID 15833847

• Adaptive myogenesis under hypoxia MOLECULAR AND CELLULAR BIOLOGY Yun, Z., Lin, Q., Giaccia, A. J. 2005; 25 (8): 3040-3055

  Abstract

  Previous studies have indicated that myoblasts can differentiate and repair muscle injury after an ischemic insult. However, it is unclear how hypoxia or glucose deprivation in the ischemic microenvironment affects myoblast differentiation. We have found that myogenesis can adapt to hypoxic conditions. This adaptive mechanism is accompanied by initial inhibition of the myoD, E2A, and myogenin genes followed by resumption of their expression in an oxygen-dependent manner. The regulation of myoD transcription by hypoxia is correlated with transient deacetylation of histones associated with the myoD promoter. It is noteworthy that, unlike the differentiation of other cell types such as preadipocytes or chondroblasts, the effect of hypoxia on myogenesis is independent of HIF-1, a ubiquitous regulator of transcription under hypoxia. While myogenesis can also adapt to glucose deprivation, the combination of severe hypoxia and glucose deprivation found in an ischemic environment results in pronounced loss of myoblasts. Our studies indicate that the ischemic muscle can be repaired via the adaptive differentiation of myogenic precursors, which depends on the levels of oxygen and glucose in the ischemic microenvironment.

  View details for DOI 10.1128/MCB.25.8.3040-3055.2005

  View details for Web of Science ID 000228138500015

  View details for PubMedID 15798192

  View details for PubMedCentralID PMC1069592

• OS-9 interacts with hypoxia-inducible factor 1 alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1 alpha MOLECULAR CELL Baek, J. H., Mahon, P. C., Oh, J., Kelly, B., Krishnamachary, B., Pearson, M., Chan, D. A., Giaccia, A. J., Semenza, G. L. 2005; 17 (4): 503-512

  Abstract

  Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen homeostasis in metazoan species. HIF-1 mediates changes in gene transcription in response to changes in cellular oxygenation. The half-life of the HIF-1alpha subunit is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that OS-9, the protein product of a widely expressed gene, interacts with both HIF-1alpha and HIF-1alpha prolyl hydroxylases. OS-9 gain-of-function promotes HIF-1alpha hydroxylation, VHL binding, proteasomal degradation of HIF-1alpha, and inhibition of HIF-1-mediated transcription. OS-9 loss-of-function caused by RNA interference increases HIF-1alpha protein levels, HIF-1-mediated transcription, and VEGF mRNA expression under nonhypoxic conditions. These data indicate that OS-9 is an essential component of a multiprotein complex that regulates HIF-1alpha levels in an O2-dependent manner.

  View details for DOI 10.1016/j.molcel.2005.01.011

  View details for Web of Science ID 000227143400007

  View details for PubMedID 15721254

• The p53(QS) transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality NATURE GENETICS Johnson, T. M., Hammond, E. M., Giaccia, A., Attardi, L. D. 2005; 37 (2): 145-152

  Abstract

  The role of transcriptional activation in p53 function is highly controversial. To define this role in vivo, we generated a Trp53 knock-in construct encoding a protein carrying mutations of two residues that are crucial for transactivation (L25Q,W26S). Here we show that these mutations have selective effects on the biological functions of p53. Although its ability to activate various p53 target genes is largely compromised, the p53(QS) protein retains the ability to transactivate the gene Bax. The ability of the p53(QS) mutant protein to elicit a DNA damage-induced G1 cell cycle-arrest response is also partially impaired. p53(QS) has selective defects in its ability to induce apoptosis: it is completely unable to activate apoptosis in response to DNA damage, is partially unable to do so when subjected to serum deprivation and retains substantial apoptotic activity upon exposure to hypoxia. These findings suggest that p53 acts through distinct, stimulus-specific pathways to induce apoptosis. The importance of the biological activity of p53(QS) in vivo is underscored by our finding that expression of p53(QS), which cannot bind mdm2, induces embryonic lethality. Taken together, these results suggest that p53 has different mechanisms of action depending on specific contextual cues, which may help to clarify the function of p53 in preventing cancer.

  View details for DOI 10.1038/ng1498

  View details for Web of Science ID 000226690100020

  View details for PubMedID 15654339

• Molecular imaging of HIF-1 alpha and pVHL interaction in vivo: The study of structure/function relationship guiding HIF-1 alpha and pVHL 47th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology Choi, C. Y., Paulmurugan, R., Ghambir, S., Koong, A. C., Le, Q. T., Giaccia, A. J. ELSEVIER SCIENCE INC. 2005: S133–S134

  View details for Web of Science ID 000232083300224

• Identification of hypoxia-regulated proteins in head and neck cancer by proteomic and tissue array profiling CANCER RESEARCH Chen, Y. J., Shi, G. Y., Wei, X., Kong, C., Zhao, S. C., Gaw, A. F., CHEN, E. Y., Yang, G. P., Giaccia, A. J., Le, Q. T., Koong, A. C. 2004; 64 (20): 7302-7310

  Abstract

  Hypoxia within solid tumors decreases therapeutic efficacy, and identification of hypoxia markers may influence the choice of therapeutic modality. Here, we used a proteomic approach to identify hypoxia-regulated proteins and validated their use as endogenous indicators of tumor hypoxia. Using two-dimensional gel electrophoresis and PowerBlot (antibody-based array), we identified a group of 20 proteins that are increased >/=1.5-fold during hypoxia. The majority of these proteins such as IkappaB kinase beta (IKKbeta), MKK3b, highly expressed in cancer (HEC), density-regulated protein 1, P150(glued), nuclear transport factor 2, binder of ARL 2, Paxillin, and transcription termination factor I have not been previously reported to be hypoxia inducible. The increase in these proteins under hypoxia was mediated through posttranscriptional mechanisms. We additionally characterized the role of IKKbeta, a regulator of the nuclear factor-kappaB transcription factor, during hypoxia. We demonstrated that IKKbeta mediates cell survival during hypoxia and is induced in a variety of squamous cell carcinoma cell lines. Furthermore, we showed that IKKbeta expression from tumor specimens correlated with tumor oxygenation in patients with head and neck squamous cell carcinomas. These data suggest that IKKbeta is a novel endogenous marker of tumor hypoxia and may represent a new target for anticancer therapy.

  View details for Web of Science ID 000224522200021

  View details for PubMedID 15492250

• JunD reduces tumor angiogenesis by protecting cells from oxidative stress CELL Gerald, D., Berra, E., Frapart, Y. M., Chan, D. A., Giaccia, A. J., Mansuy, D., Pouyssegur, J., Yaniv, M., Mechta-Grigoriou, F. 2004; 118 (6): 781-794

  Abstract

  Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.

  View details for Web of Science ID 000223992400013

  View details for PubMedID 15369676

• Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts JOURNAL OF BIOLOGICAL CHEMISTRY Salim, A., Nacamuli, R. P., Morgan, E. F., Giaccia, A. J., Longaker, M. T. 2004; 279 (38): 40007-40016

  Abstract

  Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

  View details for DOI 10.1074/jbc.M403715200

  View details for Web of Science ID 000223791500098

  View details for PubMedID 15263007

• The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease GENES & DEVELOPMENT Giaccia, A. J., Simon, M. C., Johnson, R. 2004; 18 (18): 2183-2194

  Abstract

  The ability to sense and respond to changes in oxygen is essential for the survival of prokaryotic and eukaryotic organisms. Oxygen-sensing mechanisms have been developed to maintain cell and tissue homeostasis, as well as to adapt to the chronic low-oxygen conditions found in diseases such as cancer. This report on the first Keystone Meeting on the Biology of Hypoxia will summarize our current understanding of key genes and pathways involved in oxygen sensing that are required for normal development and that are dysregulated in disease states. It will also comment on future directions for this exciting field.

  View details for DOI 10.1101/gad.1243304

  View details for Web of Science ID 000223835400001

  View details for PubMedID 15371333

  View details for PubMedCentralID PMC517513

• Inhibition of ATR leads to increased sensitivity to hypoxia/reoxygenation CANCER RESEARCH Hammond, E. M., Dorie, M. J., Giaccia, A. J. 2004; 64 (18): 6556-6562

  Abstract

  The transient opening and closing of tumor vasculature result in periods of severe oxygen deprivation (hypoxia) followed by reoxygenation. This exerts a positive selective pressure for cells that have lost their sensitivity to killing by reduced oxygen levels. These cells are effectively resistant to hypoxia-induced apoptosis and conventional therapeutic approaches. Here we show hypoxia-induced S-phase arrest results in regions of single-stranded DNA in stalled replication forks and signals the activation of ATR. S-phase cells represent the most sensitive phase of the cell cycle to the stress of hypoxia/reoxygenation. Loss of ATR or inhibition of ATR kinase activity results in a further loss of reproductive viability in S-phase cells when exposed to hypoxic conditions followed by reoxygenation but has little effect on the inhibition of DNA synthesis. This is, at least in part, mediated via Chk1 signaling because loss of Chk1 also results in increased sensitivity to hypoxia/reoxygenation. The observed decrease in reproductive survival is in part because of the accumulation of DNA damage in S-phase cells during hypoxia exposure in the absence of full ATR activity. Therefore, ATR acts to protect stalled replication forks during hypoxia exposure. In conclusion, ATR and Chk1 play critical roles in the cellular response to hypoxia/reoxygenation, and inhibitors of ATR and Chk1 represent new hypoxic cell cytotoxins.

  View details for Web of Science ID 000224089700031

  View details for PubMedID 15374968

• Developmental response to hypoxia FASEB JOURNAL Huang, S. T., Vo, K. C., Lyell, D. J., Faessen, G. H., Tulac, S., Tibshirani, R., Giaccia, A. J., Giudice, L. C. 2004; 18 (12): 1348-1365

  Abstract

  Molecular mechanisms underlying fetal growth restriction due to placental insufficiency and in utero hypoxia are not well understood. In the current study, time-dependent (3 h-11 days) changes in fetal tissue gene expression in a rat model of in utero hypoxia compared with normoxic controls were investigated as an initial approach to understand molecular events underlying fetal development in response to hypoxia. Under hypoxic conditions, litter size was reduced and IGFBP-1 was up-regulated in maternal serum and in fetal liver and heart. Tissue-specific, distinct regulatory patterns of gene expression were observed under acute vs. chronic hypoxic conditions. Induction of glycolytic enzymes was an early event in response to hypoxia during organ development; consistently, tissue-specific induction of calcium homeostasis-related genes and suppression of growth-related genes were observed, suggesting mechanisms underlying hypoxia-related fetal growth restriction. Furthermore, induction of inflammation-related genes in placentas exposed to long-term hypoxia (11 days) suggests a mechanism for placental dysfunction and impaired pregnancy outcome accompanying in utero hypoxia.

  View details for DOI 10.1096/fj.03-1377com

  View details for Web of Science ID 000224243200054

  View details for PubMedID 15333578

• Timeline - Radiation oncology: a century of achievements NATURE REVIEWS CANCER Bernier, J., Hall, E. J., Giaccia, A. 2004; 4 (9): 737-U15

  Abstract

  Over the twentieth century the discipline of radiation oncology has developed from an experimental application of X-rays to a highly sophisticated treatment of cancer. Experts from many disciplines - chiefly clinicians, physicists and biologists - have contributed to these advances. Whereas the emphasis in the past was on refining techniques to ensure the accurate delivery of radiation, the future of radiation oncology lies in exploiting the genetics or the microenvironment of the tumour to turn cancer from an acute disease to a chronic disease that can be treated effectively with radiation.

  View details for DOI 10.1038/nrc1451

  View details for Web of Science ID 000223657800018

  View details for PubMedID 15343280

• XBP1 is essential for survival under hypoxic conditions and is required for tumor growth CANCER RESEARCH Romero-Ramirez, L., Cao, H. B., Nelson, D., Hammond, E., Lee, A. H., Yoshida, H., Mori, K., Glimcher, L. H., Denko, N. C., Giaccia, A. J., Le, Q. T., Koong, A. C. 2004; 64 (17): 5943-5947

  Abstract

  Hypoxia within solid tumors is a major determinant of outcome after anticancer therapy. Analysis of gene expression changes during hypoxia indicated that unfolded protein response genes were one of the most robustly induced groups of genes. In this study, we investigated the hypoxic regulation of X-box binding protein (XBP1), a major transcriptional regulator of the unfolded protein response. Hypoxia induced XBP1 at the transcriptional level and activated splicing of its mRNA, resulting in increased levels of activated XBP1 protein. After exposure to hypoxia, apoptosis increased and clonogenic survival decreased in XBP1-deficient cells. Loss of XBP1 severely inhibited tumor growth due to a reduced capacity for these transplanted tumor cells to survive in a hypoxic microenvironment. Taken together, these studies directly implicate XBP1 as an essential survival factor for hypoxic stress and tumor growth.

  View details for PubMedID 15342372

• Hypoxic gene expression and metastasis CANCER AND METASTASIS REVIEWS Le, Q. T., Denko, N. C., Giaccia, A. J. 2004; 23 (3-4): 293-310

  Abstract

  Solid tumors possess malformed vasculature that results in the exposure of tumor cells to a low oxygen environment. Tumor hypoxia has been demonstrated in human and mouse tumors through the use of oxygen microelectrodes, hypoxic specific biomarkers, specific transcriptional changes induced by hypoxia, and secreted proteins. While many elegant experiments have demonstrated that hypoxia enhances metastatic potential, it is still unknown what mechanisms are involved in this enhancement. In this review, we discuss the clinical and basic science studies that support an important role for hypoxia in increasing the metastatic potential of tumor cells by promoting tissue remodeling, inducing angiogenesis and reducing apoptosis. Particular emphasis is given to recent findings that provide insight to the role of hypoxia in the metastatic process.

  View details for Web of Science ID 000222017400009

  View details for PubMedID 15197330

• The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation DNA REPAIR Hammond, E. M., Giaccia, A. J. 2004; 3 (8-9): 1117-1122

  Abstract

  ATM and ATR are stress-response kinases which respond to a variety of insults including ionizing radiation, replication arrest, ultraviolet radiation and hypoxia/re-oxygenation. Hypoxia occupies a unique niche in the study of both ATR- and ATM-mediated checkpoint pathways. Hypoxia is a physiologically significant stress that occurs in virtually all solid tumors and differs from most other stresses in that it does not induce DNA damage. Previous studies have indicated that hypoxia provides a unique way to induce ATR in response to inhibition of DNA replication. During tumor expansion hypoxia is inevitably followed by periods of re-oxygenation which in vitro has been shown to induce significant levels of DNA damage and an ATM response. Therefore both ATR and ATM have a role to play in hypoxia/re-oxygenation.

  View details for DOI 10.1016/j.dnarep.2004.03.035

  View details for Web of Science ID 000223650700038

  View details for PubMedID 15279800

• The use of plasma surface-enhanced laser desorption/ionization time-of-flight mass spectrometry proteomic patterns for detection of head and neck squamous cell cancers 45th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Soltys, S. G., Le, Q. T., Shi, G. Y., Tibshirani, R., Giaccia, A. J., Koong, A. C. AMER ASSOC CANCER RESEARCH. 2004: 4806–12

  Abstract

  Our study was undertaken to determine the utility of plasma proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry for the detection of head and neck squamous cell carcinomas (HNSCCs).Pretreatment plasma samples from HNSCC patients or controls without known neoplastic disease were analyzed on the Protein Biology System IIc SELDI-TOF mass spectrometer (Ciphergen Biosystems, Fremont, CA). Proteomic spectra of mass:charge ratio (m/z) were generated by the application of plasma to immobilized metal-affinity-capture (IMAC) ProteinChip arrays activated with copper. A total of 37356 data points were generated for each sample. A training set of spectra from 56 cancer patients and 52 controls were applied to the "Lasso" technique to identify protein profiles that can distinguish cancer from noncancer, and cross-validation was used to determine test errors in this training set. The discovery pattern was then used to classify a separate masked test set of 57 cancer and 52 controls. In total, we analyzed the proteomic spectra of 113 cancer patients and 104 controls.The Lasso approach identified 65 significant data points for the discrimination of normal from cancer profiles. The discriminatory pattern correctly identified 39 of 57 HNSCC patients and 40 of 52 noncancer controls in the masked test set. These results yielded a sensitivity of 68% and specificity of 73%. Subgroup analyses in the test set of four different demographic factors (age, gender, and cigarette and alcohol use) that can potentially confound the interpretation of the results suggest that this model tended to overpredict cancer in control smokers.Plasma proteomic profiling with SELDI-TOF mass spectrometry provides moderate sensitivity and specificity in discriminating HNSCC. Further improvement and validation of this approach is needed to determine its usefulness in screening for this disease.

  View details for Web of Science ID 000222840700027

  View details for PubMedID 15269156

• Stem cell differentiation NATURE BIOTECHNOLOGY Salim, A., Giaccia, A. J., Longaker, M. T. 2004; 22 (7): 804-805

  View details for DOI 10.1038/nbt0704-803d

  View details for Web of Science ID 000222533000013

  View details for PubMedID 15229534

• The hypoxic tumor microenvironment and gene expression SEMINARS IN RADIATION ONCOLOGY Leo, C., Giaccia, A. J., Denko, N. C. 2004; 14 (3): 207-214

  Abstract

  Solid tumors are not static entities but are constantly responding to environmental signals as they grow and develop. One mechanism by which they respond to the adverse conditions of the tumor microenvironment is through coordinated changes in gene expression. The synchronized turning of genes on and off leads to biologic adaption to the adverse oxygen-poor environment. Because tumor hypoxia can be found in almost every solid tumor, it represents one of the most pervasive microenvironmental stresses that can impact malignant progression and therapeutic response. Interestingly, tumors that exhibit robust induction of hypoxia-responsive gene expression networks show a clinically more aggressive natural history. The contribution of hypoxia-responsive gene networks to malignant response is currently under investigation. An understanding of the coordinated functions of hypoxia induced and repressed genes can lead to a better understanding of the clinical significance of the hypoxic tumor phenotype.

  View details for DOI 10.1016/j.semradonc.2004.04.007

  View details for Web of Science ID 000223088200003

  View details for PubMedID 15254863

• Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development DEVELOPMENT Pfander, D., Kobayashi, T., Knight, M. C., Zelzer, E., Chan, D. A., Olsen, B. R., Giaccia, A. J., Johnson, R. S., Haase, V. H., Schipani, E. 2004; 131 (10): 2497-2508

  Abstract

  The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1alpha (HIF1alpha), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL (Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1alpha leads to increased expression levels of HIF1alpha target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1a genes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1alpha. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.

  View details for DOI 10.1242/dev.01138

  View details for Web of Science ID 000222127700025

  View details for PubMedID 15128677

• Proceedings of the Oxygen Homeostasis/Hypoxia Meeting. Cancer research Kaufman, B., Scharf, O., Arbeit, J., Ashcroft, M., Brown, J. M., Bruick, R. K., Chapman, J. D., Evans, S. M., Giaccia, A. J., Harris, A. L., Huang, E., Johnson, R., Kaelin, W., Koch, C. J., Maxwell, P., Mitchell, J., Neckers, L., Powis, G., Rajendran, J., Semenza, G. L., Simons, J., Storkebaum, E., Welch, M. J., Whitelaw, M., Melillo, G., Ivy, S. P. 2004; 64 (9): 3350-3356

  View details for PubMedID 15126380

• Topical treatment with inhibitors of the phosphatidylinositol 3 '-kinase/akt and raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways reduces melanoma development in severe combined immunodericient mice CANCER RESEARCH Bedogni, B., O'Neill, M. S., Welford, S. M., Bouley, D. M., Giaccia, A. J., Denko, N. C., Powell, M. B. 2004; 64 (7): 2552-2560

  Abstract

  Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways inhibited the growth of TPras transgenic melanomas in severe combined immunodeficient mice, blocked invasive behavior, and reduced angiogenesis. The inhibitor Ly294002, which is specific for phosphatidylinositol 3'-kinase, effectively reduced melanoma cell growth both in vitro and in vivo. Both Ly294002 and U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, reduced invasion, which correlated with reduction of the metalloproteinase matrix metalloproteinase 2. Tumor angiogenesis was disrupted through inhibition of vascular endothelial growth factor production from the tumor cells and antiangiogenic effects on endothelial cells. Observations with TPras melanoma cells that express dominant negative Deltap85 or kinase-inactive Raf(301) supported the specificity of the phenomena observed with the chemical inhibitors. These studies demonstrate that topical treatment targeting Ras effectors is efficacious, without systemic toxicities, and may prove to be useful in treating and preventing the progression of cutaneous melanoma.

  View details for Web of Science ID 000220586500035

  View details for PubMedID 15059911

• Distinct aerobic and hypoxic mechanisms of HIF-alpha regulation by CSN5 GENES & DEVELOPMENT Bemis, L., Chan, D. A., Finkielstein, C. V., Qi, L., Sutphin, P. D., Chen, X. J., Stenmark, K., Giaccia, A. J., Zundel, W. 2004; 18 (7): 739-744

  Abstract

  Mammalian oxygen homeostasis is dependent on the HIF family of transcription factors. The CSN subunit, CSN5, binds both the CODD of HIF-1 alpha and the pVHL tumor suppressor. High CSN5 expression generates a pVHL-independent form of CSN5 that stabilizes HIF-1 alpha aerobically by inhibiting HIF-1 alpha prolyl-564 hydroxylation. Aerobic CSN5 association with HIF-1 alpha occurs independently of the CSN holocomplex, leading to HIF-1 alpha stabilization independent of Cullin 2 deneddylation. CSN5 weakly associates with HIF-1 alpha under hypoxia, but is required for optimal hypoxia-mediated HIF-1 alpha stabilization. These results indicate that CSN5 regulates aerobic as well as hypoxic HIF-1 alpha stability by different mechanisms during oncogenesis.

  View details for DOI 10.1101/gad.1180104

  View details for Web of Science ID 000220923100002

  View details for PubMedID 15082527

• Dead cells don't form tumors - HIF-dependent cytotoxins CELL CYCLE Sutphin, P. D., Chan, D. A., Giaccia, A. J. 2004; 3 (2): 160-163

  Abstract

  Elimination or reduction of tumor burden is the primary goal of cancer therapy. Strategies to achieve this goal with the fewest adverse effects to the patient are an area of intense investigation. Elevated protein levels of hypoxia-inducible factor (HIF) are commonly found in solid tumors, while rarely found in healthy tissue. Numerous studies have suggested that HIF activity is essential for the development of solid tumors. Thus, inhibition of HIF represents an attractive therapeutic target for eradicating tumors. The search for small molecules that target and inhibit HIF activity is currently underway. We propose an alternate approach: to directly target and kill HIF-activated tumor cells. This approach is advantageous in that cells with activated HIF will be eliminated directly. Specific elimination of HIF-activated cells represents a potential mechanism for inhibiting tumor growth, with the potential advantage of sparing the patient of the normal tissue toxicity associated with current treatment options.

  View details for Web of Science ID 000222361200020

  View details for PubMedID 14712081

• Regulation of in vitro adipogenesis by estradiol. 51st Annual Meeting of the Society-for-Gynecologic-Investigation Swiersz, L. M., Yun, Z., Theologis, A., Pisarska, M. D., Giaccia, A. J. ELSEVIER SCIENCE INC. 2004: 314A–314A

  View details for Web of Science ID 000220184500708

• Oxygen-dependent regulation of adipogenesis OXYGEN SENSING Swiersz, L. M., Giaccia, A. J., Yun, Z. 2004; 381: 387-?

  View details for Web of Science ID 000221513700027

  View details for PubMedID 15063688

• XBP1 is essential for survival under hypoxic conditions and is required for tumor growth 46th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology Romero, L., Cao, H., Hammond, E., Giaccia, A. J., Le, Q. T., Koong, A. C. ELSEVIER SCIENCE INC. 2004: S192–S193

  View details for Web of Science ID 000223854700105

• Identification of Galectin-1 as a novel hypoxia-regulated protein via proteomic approaches 46th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology Le, Q., Shi, G., Nelson, D., Patel, D., Zhao, S., Giaccia, A. J., Kong, C., Koong, A. C. ELSEVIER SCIENCE INC. 2004: S192–S192

  View details for Web of Science ID 000223854700104

• Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS Hammond, E. M., GREEN, S. L., Giaccia, A. J. 2003; 532 (1-2): 205-213

  Abstract

  Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.

  View details for DOI 10.1016/j.mrfmmm.2003.08.017

  View details for Web of Science ID 000187355500014

  View details for PubMedID 14643437

• Therapeutic exploitation of the physiological and molecular genetic alterations in head and neck cancer CLINICAL CANCER RESEARCH Le, Q. T., Giaccia, A. J. 2003; 9 (12): 4287-4295

  Abstract

  Despite improvements in the diagnosis and management of head and neck squamous cell carcinomas, there has been minimal increase in the long-term survival in these patients over the last 30 years. Treatment intensification with concurrent chemoradiotherapy has been shown to increase survival and improve organ preservation over radiotherapy alone in patients with locally advanced tumor; however, at a cost of increased long-term toxicity. Recent advances in molecular technology have ushered in a new age of targeted therapy, which holds promise for a better outcome for these patients with potentially less normal tissue toxicity. Some of the new approaches aim to specifically inhibit tumor growth and metastasis by targeting the tumor microenvironment or vasculature, whereas others focus on specific protein or signal transduction pathways. This review will summarize these new molecular and physiological based strategies that can be used for both treatment and chemoprevention of head and neck squamous cell carcinoma.

  View details for Web of Science ID 000185830700001

  View details for PubMedID 14555497

• HIF-1 as a target for drug development NATURE REVIEWS DRUG DISCOVERY Giaccia, A., Siim, B. G., Johnson, R. S. 2003; 2 (10): 803-811

  Abstract

  Sensing and responding to fluxes in oxygen tension is perhaps the single most important variable in physiology, and animal tissues have developed a number of essential mechanisms to cope with the stress of low physiological oxygen levels, or hypoxia. Among these coping mechanisms is the response mediated by the hypoxia-inducible transcription factor, or HIF-1. HIF-1 is an essential component in changing the transcriptional repertoire of tissues as oxygen levels drop, and could prove to be a very important target for drug development, as treatments evolve for diseases, such as cancer, heart disease and stroke, in which hypoxia is a central aspect.

  View details for DOI 10.1038/nrd1199

  View details for Web of Science ID 000185920400013

  View details for PubMedID 14526383

• Investigating hypoxic tumor physiology through gene expression patterns ONCOGENE Denko, N. C., Fontana, L. A., Hudson, K. M., Sutphin, P. D., Raychaudhuri, S., Altman, R., Giaccia, A. J. 2003; 22 (37): 5907-5914

  Abstract

  Clinical evidence shows that tumor hypoxia is an independent prognostic indicator of poor patient outcome. Hypoxic tumors have altered physiologic processes, including increased regions of angiogenesis, increased local invasion, increased distant metastasis and altered apoptotic programs. Since hypoxia is a potent controller of gene expression, identifying hypoxia-regulated genes is a means to investigate the molecular response to hypoxic stress. Traditional experimental approaches have identified physiologic changes in hypoxic cells. Recent studies have identified hypoxia-responsive genes that may define the mechanism(s) underlying these physiologic changes. For example, the regulation of glycolytic genes by hypoxia can explain some characteristics of the Warburg effect. The converse of this logic is also true. By identifying new classes of hypoxia-regulated gene(s), we can infer the physiologic pressures that require the induction of these genes and their protein products. Furthermore, these physiologically driven hypoxic gene expression changes give us insight as to the poor outcome of patients with hypoxic tumors. Approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. However, there is significant heterogeneity in the transcriptional response to hypoxia between different cell types. Moreover, the coordinated change in the expression of families of genes supports the model of physiologic pressure leading to expression changes. Understanding the evolutionary pressure to develop a 'hypoxic response' provides a framework to investigate the biology of the hypoxic tumor microenvironment.

  View details for DOI 10.1038/sj.onc.1206703

  View details for Web of Science ID 000185086100017

  View details for PubMedID 12947397

• Hypoxia-induced gene expression occurs solely through the action of hypoxia-inducible factor 1 alpha (HIF-1 alpha): Role of cytoplasmic trapping of HIF-2 alpha MOLECULAR AND CELLULAR BIOLOGY Park, S. K., Dadak, A. M., Haase, V. H., Fontana, L., Giaccia, A. J., Johnson, R. S. 2003; 23 (14): 4959-4971

  Abstract

  The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. Here we report on the differential regulation of protein expression and transcriptional activity of HIF-1alpha and -2alpha by hypoxia in immortalized mouse embryo fibroblasts (MEFs). We show that oxygen-dependent protein degradation is restricted to HIF-1alpha, as HIF-2alpha protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2alpha remained transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2alpha translocated into the nucleus and could stimulate expression of hypoxia-inducible genes. We show that the factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1alpha but has no effect on HIF-2alpha-mediated transcription in MEFs. We propose that HIF-2alpha is not a redundant transcription factor of HIF-1alpha for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1alpha but not HIF-2alpha in response to low-oxygen stress.

  View details for DOI 10.1128/MCB.23.14.4959-4971.2003

  View details for Web of Science ID 000183958100020

  View details for PubMedID 12832481

• HIF-alpha, a gender independent transcription factor CLINICAL CANCER RESEARCH Le, Q. T., Giaccia, A. J. 2003; 9 (7): 2391-2393

  View details for Web of Science ID 000184108700002

  View details for PubMedID 12855609

• ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation JOURNAL OF BIOLOGICAL CHEMISTRY Hammond, E. M., Dorie, M. J., Giaccia, A. J. 2003; 278 (14): 12207-12213

  Abstract

  The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.

  View details for DOI 10.1074/jbc.M212360200

  View details for Web of Science ID 000182015700064

  View details for PubMedID 12519769

• Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas CLINICAL CANCER RESEARCH Le, Q. T., Sutphin, P. D., Raychaudhuri, S., Yu, S. C., Terris, D. J., Lin, H. S., Lum, B., Pinto, H. A., Koong, A. C., Giaccia, A. J. 2003; 9 (1): 59-67

  Abstract

  Tumor hypoxia modifies treatment efficacy and promotes tumor progression. Here, we investigated the relationship between osteopontin (OPN), tumor pO(2), and prognosis in patients with head and neck squamous cell carcinomas (HNSCC).We performed linear discriminant analysis, a machine learning algorithm, on the NCI-60 cancer cell line microarray expression database to identify a gene profile that best distinguish cell lines with high Von-Hippel Lindau (VHL) gene expression, an important regulator of hypoxia-related genes, from those with low expression. Plasma OPN levels in 15 volunteers, 31 VHL patients, and 54 HNSCC patients were quantitatively measured by ELISA. The relationships between plasma OPN levels, tumor pO(2) as measured by the Eppendorf microelectrode, freedom from relapse (FFR), and survival in HNSCC patients were evaluated.Microarray analysis indicated that OPN gene expression inversely correlated with that of VHL. These findings were confirmed by Northern blot analysis. ELISA studies and Western blot in a HNSCC cell line demonstrated that hypoxia exposure resulted in increased OPN secretion. Patients with VHL syndrome had significantly higher plasma OPN levels than healthy volunteers. Plasma OPN level inversely correlated with tumor pO(2) (P = 0.003, r = -0.42). OPN levels correlated with clinical outcomes. The 1-year FFR and survival rates were 80 and 100%, respectively, for patients with OPN levels 450 ng/ml (P = 0.002 and 0.0005). Multivariate analysis revealed that OPN was an independent predictor for FFR and survival.Plasma OPN levels appeared to correlate with tumor hypoxia in HNSCC patients and may serve as noninvasive tests to identify patients at high risk for tumor recurrence.

  View details for Web of Science ID 000180430600008

  View details for PubMedID 12538452

• Hypoxia and anoxia induce different metabolic and growth factor responses in osteoblasts 10th International Congress of the International-Society-of-Craniofacial-Surgery Salim, A., Nacamuli, R. P., Fong, K. D., Shi, Y. Y., Giaccia, A. J., Longaker, M. T. MEDIMOND PUBLISHING CO. 2003: 477–479

  View details for Web of Science ID 000227470500099

• Tumor deprivation of oxygen and tumor suppressor gene function. Methods in molecular biology (Clifton, N.J.) Yun, Z., Giaccia, A. J. 2003; 223: 485-504

  View details for PubMedID 12777746

• Hypoxia-Mediated Signaling Pathways Handbook of Cell Signaling Koong AC, Giaccia AJ 2003; 3: 277-282
• Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1 alpha JOURNAL OF BIOLOGICAL CHEMISTRY Chan, D. A., Sutphin, P. D., Denko, N. C., Giaccia, A. J. 2002; 277 (42): 40112-40117

  Abstract

  Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.

  View details for DOI 10.1074/jbc.M206922200

  View details for Web of Science ID 000178662500133

  View details for PubMedID 12186875

• Regulation of hypoxia-inducible factor 1 alpha expression and function by the mammalian target of rapamycin MOLECULAR AND CELLULAR BIOLOGY Hudson, C. C., Liu, M., Chiang, G. G., Otterness, D. M., Loomis, D. C., Kaper, F., Giaccia, A. J., Abraham, R. T. 2002; 22 (20): 7004-7014

  Abstract

  Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1alpha subunit and a constititutively expressed HIF-1beta subunit. Under hypoxic conditions, the HIF-1alpha subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1alpha-HIF-1beta heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1alpha and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1alpha stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1alpha fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1alpha stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.

  View details for DOI 10.1128/MCB.22.20.7004-7014.2002

  View details for Web of Science ID 000178266400007

  View details for PubMedID 12242281

• Sex hormones affect adipogenesis. 58th Annual Meeting of the American-Society-for-Reproductive-Medicine Swiersz, L. M., Yun, Z., Giaccia, A. ELSEVIER SCIENCE INC. 2002: S101–S102

  View details for Web of Science ID 000178239400269

• Epigenetic changes in tumor Fas levels determine immune escape and response to therapy CANCER CELL Maecker, H. L., Yun, Z., Maecker, H. T., Giaccia, A. J. 2002; 2 (2): 139-148

  Abstract

  Epigenetic regulation of gene expression significantly influences cell growth and differentiation. Here we show that epigenetic silencing of Fas determines tumor growth in vivo and apoptotic sensitivity in vitro. In established tumors with epigenetically repressed Fas, restoration of Fas activity either by transfection of fas or treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase, suppresses tumor growth and restores chemosensitivity. The TSA-dependent chemosensitivity and tumor growth control require both tumor Fas and the host NK (natural killer) cell functions. This work demonstrates the importance of epigenetic modification of Fas in tumor progression and immune evasion, and emphasizes the essential interplay between Fas and innate immunity in the control of chemoresistant tumors.

  View details for Web of Science ID 000178352600009

  View details for PubMedID 12204534

• Antiangiogenic therapy and p53 SCIENCE Hammond, E. M., Giaccia, A. J. 2002; 297 (5581)

  View details for Web of Science ID 000177054200001

  View details for PubMedID 12142499

• Effects of perillyl alcohol on melanoma in the TPras mouse model CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Lluria-Prevatt, M., Morreale, J., Gregus, J., ALBERTS, D. S., Kaper, F., Giaccia, A., Powell, M. B. 2002; 11 (6): 573-579

  Abstract

  This study evaluates the chemopreventive effects of topically applied perillyl alcohol on the development of melanoma in TPras transgenic mice. Our strategy was to target critical pathways in the development of melanoma, in particular, the ras pathway. Ras has been shown in our experimental mouse model, as well as others, to be important in the development and maintenance of melanomas. Perillyl alcohol (POH), a naturally occurring monoterpene, inhibits the isoprenylation of small G protein, including Ras. POH (10 mM) was applied to the shaved dorsal skin of TPras mice starting 1 week before five treatments of dimethylbenz[a]anthracene (50 microg) and was continued for 38 weeks. We observed a delay in the appearance of tumors and a 25-35% reduction in melanoma incidence. POH treatment of melanoma cells in vitro reduced the levels of detectable Ras protein and inhibits the activation of downstream targets, mitogen-activated protein kinases and Akt. POH only minimally induced apoptosis in this system. Pretreatment but not post-treatment of the melanoma cells with POH, however, markedly reduced levels of UV-induced reactive oxygen species. These studies suggest that POH inhibition of the Ras signaling pathway may be an effective target for chemoprevention of melanoma.

  View details for Web of Science ID 000176142600010

  View details for PubMedID 12050099

• Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: A mechanism for regulation of adipogenesis by hypoxia DEVELOPMENTAL CELL Yun, Z., Maecker, H. L., Johnson, R. S., Giaccia, A. J. 2002; 2 (3): 331-341

  Abstract

  Cellular differentiation involves transcriptional responses to environmental stimuli. Adipocyte differentiation is inhibited under hypoxic conditions, indicating that oxygen (O(2)) is an important physiological regulator of adipogenesis. Hypoxia inhibits PPAR gamma 2 nuclear hormone receptor transcription, and overexpression of PPAR gamma 2 or C/EBP beta stimulates adipogenesis under hypoxia. Mouse embryonic fibroblasts deficient in hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) are refractory to hypoxia-mediated inhibition of adipogenesis. The HIF-1-regulated gene DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split transcription repressor family, represses PPAR gamma 2 promoter activation and functions as an effector of hypoxia-mediated inhibition of adipogenesis. These data indicate that an O(2)-sensitive signaling mechanism regulates adipogenesis. Thus, agents that regulate HIF-1 activity or O(2) sensing may be used to inhibit adipogenesis and control obesity.

  View details for Web of Science ID 000175387900012

  View details for PubMedID 11879638

• Hypoxia links ATR and p53 through replication arrest MOLECULAR AND CELLULAR BIOLOGY Hammond, E. M., Denko, N. C., Dorie, M. J., Abraham, R. T., Giaccia, A. J. 2002; 22 (6): 1834-1843

  Abstract

  Previous studies have demonstrated that phosphorylation of human p53 on serine 15 contributes to protein stabilization after DNA damage and that this is mediated by the ATM family of kinases. However, cellular exposure to hypoxia does not induce any detectable level of DNA lesions compared to ionizing radiation, and the oxygen dependency of p53 protein accumulation differs from that of HIF-1, the hypoxia-inducible transcription factor. Here we show that, under severe hypoxic conditions, p53 protein accumulates only in S phase and this accumulation correlates with replication arrest. Inhibition of ATR kinase activity substantially reduces hypoxia-induced phosphorylation of p53 protein on serine 15 as well as p53 protein accumulation. Thus, hypoxia-induced cell growth arrest is tightly linked to an ATR-signaling pathway that is required for p53 modification and accumulation. These studies indicate that the ATR kinase plays an important role during tumor development in responding to hypoxia-induced replication arrest, and hypoxic conditions could select for the loss of key components of ATR-dependent checkpoint controls.

  View details for DOI 10.1128/MCB.22.6.1834-1843.2002

  View details for Web of Science ID 000174035500020

  View details for PubMedID 11865061

  View details for PubMedCentralID PMC135616

• Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis GENE THERAPY Liu, S. C., Minton, N. P., Giaccia, A. J., Brown, J. M. 2002; 9 (4): 291-296

  Abstract

  A major obstacle in cancer gene therapy is selective tumor delivery. Previous studies have suggested that genetically engineered anaerobes of the genus Clostridium might be gene therapy vectors because of their ability to proliferate selectively in the hypoxic/necrotic regions common to solid tumors. However, the tumor colonization efficiency of the strain previously used was insufficient to produce any antitumor effect. Here we describe for the first time the successful transformation of C. sporogenes, a clostridial strain with the highest reported tumor colonization efficiency, with the E. coli cytosine deaminase (CD) gene and show that systemically injected spores of these bacteria express CD only in the tumor. This enzyme can convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). Furthermore, systemic delivery of 5-FC into mice previously injected with CD-transformed spores of C. sporogenes produced greater antitumor effect than maximally tolerated doses of 5-FU. Since most human solid tumors have hypoxic and necrotic areas this vector system has considerable promise for tumor-selective gene therapy.

  View details for DOI 10.1038/sj/gt/3301659

  View details for Web of Science ID 000174314000007

  View details for PubMedID 11896468

• Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy NEOPLASIA Shibata, T., Giaccia, A. J., Brown, J. M. 2002; 4 (1): 40-48

  Abstract

  Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase (NTR) gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% 02 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

  View details for Web of Science ID 000173412000006

  View details for PubMedID 11922390

• Genetic Basis of Altered Responsiveness of Cancer Cells to Their Microenvironment Oncogene Directed Therapies Giaccia AJ 2002: 113-129
• Hypoxia and Cell Cycle Cell Cycle Checkpoints and Cancer Freiberg RA, Green SL, Giaccia AJ 2002: 143-154
• Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: Suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Popovici, R. M., Lu, M., Bhatia, S., Faessen, G. H., Giaccia, A. J., Giudice, L. C. 2001; 86 (6): 2653-2659

  Abstract

  Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

  View details for Web of Science ID 000169412000049

  View details for PubMedID 11397868

• Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation CANCER RESEARCH CHEN, E. Y., Mazure, N. M., Cooper, J. A., Giaccia, A. J. 2001; 61 (6): 2429-2433

  Abstract

  Hypoxia initiates numerous intracellular signaling pathways important in regulating cell proliferation, differentiation, and death. In this study, we investigated the pathway that hypoxia uses to activate Akt and inactivate glycogen synthase kinase-3 (GSK-3), two proteins the functions of which are important in cell survival and energy metabolism. Severe hypoxia (0.01% oxygen) initiated a signaling cascade by inducing the tyrosine phosphorylation of the platelet-derived growth factor (PDGF) receptor within 1 h of treatment and increasing receptor association with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). Hypoxia-induced signaling also resulted in PI 3-K-dependent phosphorylation of Akt on Ser-473, a modification of Akt that is important for its activation. This activation of Akt by hypoxia was substantially diminished in cells that possessed mutations in their PDGF receptor-PI 3-K interaction domain. In addition, Akt activation by hypoxia was resistant to treatment with the growth factor receptor poison suramin but was sensitive to treatment with the PI 3-K inhibitor wortmannin. Activation of Akt by hypoxia resulted in the phosphorylation of GSK-3alpha and GSK-3beta at Ser-9 and Ser-21, two well-documented Akt phosphorylation sites, respectively, that are inactivating modifications of each GSK-3 isoform. In support of the phosphorylation data, GSK-3 activity was significantly reduced under hypoxia. In conclusion, we propose that hypoxia activates a growth factor receptor/PI 3-K/Akt cascade that leads to GSK-3 inactivation, a pathway that can impact cell survival, proliferation, and metabolism.

  View details for Web of Science ID 000167697500017

  View details for PubMedID 11289110

• Tumor hypoxia, the physiological link between Trousseau's syndrome (carcinoma-induced coagulopathy) and metastasis CANCER RESEARCH Denko, N. C., Giaccia, A. J. 2001; 61 (3): 795-798

  View details for Web of Science ID 000167020100001

  View details for PubMedID 11221857

• Regulation of p53 by hypoxia: Dissociation of transcriptional repression and apoptosis from p53-dependent transactivation MOLECULAR AND CELLULAR BIOLOGY Koumenis, C., Alarcon, R., Hammond, E., Sutphin, P., Hoffman, W., Murphy, M., Derr, J., Taya, Y., Lowe, S. W., Kastan, M., Giaccia, A. 2001; 21 (4): 1297-1310

  Abstract

  Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

  View details for Web of Science ID 000166651800030

  View details for PubMedID 11158315

• p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest MOLECULAR AND CELLULAR BIOLOGY GREEN, S. L., Freiberg, R. A., Giaccia, A. J. 2001; 21 (4): 1196-1206

  Abstract

  We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.

  View details for Web of Science ID 000166651800021

  View details for PubMedID 11158306

• Genetic determinants that influence hypoxia-induced apoptosis. Novartis Foundation symposium Alarcon, R. M., Denko, N. C., Giaccia, A. J. 2001; 240: 115-128

  Abstract

  The p53 tumour suppressor gene is capable of activating both death receptor and mitochondrial-signalled forms of apoptotic cell death in response to diverse stimuli. Studies have suggested that impairment of the mitochondrial-signalled Apaf/caspase 9 pathway and not the death receptor Fas pathway results in almost complete resistance to apoptotic cell death induced by a low oxygen environment. However, it is unclear how p53 signals the activation of this pathway and whether it is through already identified p53 effector genes such as the pro-apoptotic gene bax, or through novel effectors such as BNIP-3/BNIP-3L. Comparison of cell lines genetically matched at the bax, cytochrome c, apaf, caspase 9 and caspase 3 loci indicated that except for bax, all of these genes were essential for hypoxia induced apoptosis both in cell culture and in transplanted tumours. These data imply that cytochrome c plays a pivotal role in signalling cell death by apoptosis under hypoxic conditions, and that the release of cytochrome c is independent of both Bax and p53. In contrast to cytochrome c, p53 modulates the magnitude of apoptosis under hypoxic conditions, but in itself is not required for the activation of the caspase cascade.

  View details for PubMedID 11727925

• Regulation of insulin-like growth factor-binding protein 1 by hypoxia and 3 ',5 '-cyclic adenosine monophosphate is additive in HepG2 cells 80th Annual Meeting of the Endocrine-Society Sugawara, J., Tazuke, S. I., F-Suen, L., Powell, D. R., Kaper, F., Giaccia, A. J., Giudice, L. C. ENDOCRINE SOC. 2000: 3821–27

  Abstract

  Insulin-like growth factor-binding protein 1 (IGFBP-1) is important in regulating minute-to-minute IGF bioavailability in the circulation and is primarily an inhibitor of IGF action systemically and in most cellular systems. Understanding regulation of IGFBP-1 is, thus, important in understanding regulation of IGF actions. The IGFBP-1 promoter contains a cAMP response element, and cAMP stimulates IGFBP-1 gene expression at the transcriptional level. Recently, we have found three consensus sequences for the hypoxia response element in intron 1 of the IGFBP-1 gene. Herein, we have investigated the effects of hypoxia and a cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP), on IGFBP-1 expression in HepG2 cells, a model system for IGFBP-1 gene regulation. HepG2 cells were exposed to normoxia (20% pO2) or hypoxia (2% pO2) for 24 h in the absence or presence of 8-Br-cAMP (0.1, 0.5, and 1 mM). Western ligand blotting revealed IGFBP-1 as the predominant IGFBP in HepG2-conditioned media, which increased in a dose-dependent manner after incubation with 8-Br-cAMP in normoxia and hypoxia (3-fold and 7-fold at 1 mM, respectively). Under hypoxic, compared with normoxic, conditions, IGFBP-1 protein and messenger RNA (mRNA) levels increased approximately 10-fold and 20-fold, respectively. In normoxia, 8-Br-cAMP stimulated IGFBP-1 protein and mRNA levels in a dose-dependent manner (7-fold and 10-fold at 1 mM). Hypoxia and 8-Br-cAMP showed additive stimulatory effects on IGFBP-1 protein and mRNA levels (35-fold and 50-fold at 1 mM) that were time and dose dependent. Primary transcripts of IGFBP-1 mRNA were increased concordantly with IGFBP-1 mRNA. The half-life of the IGFBP-1 mRNA was markedly increased (approximately 6-fold) by hypoxia, and cAMP minimally enhanced this effect. These results demonstrate that hypoxia and compounds that increase intracellular cAMP additively regulate IGFBP-1 gene expression by transcriptional and posttranscriptional mechanisms. Regulation of IGFBP-1 mRNA and protein by cAMP and hypoxia may be important for understanding the physiologic and pathophysiologic roles of IGFBP-1.

  View details for Web of Science ID 000089820800059

  View details for PubMedID 11061545

• p53 promotes selection for Fas-mediated apoptotic resistance CANCER RESEARCH Maecker, H. L., Koumenis, C., Giaccia, A. J. 2000; 60 (16): 4638-4644

  Abstract

  Although p53 inactivation is implicated as a mechanism to explain diminished apoptotic response, it is clear that tumor cells that possess transcriptionally functional p53 can also be resistant to diverse apoptotic stimuli. We hypothesize that oncogenic activation and DNA damage are sufficient stimuli to increase the p53-dependent transcription of Fas and thereby establish a situation in which cell to cell contact could be a selective pressure to either lose p53 function or inactivate components of the Fas death pathway. Examination of genetically matched tumor cell lines that possessed either wild-type or null p53 loci indicated that cells possessing functional p53 increased their surface levels of Fas and Fas ligand (FasL) in response to DNA damage. In contrast, stress induced by changes in the tumor microenvironment such as decreased oxygen did not up-regulate Fas or FasL. Cells with wild-type p53 underwent Fas-mediated killing in the presence of either FasL-expressing killer cells or activating Fas antibodies, whereas cells in which p53 was deleted or inactivated were protected from such killing. Furthermore, Fas and FasL expression and induction became transcriptionally repressed in transformed cells with wild-type p53 with increasing passage, whereas other p53 downstream targets and functions, such as p21 inducibility and cell cycle arrest, remained intact. Repression of the Fas locus could be reverted by treatment with the histone deacetylase inhibitor trichostatin A. These results support a model of tumor progression in which oncogenic transformation drives tumor cells to lose either p53 or their Fas sensitivity as a means of promoting their survival and evade immune surveillance.

  View details for Web of Science ID 000088961100055

  View details for PubMedID 10969818

• Regulation of insulin-like growth factor-binding protein-1 by nitric oxide under hypoxic conditions JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Sugawara, J., Suh, D. S., Faessen, G. H., Suen, L. F., Shibata, T., Kaper, F., Giaccia, A. J., Giudice, L. C. 2000; 85 (8): 2714-2721

  Abstract

  Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.

  View details for Web of Science ID 000165784500013

  View details for PubMedID 10946870

• p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia EXPERIMENTAL CELL RESEARCH Denko, N. C., GREEN, S. L., Edwards, D., Giaccia, A. J. 2000; 258 (1): 82-91

  Abstract

  X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.

  View details for DOI 10.1006/excr.2000.4928

  View details for Web of Science ID 000088156200009

  View details for PubMedID 10912790

• Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor CLINICAL CANCER RESEARCH Laderoute, K. R., Alarcon, R. M., Brody, M. D., Calaoagan, J. M., CHEN, E. Y., KNAPP, A. M., Yun, Z., Denko, N. C., Giaccia, A. J. 2000; 6 (7): 2941-2950

  Abstract

  Tumor angiogenesis, the development of new blood vessels during malignant progression, is a regulated process that has both genetic and physiological controls. Physiologically, angiogenesis is stimulated by decreases in tissue oxygenation (i.e., hypoxia). We investigated the effect of hypoxia on the expression of two angiogenic factors reported to be genetically regulated by the p53 tumor suppressor gene: (a) the angiogenic inhibitor thrombospondin 1 (TSP-1); and (b) the angiogenic inducer vascular endothelial growth factor (VEGF). Analysis of rodent cells that differ in their p53 genotype (p53+/+ or p53-/-) indicated that in vitro exposure to hypoxia simultaneously suppressed TSP-1 and induced VEGF expression, regardless of the p53 genotype. On transformation of these cells with E1A and oncogenic H-ras, the basal level of TSP-1 expression was strongly diminished, whereas that of VEGF could still be induced by hypoxia. Consistent with these in vitro findings, sections of tumors derived from the transformed p53+/+ and p53-/- cells showed that VEGF protein overlapped with regions of hypoxia, whereas TSP-1 protein was below the limits of detection in tumor tissue. Using a panel of normal/immortalized and transformed human cells, it was found that the ability of hypoxia to inhibit TSP-1 expression depends on the cell type and/or the degree of transformation. In contrast, VEGF expression was induced by hypoxia in all of the human cell types examined. Together, these findings suggest that hypoxic and oncogenic signals could interact in the tumor microenvironment to inhibit TSP-1 and induce VEGF expression, promoting the switch to the angiogenic phenotype.

  View details for Web of Science ID 000088147500047

  View details for PubMedID 10914744

• Development of a hypoxia-responsive vector for tumor-specific gene therapy GENE THERAPY Shibata, T., Giaccia, A. J., Brown, J. M. 2000; 7 (6): 493-498

  Abstract

  We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. As vascular endothelial growth factor (VEGF) is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with five copies of hypoxia-responsive elements (HREs) derived from the 5'-untranslated region (UTR) of the human VEGF showed excellent transcriptional activation at low oxygen tension relevant to tumor hypoxia. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. In addition, we also investigated whether the 3' UTR of the VEGF gene would confer increased post-transcriptional mRNA stability under hypoxic conditions. However, despite increases in the hypoxic/aerobic ratio of luciferase activity, gene expression with 3' UTR was lower due to mRNA destabilization by AU-rich elements (AREs). Thus, we found no benefit from the inclusion of the 3' UTR in our vectors. Of all the vectors tested, the combination of 5HRE and a CMV minimal promoter exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter. We propose that this vector would be useful for tumor selective gene therapy.

  View details for Web of Science ID 000085785500006

  View details for PubMedID 10757022

• Caveolin 1-mediated regulation of receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by ceramide MOLECULAR AND CELLULAR BIOLOGY Zundel, W., Swiersz, L. M., Giaccia, A. 2000; 20 (5): 1507-1514

  Abstract

  Previous studies have indicated that proapoptotic stresses downregulate the phosphatidylinositol 3-kinase [PI(3)K]/Akt survival pathway via the activation of acid-sphingomyelinase (A-SMase) and ceramide production. Ceramide induces apoptosis and inhibits PI(3)K activity without altering expression, association, or phosphorylation of receptors, adapter proteins, or PI(3)K subunits. PI(3)K inhibition by ceramide is associated with recruitment of caveolin 1 to PI(3)K-associated receptor complexes within lipid raft microdomains. Overexpression of caveolin 1 alone is sufficient to alter PI(3)K activity and sensitizes fibroblasts to ceramide-induced cell death. Most importantly, antisense expression of caveolin 1 dramatically reduces ceramide-induced PI(3)K deregulation and results in a loss-of-function stress response similar to that in A-SMase-deficient cells. Stress-induced recruitment of caveolin 1 to receptor complexes was found to be dependent on A-SMase since cell lines deficient in A-SMase did not exhibit caveolin 1 association with PI(3)K receptor complexes. Thus, a genetic link between A-SMase activation and caveolin 1-induced inhibition of PI(3)K activity exists. These results led us to propose that stress-induced changes in raft microdomains lead to altered receptor tyrosine kinase signal transduction through the modulation of caveolin 1 by ceramide.

  View details for Web of Science ID 000085342200005

  View details for PubMedID 10669728

• Loss of PTEN facilitates HIF-1-mediated gene expression GENES & DEVELOPMENT Zundel, W., Schindler, C., Haas-Kogan, D., Koong, A., Kaper, F., Chen, E., Gottschalk, A. R., Ryan, H. E., Johnson, R. S., Jefferson, A. B., Stokoe, D., Giaccia, A. J. 2000; 14 (4): 391-396

  Abstract

  In glioblastoma-derived cell lines, PTEN does not significantly alter apoptotic sensitivity or cause complete inhibition of DNA synthesis. However, in these cell lines PTEN regulates hypoxia- and IGF-1-induced angiogenic gene expression by regulating Akt activation of HIF-1 activity. Restoration of wild-type PTEN to glioblastoma cell lines lacking functional PTEN ablates hypoxia and IGF-1 induction of HIF-1-regulated genes. In addition, Akt activation leads to HIF-1alpha stabilization, whereas PTEN attenuates hypoxia-mediated HIF-1alpha stabilization. We propose that loss of PTEN during malignant progression contributes to tumor expansion through the deregulation of Akt activity and HIF-1-regulated gene expression.

  View details for PubMedID 10691731

• Candidate genes for the hypoxic tumor phenotype CANCER RESEARCH Koong, A. C., Denko, N. C., Hudson, K. M., Schindler, C., Swiersz, L., Koch, C., Evans, S., Ibrahim, H., Le, Q. T., Terris, D. J., Giaccia, A. J. 2000; 60 (4): 883-887

  Abstract

  In this study, we have analyzed changes induced by hypoxia at the transcriptional level of genes that could be responsible for a more aggressive phenotype. Using a series of DNA array membranes, we identified a group of hypoxia-induced genes that included plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor-binding protein 3 (IGFBP-3), endothelin-2, low-density lipoprotein receptor-related protein (LRP), BCL2-interacting killer (BIK), migration-inhibitory factor (MIF), matrix metalloproteinase-13 (MMP-13), fibroblast growth factor-3 (FGF-3), GADD45, and vascular endothelial growth factor (VEGF). The induction of each gene was confirmed by Northern blot analysis in two different squamous cell carcinoma-derived cell lines. We also analyzed the kinetics of PAI-1 induction by hypoxia in more detail because it is a secreted protein that may serve as a useful molecular marker of hypoxia. On exposure to hypoxia, there was a gradual increase in PAI-1 mRNA between 2 and 24 h of hypoxia followed by a rapid decay after 2 h of reoxygenation. PAI-1 levels were also measured in the serum of a small group of head and neck cancer patients and were found to correlate with the degree of tumor hypoxia found in these patients.

  View details for Web of Science ID 000085503100022

  View details for PubMedID 10706099

• Epigenetic regulation of gene expression in cervical cancer cells by the tumor microenvironment CLINICAL CANCER RESEARCH Denko, N., Schindler, C., Koong, A., Laderoute, K., Green, C., Giaccia, A. 2000; 6 (2): 480-487

  Abstract

  Evidence is accumulating that the adverse tumor microenvironment both modifies the malignant progression of tumor cells and contributes to chemotherapy and radiation resistance. We hypothesized that some of the effects on malignant progression are mediated through the transcriptional regulation of genes responsive to the stresses of the microenvironment, such as low oxygen or low glucose conditions. To determine epigenetic changes in gene expression that were consistent with that hypothesis, we used an in vitro subtractive hybridization method, representational difference analysis, to identify hypoxia-induced cDNAs from cultured human cervical epithelial cells. We identified 12 induced genes: two novel genes (HIG1 and HIG2), three genes known to be hypoxia-inducible (tissue factor, GAPDH, thioredoxin), and seven genes not previously identified as hypoxia-inducible [HNRNP(a1), ribosomal L7, annexin V, lipocortin 2, Ku(70), PRPP synthase, and acetoacetyl-CoA thiolase]. In cultured cells, HIG1 and HIG2 expression is induced by hypoxia and by glucose deprivation, but their expression is not induced by serum deprivation, UV, or ionizing radiation. The putative HIG1 and HIG2 open reading frames are expressed in cells, as confirmed by epitope tagging. In addition, tumor xenografts derived from human cervical cancer cells display increased expression of HIG1 and HIG2 when they are deprived of oxygen. Taken together, these data suggest a coordinated transcriptional response of eukaryotic cells to microenvironmental stresses found in the solid tumor.

  View details for Web of Science ID 000085502600023

  View details for PubMedID 10690527

• Genetically modified clostridium for gene therapy of tumors. Methods in molecular medicine Fox, M. E., LEMMON, M. J., Giaccia, A. J., Minton, N. P., Brown, J. M. 2000; 35: 413-418

  Abstract

  Many murine and human tumors contain hypoxic or necrotic regions in which the oxygen tension is abnormally low. For example,>50% of primary tumors of the breast, cervix, and head and neck contain areas that are hypoxic. Because hypoxic regions are not present in normal tissue, this provides the potential for selectively targeting gene therapy to tumor cells.

  View details for DOI 10.1385/1-59259-086-1:413

  View details for PubMedID 21390820

• Hypoxia induces p53 accumulation through MDM2 down-regulation and inhibition of E6-mediated degradation CANCER RESEARCH Alarcon, R., Koumenis, C., Geyer, R. K., Maki, C. G., Giaccia, A. J. 1999; 59 (24): 6046-6051

  Abstract

  Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.

  View details for Web of Science ID 000084530400007

  View details for PubMedID 10626788

• A p53 and apoptotic independent role for p21(waf1) in tumour response to radiation therapy ONCOGENE Wouters, B. G., Denko, N. C., Giaccia, A. J., Brown, J. M. 1999; 18 (47): 6540-6545

  Abstract

  Loss of p21 in human cancer cells results in checkpoint failure, induction of polyploidy and subsequent apoptosis following DNA damage. Tumours in immunodeficient mice derived from cells lacking p21 are also more sensitive to ionizing radiation than their wild-type counterparts. Abrogation of p53 in the p21+/+ parental cells results in an in vitro phenotype that is indistinguishable from that of the p21 knockout cells. Thus, the in vitro phenotype resulting from loss of p21 is consistent with its well-established role in the p53/p21 damage response pathway. However, despite the similar in vitro phenotype, p21+/+ cells with abrogated p53 show no evidence of the sensitivity observed in the p21-/- cells when grown as tumours in immunodeficient mice. The increased radio-sensitization stabilization of p21-/- tumours is also unrelated to the increase in apoptosis observed in these tumours following radiation treatment. Apoptosis in the p21-/- tumours was significantly reduced by expression of bcl-2 without any corresponding change in the overall response of the tumour. Similarly, abrogation of p53 in the p21+/+ tumours substantially increased radiation-induced apoptosis within the tumours without increasing their radiation sensitivity. Dissociation of these in vivo and in vitro phenotypes indicates that p21 participates in a novel in vivo specific damage response pathway that is distinct from its role in the p53 pathway, and therefore that it may be an effective therapeutic target for cancer therapy.

  View details for Web of Science ID 000083709200017

  View details for PubMedID 10597257

• Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development TERATOLOGY CHEN, E. Y., Fujinaga, M., Giaccia, A. J. 1999; 60 (4): 215-225

  Abstract

  Recent studies have suggested the importance of hypoxia-inducible transcription factors in development, yet the questions of whether hypoxia actually exists in a developing embryo in vivo and, if so, what role it plays in development remain unanswered. In this study, we directly demonstrate that regions of hypoxia, most prominently the hindbrain, otic vesicle, and first branchial arch, exist in a gestational day (GD) 11 rat embryo grown in utero. We also show that varying the oxygen environment of an embryo affects its morphological development. Rat embryos which were grown at 45% oxygen from GD 9-11 showed gross morphological abnormalities, including defective cranial neural tube closure, incomplete otic vesicle invagination, and abnormal somite formation and embryo turning. These embryos, in addition, exhibited reduced cell death. On the other hand, embryos which were grown at 5% oxygen during the same period were stunted in overall growth, yet morphologically normal, and displayed prominent areas of apoptosis. In this study, we propose that embryonic development, like tumor development, requires two different but interactive sets of signals. One set exists in the genetic program for development; the other set arises from changes in the microenvironment of the embryo. Therefore, it is the interplay between these two sets of cues that drives normal embryonic development. The requirement for hypoxia to activate apoptotic cell death is but one example of such interactions.

  View details for Web of Science ID 000082964400006

  View details for PubMedID 10508975

• Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: Potential relevance to menstruation and endometrial regeneration JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Popovici, R. M., Irwin, J. C., Giaccia, A. J., Giudice, L. C. 1999; 84 (6): 2245-2248

  Abstract

  The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.

  View details for Web of Science ID 000080674000090

  View details for PubMedID 10372740

• Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments - A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity JOURNAL OF BIOLOGICAL CHEMISTRY Laderoute, K. R., Mendonca, H. L., Calaoagan, J. M., KNAPP, A. M., Giaccia, A. J., Stork, P. J. 1999; 274 (18): 12890-12897

  Abstract

  Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.

  View details for Web of Science ID 000080056800098

  View details for PubMedID 10212278

• Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition SCIENCE Soengas, M. S., Alarcon, R. M., Yoshida, H., Giaccia, A. J., HAKEM, R., Mak, T. W., Lowe, S. W. 1999; 284 (5411): 156-159

  Abstract

  The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.

  View details for Web of Science ID 000079509000057

  View details for PubMedID 10102818

• Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1 CANCER RESEARCH MURPHY, B. J., Andrews, G. K., Bittel, D., Discher, D. J., McCue, J., Green, C. J., Yanovsky, M., Giaccia, A., SUTHERLAND, R. M., LADEROUTE, K. R., Webster, K. A. 1999; 59 (6): 1315-1322

  Abstract

  Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.

  View details for Web of Science ID 000079125200025

  View details for PubMedID 10096565

• Multiple roles for myc deregulation in oncogenesis DNA Alternations in Cancer: Genetic and Epigenetic Changes Giaccia AJ, Denko NC 1999: 105-119
• The influence of tumor hypoxia on malignant progression International Symposium on Tumor Oxygenation Giaccia, A. J., Koumenis, C., Denko, N. WISSENSCHAFTLICHE verlagsgesellschaft mbh. 1999: 115–124

  View details for Web of Science ID 000089455600014

• Chemotherapy, tumor microenvironment and apoptosis Apoptosis and Cancer Chemotherapy Koumenis C, Denko N, Giaccia A 1999: 223-240
• The influence of tumor hypoxia on malignant progression Tumor Hypoxia: Pathophysiology, Clinical Significance and Therapeutic Perspectives Giaccia AJ, Koumenis C, Denko N 1999: 115-124
• Highly conserved RNA sequences that are sensors of environmental stress MOLECULAR AND CELLULAR BIOLOGY Spicher, A., Guicherit, O. M., Duret, L., Aslanian, A., Sanjines, E. M., Denko, N. C., Giaccia, A. J., Blau, H. M. 1998; 18 (12): 7371-7382

  Abstract

  The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.

  View details for Web of Science ID 000077099700049

  View details for PubMedID 9819424

• The complexity of p53 modulation: emerging patterns from divergent signals GENES & DEVELOPMENT Giaccia, A. J., Kastan, M. B. 1998; 12 (19): 2973-2983

  View details for Web of Science ID 000076461700001

  View details for PubMedID 9765199

• Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: A possible model for IGFBP-1 expression in fetal hypoxia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tazuke, S. I., Mazure, N. M., Sugawara, J., Carland, G., Faessen, G. H., Suen, L. F., Irwin, J. C., Powell, D. R., Giaccia, A. J., Giudice, L. C. 1998; 95 (17): 10188-10193

  Abstract

  IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.

  View details for Web of Science ID 000075475200085

  View details for PubMedID 9707622

• Heat shock protein 72 modulates pathways of stress-induced apoptosis JOURNAL OF BIOLOGICAL CHEMISTRY Buzzard, K. A., Giaccia, A. J., Killender, M., Anderson, R. L. 1998; 273 (27): 17147-17153

  Abstract

  The resistance to stress-induced apoptosis conferred by the thermotolerant state or by exogenous expression of HSP72 was measured in mouse embryo fibroblasts. The induction of thermotolerance protects cells from heat, tumor necrosis factor alpha (TNFalpha), and ceramide-induced apoptosis but not from ionizing radiation. Because the development of thermotolerance is associated with increased levels of heat shock proteins, we determined whether constitutive expression of one of the major inducible heat shock proteins, HSP72, could also protect cells from stress-induced apoptosis. Cells expressing constitutive HSP72 were shown to have significantly reduced levels of apoptosis after heat, TNFalpha, and ceramide but not after ionizing radiation. Activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was found to be strongly inhibited in thermotolerant cells after heat shock but not after other stresses. Cells that constitutively express HSP72 did not demonstrate decreased SAPK/JNK activation after any of these stresses. Thus, factors other than HSP72 that are induced in the thermotolerant state are able to reduce activation of SAPK/JNK after heat stress. Notably, the level of activation of SAPK/JNK did not correlate with the amount of apoptosis detected after different stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose) polymerase cleavage in cells after heat shock and TNFalpha but not after ceramide or ionizing radiation. The results suggest either that SAPK/JNK activation is not required for apoptosis in mouse embryo fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the data support the concept that caspase activity, which can be down-regulated by HSP72, is a crucial step in stress-induced apoptosis. Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.

  View details for Web of Science ID 000074545200072

  View details for PubMedID 9642282

• Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress GENES & DEVELOPMENT Zundel, W., Giaccia, A. 1998; 12 (13): 1941-1946

  Abstract

  The initiation of apoptosis often transpires in the presence of agents that regulate cell survival. This study evaluated the effects of stress-induced ceramide on the anti-apoptotic activity of the phosphoinositide-3 kinase [PI(3)K] pathway. PI(3)K activity is directly down-regulated by stress-induced ceramide in a dose-dependent manner with rapid kinetics and high specificity. Ceramide inhibition of PI(3)K is dependent on acid-sphingomyelinase. Down-regulation of PI(3)K by ceramide results in inhibition of the kinase Akt and decreased phosphorylation of the death effector Bad. Thus, ceramide levels could act as a general apoptotic rheostat controlling cell survival by regulating PI(3)K anti-apoptotic effector mechanisms.

  View details for Web of Science ID 000074700100002

  View details for PubMedID 9649498

• Tumor hypoxia and the cell cycle: Implications for malignant progression and response to therapy CANCER JOURNAL GREEN, S. L., Giaccia, A. J. 1998; 4 (4): 218-223

  View details for Web of Science ID 000075038300002

  View details for PubMedID 9689978

• The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury BRAIN RESEARCH Lee, J. E., Kim, C. Y., Giaccia, A. J., Giffard, R. G. 1998; 795 (1-2): 10-16

  Abstract

  Many oncogenes are implicated in the regulation of apoptosis as well as in control of the cell cycle, and several have been shown to protect cells from injury. We tested whether the expression of human papillomavirus type 16 genes E6 and E7 could protect primary astrocytes from injury. Retroviral vectors were used to express E6, E7, or E6E7 in primary murine astrocyte cultures. Astrocytes expressing E6E7 suffered less than half the injury seen in controls after exposure to 400 microM H2O2. When we compared astrocytes expressing only E6 or E7, cells expressing E7 alone were protected to a greater extent and from more severe injury than those expressing E6. E6E7, E6 and E7 all provided protection from 30 h glucose deprivation, but again E7 provided the best protection, reducing injury to less than a third of that seen in controls. Expression of E7 alone decreases vulnerability to both hydrogen peroxide and glucose deprivation injury while E6 alone primarily decreases glucose deprivation injury. These results demonstrate that expression of human papillomavirus oncogenes can reduce the vulnerability of astrocytes to oxidative stress and nutrient deprivation.

  View details for PubMedID 9622584

• Hypoxia-induced apoptosis is required for proper embryonic CHEN, E. Y., Fujinaga, M., Giaccia, A. J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 1998: 195–95

  View details for Web of Science ID 000074281900220

• Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy CANCER RESEARCH Rupnow, B. A., Murtha, A. D., Alarcon, R. M., Giaccia, A. J., Knox, S. J. 1998; 58 (9): 1779-1784

  Abstract

  Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.

  View details for Web of Science ID 000073344700003

  View details for PubMedID 9581811

• The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy CANCER RESEARCH Brown, J. M., Giaccia, A. J. 1998; 58 (7): 1408-1416

  Abstract

  The physiology of solid tumors differs from that of normal tissues in a number of important aspects, the majority of which stem from differences between the two vasculatures. Compared with the regular, ordered vasculature of normal tissues, blood vessels in tumors are often highly abnormal, distended capillaries with leaky walls and sluggish flow. Tumor growth also requires continuous new vessel growth, or angiogenesis. These physiological differences can be problems for cancer treatment; for example, hypoxia in solid tumors leads to resistance to radiotherapy and to some anticancer drugs. However, these differences can also be exploited for selective cancer treatment. Here we review four such areas that are under active investigation: (a) hypoxia-selective cytotoxins take advantage of the unique low oxygen tension in the majority of human solid tumors. Tirapazamine, a drug in the final stages of clinical trials, is one of the more promising of these agents; (b) leaky tumor blood vessels can be exploited using liposomes that have been sterically stabilized to have a long intravascular half-life, allowing them to selectively accumulate in solid tumors; (c) the tumor microenvironment is a stimulus to angiogenenesis, and inhibition of angiogenesis can be a powerful anticancer therapy not susceptible to acquired drug resistance; and (d) we discuss attempts to use gene therapy activated either by the low oxygen environment or by necrotic regions of tumors.

  View details for Web of Science ID 000072780200020

  View details for PubMedID 9537241

• Acidosis reduces neuronal apoptosis NEUROREPORT Xu, L. J., Glassford, A. J., Giaccia, A. J., Giffard, R. G. 1998; 9 (5): 875-879

  Abstract

  Acidosis is a well established concomitant of tissue ischemia. Acidosis in the pH range 6.0-7.0 is seen in cerebral ischemia and within solid tumors. Extracellular acidosis of pH 6.0 and 6.4 provided essentially complete protection from 48 h serum deprivation induced apoptotic death of cultured primary murine neurons. We tested the effect of p53 using transformed mouse embryo fibroblasts of either p53+/+ or p53-/- genotype. Both were markedly protected from serum deprivation by acidity. Hypoxia induced fibroblast injury was also reduced at pH 6.8. Lower pH resulted in a shift from apoptotic to necrotic morphology after 42 h hypoxia. Acidosis reduces apoptosis of both normal and transformed cells, irrespective of p53 status.

  View details for Web of Science ID 000072808900021

  View details for PubMedID 9579683

• p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts CELL DEATH AND DIFFERENTIATION Rupnow, B. A., Alarcon, R. M., Giaccia, A. J., Knox, S. J. 1998; 5 (2): 141-147

  Abstract

  Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.

  View details for Web of Science ID 000071647200003

  View details for PubMedID 10200458

• Cancer therapy and tumor physiology SCIENCE Giaccia, A. J., Brown, J. M., Wouters, B., Denko, N., Koumenis, C. 1998; 279 (5347): 12-13

  View details for Web of Science ID 000071323900006

  View details for PubMedID 9441397

• Transformed cells require continuous activity of RNA polymerase II to resist oncogene-induced apoptosis MOLECULAR AND CELLULAR BIOLOGY Koumenis, C., Giaccia, A. 1997; 17 (12): 7306-7316

  Abstract

  Studies have indicated that deregulated oncogene expression can result in either programmed cell death or proliferation, depending on the cellular microenvironment. However, little is known about whether oncogenic signals in themselves are able to activate a cellular apoptotic program. We have tested the hypothesis that oncogenic signals in the absence of gene expression are sufficient to induce cell death, which would indicate that constitutive expression of antiapoptotic genes is necessary for maintenance of the transformed state. Using two highly specific RNA polymerase (RNAP) II inhibitors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin, which inhibit RNAP II function by two distinct mechanisms, we found that inhibition of gene expression substantially increased apoptosis in a time- and dose-dependent manner in p53+/+- and p53(-/-)-transformed mouse embryonic fibroblasts and in HeLa cells, demonstrating that this type of apoptosis does not require wild-type p53. Engineered expression of an alpha-amanitin resistance RNAP II gene rendered cells resistant to induction of apoptosis by alpha-amanitin without affecting their sensitivity to DRB, indicating that alpha-amanitin induces apoptosis solely by inhibiting RNAP II function and not by a nonspecific mechanism. DRB-induced apoptosis was independent of the cell cycle or ongoing DNA replication, since DRB induced similar levels of apoptosis in asynchronous cells and cells synchronized by collection at mitosis. Inhibition of RNAP II in untransformed cells like Rat-1 or human AG1522 fibroblasts resulted not in apoptosis but in growth arrest. In contrast, deregulated expression of c-Myc in Rat-1 cells dramatically increased their sensitivity to DRB, directly demonstrating that apoptosis following inhibition of RNAP II function is greatly enhanced by oncogenic expression. The requirement for RNAP II function to prevent oncogene-induced apoptosis implies the need for the constitutive expression of an antiapoptotic gene(s) to maintain the transformed state. The differential sensitivities of untransformed and transformed cells to induction of apoptosis by transcriptional inhibition, coupled with the finding that this type of apoptosis is independent of p53 status, suggest that inhibition of RNAP II may be exploited therapeutically for the design of successful antitumor agents.

  View details for Web of Science ID A1997YG44100055

  View details for PubMedID 9372962

• Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element BLOOD Mazure, N. M., CHEN, E. Y., LADEROUTE, K. R., Giaccia, A. J. 1997; 90 (9): 3322-3331

  Abstract

  Tumor angiogenesis, the development of new blood vessels, is a highly regulated process that is controlled genetically by alterations in oncogene and tumor suppressor gene expression and physiologically by the tumor microenvironment. Previous studies indicate that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF). In this report, we show Ras-transformed cells do not use the downstream effectors c-Raf-1 or mitogen activated protein kinases (MAPK) in signaling VEGF induction by hypoxia as overexpression of kinase-defective alleles of these genes does not inhibit VEGF induction under low oxygen conditions. In contrast to the c-Raf-1/MAP kinase pathway, hypoxia increases phosphatidylinositol 3-kinase (PI 3-kinase) activity in a Ras-dependent manner, and inhibition of PI 3-kinase activity genetically and pharmacologically results in inhibition of VEGF induction. We propose that hypoxia modulates VEGF induction in Ras-transformed cells through the activation of a stress inducible PI 3-kinase/Akt pathway and the hypoxia inducible factor-1 (HIF-1) transcriptional response element.

  View details for Web of Science ID A1997YD10800006

  View details for PubMedID 9345014

• Loss of p21(Waf1/Cip1) sensitizes tumors to radiation by an apoptosis-independent mechanism CANCER RESEARCH Wouters, B. G., Giaccia, A. J., Denko, N. C., Brown, J. M. 1997; 57 (21): 4703-4706

  Abstract

  Cellular checkpoints are important mediators of the response of normal cells following genotoxic damage, and interruption of these checkpoints is a common feature of many solid tumors. Although the effects of loss in checkpoint function in tumor cells are well understood in terms of cell cycle control, there is little information on their role in determining treatment efficacy in vivo. We have examined both the in vitro and in vivo responses of isogenic lines differing only in the p53-transactivated checkpoint gene, p21Waf1/Cip1. When assayed in vitro, loss of p21 in human colon tumor cells results in a selective induction of apoptosis [Waldman, T., et al., Nature (Lond.), 381: 713-716, 1996.] but no difference in the clonogenic survival. However, when grown as xenografts and irradiated in situ, p21-deficient tumors were significantly more sensitive to radiation as assessed both by clonogenic survival and by regrowth of the tumors following treatment. These data indicate that loss of p21 results in increased sensitivity to killing by ionizing radiation that is independent of the induction of apoptosis and cell cycle arrest but that is specific to cells when they are grown as a solid tumor. These results have important implications for assessing both the genetic determinants of sensitivity to anticancer agents and efficacy of anticancer agents.

  View details for Web of Science ID A1997YD35800009

  View details for PubMedID 9354425

• Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions CANCER RESEARCH Kim, C. Y., Tsai, M. H., OSMANIAN, C., Graeber, T. G., Lee, J. E., Giffard, R. G., DiPaolo, J. A., Peehl, D. M., Giaccia, A. J. 1997; 57 (19): 4200-4204

  Abstract

  Since human papillomavirus (HPV) infection is strongly associated with cervical neoplasia and tumor hypoxia has prognostic significance in human cervical carcinomas, we examined the relationship between hypoxia and apoptosis in human cervical epithelial cells expressing high-risk HPV type 16 oncoproteins. In vitro, hypoxia stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes but not in cervical fibroblasts infected with E6 and E7. Furthermore, cell lines derived from HPV-associated human cervical squamous cell carcinomas were substantially less sensitive to apoptosis induced by hypoxia, indicating that these cell lines have acquired additional genetic alterations that reduced their apoptotic sensitivity. Although the process of long-term cell culturing resulted in selection for subpopulations of HPV oncoprotein-expressing cervical epithelial cells with diminished apoptotic potential, the exposure of cells to hypoxia greatly accelerated the selection process. These results provide evidence for the role of hypoxia-mediated selection of cells with diminished apoptotic potential in the progression of human tumors and can in part explain why cervical tumors that possess low pO2 values are more aggressive.

  View details for PubMedID 9331075

• Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment GENE THERAPY LEMMON, M. J., VANZIJL, P., Fox, M. E., MAUCHLINE, M. L., Giaccia, A. J., Minton, N. P., Brown, J. M. 1997; 4 (8): 791-796

  Abstract

  A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

  View details for Web of Science ID A1997XP16200007

  View details for PubMedID 9338007

• Protease inhibitor TPCK represses Ha-ras (Val12) transformation and nuclear factor-kappa B activation INTERNATIONAL JOURNAL OF ONCOLOGY Denko, N. C., CHEN, E. Y., LADEROUTE, K. R., Stambrook, P., Giaccia, A. J. 1997; 10 (5): 895-900

  Abstract

  Certain chymotrypsin-like protease inhibitors such as TPCK exhibit a well described anti-tumorigenic activity by an as yet undescribed mechanism. One potential cellular target for TPCK in transformed cells is the ms-inducible NF-kappa B family of transcription factors. We therefore used TPCK to examine the physiologic role of NF-kappa B during Ha-ras induced transformation, independent of another major downstream effector of Ha-ras, AP-1. Using a conditionally transformed NIH3T3 cell line, we found that TPCK (but not the control inhibitor TLME) inhibited the anchorage-independent growth of Ha-ras transformed cells, but not their anchorage-dependent growth on plastic tissue culture dishes. Likewise, TPCK reduced the ability of Ha-ras to stimulate DNA synthesis in growth factor depleted cells, but not the ability of serum to stimulate DNA synthesis in the same growth factor depleted cells. Gel shift analysis and reporter gene expression indicated that TPCK blocked Ha-ras-induced NF-kappa B activity, while only having minimal effects on Ha-ras-induced AP-1 activity. TPCK is therefore able to Inhibit the Ha-ras transformed phenotype of cells by inhibiting the transcriptional activity of NF-kappa B, while having little effect upon transcriptional activity of AP-1.

  View details for Web of Science ID A1997WW11900003

  View details for PubMedID 21533459

• Protease Inhibitor TPCK represses Ha-ras (Val 12) transformation and nuclear factor-&#954; B activation Int J Oncology Denko N, Chen E, Koong A, Stambrook PN, Giaccia AJ 1997; 10: 895-900
• The role of hypoxia, p53, and apoptosis in human cervical carcinoma pathogenesis Kim, C. Y., Tsai, M. H., OSMANIAN, C., CALKINS, D. P., Graeber, T. G., Greenspan, D. L., Kennedy, A. S., Rinker, L. H., Varia, M. A., DiPaolo, J. A., Peehl, D. M., Raleigh, J. A., Giaccia, A. J. ELSEVIER SCIENCE INC. 1997: 206–

  View details for Web of Science ID A1997XW28000224

• The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 induction PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fried, L. M., Koumenis, C., Peterson, S. R., GREEN, S. L., VANZIJL, P., ALLALUNISTURNER, J., Chen, D. J., Fishel, R., Giaccia, A. J., Brown, J. M., Kirchgessner, C. U. 1996; 93 (24): 13825-13830

  Abstract

  Severe combined immunodeficient (SCID) mice display an increased sensitivity to ionizing radiation compared with the parental, C.B-17, strain due to a deficiency in DNA double-strand break repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) has previously been identified as a strong candidate for the SCID gene. DNA-PK phosphorylates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells of DNA damage. To determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (human DNA-PKcs-deficient glioblastoma) cells with their respective wild-type parents following ionizing radiation. Our findings indicate that (i) p53 levels are increased in SCID cells following ionizing radiation, and (ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. The hyperphosphorylation of RPA p34 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA in crude extracts derived from both C.B-17 and SCID cells. These results suggest that DNA-PK is not the only kinase capable of phosphorylating RPA. We conclude that the DNA damage response involving p53 and RPA is not associated with the defect in DNA repair in SCID cells and that the physiological substrate(s) for DNA-PK essential for DNA repair has not yet been identified.

  View details for Web of Science ID A1996VV46700068

  View details for PubMedID 8943020

• Modulation of c-myc activity and apoptosis in vivo CANCER RESEARCH Alarcon, R. M., Rupnow, B. A., Graeber, T. G., Knox, S. J., Giaccia, A. J. 1996; 56 (19): 4315-4319

  Abstract

  We have developed an animal tumor model system to study the effects of c-Myc activation on apoptosis induction in vivo. Tumors were generated in SCID mice from Rat-1 fibroblasts that constitutively express an inactive c-Myc-estrogen receptor fusion protein (T.D. Littlewood et al, Nucleic Acids Res., 23: 1686 -1690, 1995), which is activated in vivo by the administration of 4-hydroxytamoxifen in time release pellets. We demonstrate that activation of c-Myc results in a substantial increase in the number of apoptotic tumor cells and that this apoptosis is predominant in regions of tumor hypoxia. c-Myc-induced apoptosis of hypoxic cells is inhibited in tumors that overexpress the human Bcl-2 protein. Bcl-2, however, does not prevent p53 protein accumulation or the down-regulation of the cyclin-cdk inhibitor p27 protein following c-Myc activation by 4-hydroxytamoxifen. This result suggests that Bcl-2 does not affect c-Myc function directly but acts downstream of c-Myc to inhibit apoptosis. We propose that the ability of activated c-Myc to enhance cellular proliferation might contribute to the genesis of early neoplasms that are held in check by the alternate ability of c-Myc to induce apoptosis of cells that have outgrown their supply of oxygen or other factors associated with hypoxic regions of solid tumors. Secondary genetic lesions downstream of c-Myc that suppress the apoptotic potential of tumor cells, such as Bcl-2 overexpression, might play an important role in the malignant progression of these tumors because they would disrupt the balance between apoptosis and proliferation initiated by c-Myc deregulation.

  View details for Web of Science ID A1996VK30700006

  View details for PubMedID 8813114

• Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression CANCER RESEARCH Mazure, N. M., CHEN, E. Y., Yeh, P., LADEROUTE, K. R., Giaccia, A. J. 1996; 56 (15): 3436-3440

  Abstract

  Hypoxia can select for cells that have lost their apoptotic potential, thereby making them resistant to adverse conditions. However, long-term survival of transformed cells which have diminished apoptotic sensitivity when exposed to low oxygen conditions would require the activation of their angiogenic program to compensate for an insufficient oxygen supply. In this report, we show that the activity (of oncogenic Ha-ras, either constitutively or transiently, enhances the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF), by hypoxia. Analysis of the 5' flanking region of the VEGF promoter indicates that a HIF-1-like sequence is to promote a 15-fold increase in reporter gene activity in Ha-ras-transformed cells when exposed to hypoxia, whereas mutations in the same site totally inhibited VEGF induction. Under low oxygen conditions, VEGF induction is inhibited in cells expressing a mutant inhibitory allele of Ha-ras (RasN17), indicating a direct role for Ras in modulating VEGF activity. We propose that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through VEGF induction.

  View details for Web of Science ID A1996UZ28800011

  View details for PubMedID 8758908

• Early responses of SiHa human squamous carcinoma cells to hypoxic signals: Evidence of parallel activation of NF-kappa B and AP-1 transcriptional complexes INTERNATIONAL JOURNAL OF ONCOLOGY LADEROUTE, K. R., Calaoagan, J. M., Mendonca, H. L., AUSSERER, W. A., CHEN, E. Y., Giaccia, A. J., SUTHERLAND, R. M. 1996; 8 (5): 875-882

  Abstract

  We compared the responses of c-Jun/AP-1 transcriptional complexes with those of NF-kappa B, an established hypoxia-inducible transcriptional complex, in hypoxic SiHa human squamous carcinoma cells. We observed that NF-kappa B was activated rapidly, while AP-1 activation was detectable only after prolonged hypoxia. However, in parallel with NF-kappa B activation, hypoxia induced a protein kinase activity that could phosphorylate the transactivation domain of the ATF-2 transcription factor in vitro. Taken together, these experiments indicate that NF-kappa B can rapidly transduce hypoxic signals through increased DNA-binding and transactivation activities, whereas specific AP-1 (ATF-2/c-Jun) complexes may be activated under the same hypoxic conditions by a stress-reponsive MAPK pathway.

  View details for Web of Science ID A1996UF76100006

  View details for PubMedID 21544440

• Mechanism of heat shock protein 72 induction in primary cultured astrocytes after oxygen-glucose deprivation NEUROLOGICAL RESEARCH Bergeron, M., Mivechi, N. F., Giaccia, A. J., Giffard, R. G. 1996; 18 (1): 64-72

  Abstract

  Induction of stress proteins is thought to be important in the protection of cells from a variety of environmental insults including heat, hypoxia and ischemia. The aim of this study was to compare the mechanism of induction of heat shock protein 72 (HSP72) in primary cultures of murine cortical astrocytes by heat and combined oxygen-glucose deprivation (OGD), a model of in vitro ischemia. 35S-methionine labeling and immunoblotting showed increased HSP72 synthesis and accumulation lasting for up to 24 h following heat or OGD. Heat induced a markedly greater amount of HSP72 mRNA and protein than did OGD. We then sought evidence of heat shock transcription factor-1 (HSF-1) activation. An increase in apparent molecular weight of nuclear HSF-1 after heat or OGD was observed, consistent with increased phosphorylation. To seek an explanation of the difference between heat and OGD as inducers of HSP72 we examined the binding activity of HSP72 + 73 to other proteins. More cellular protein was found to co-immunoprecipitate with HSP72 + 73, and more HSP72 + 73 was found in the pellet fraction after heat shock compared to OGD. These results suggest that HSP72 induction is regulated in astrocytes at least in part at the level of HSF activation, by both heat and OGD. Reduced availability of free HSP72 + 73 in heated cells could be responsible for the greater magnitude of HSP72 induction after heat compared to OGD.

  View details for Web of Science ID A1996TW49400013

  View details for PubMedID 8714540

• Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia GENE THERAPY Fox, M. E., LEMMON, M. J., MAUCHLINE, M. L., Davis, T. O., Giaccia, A. J., Minton, N. P., Brown, J. M. 1996; 3 (2): 173-178

  Abstract

  Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

  View details for Web of Science ID A1996TZ78700010

  View details for PubMedID 8867865

• Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours NATURE Graeber, T. G., OSMANIAN, C., Jacks, T., Housman, D. E., Koch, C. J., Lowe, S. W., Giaccia, A. J. 1996; 379 (6560): 88-91

  Abstract

  Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.

  View details for Web of Science ID A1996TN21600062

  View details for PubMedID 8538748

• Hypoxic Stress Proteins: Survival of the Fittest. Seminars in radiation oncology Giaccia, A. J. 1996; 6 (1): 46-58

  Abstract

  The importance of tumor microenvironmental in malignant progression has been largely ignored. Tumor cells protect themselves from changes in the microenvironment due to a decrease in nutrients and oxygen by reducing macromolecular synthesis and inducing genes that will promote angiogenesis and tissue remodeling. This ability of transformed cells to survive fluctuations in oxygen tensions is clinically important, as tumors with high hypoxic fractions respond poorly to many forms of cancer therapy. While it is commonly accepted that the decrease in molecular oxygen in hypoxic cells makes them more refractory to killing by agents such as ionizing radiation which use oxygen radical formation, the cessation of division and loss of apoptotic (cell suicide) potential in hypoxic cells are also important in their resistance to killing by radiotherapy and chemotherapy. In this chapter, we will discuss hypoxia-induced stress proteins in regards to three clinically relevant end points: inhibition of cell proliferation, induced of apoptosis, and regulation of genes modulating angiogenesis. Although these three end points may seem unrelated, in fact, they are intimately linked with each other in the cellular response to hypoxia and malignant progression. One of the goals of this review is to inform both clinician and scientist of these interrelationships and discuss how hypoxia selects for tumors that are clonal expansions of cells that have lost their apoptotic ability and have switched to a proangiogenic phenotype.

  View details for DOI 10.1053/SRAO0060046

  View details for PubMedID 10717161

• Early response of SiHa human squamous carcinoma cells to hypoxic signals: Evidence of parallel activation of NF-kB and AP-1 transcriptional complexes Int J Oncol Laderoute KR, Calaoagan JM, Ausserer WA, Chen EY, Giaccia AJ, Sutherland RM 1996; 8: 875-882
• New directions for radiation biology research in cancer of the uterine cervix. Journal of the National Cancer Institute. Monographs Kapp, D. S., Giaccia, A. J. 1996: 131-139

  Abstract

  A simplified model for tumorigenesis, locoregional growth, and metastases is proposed for carcinoma of the cervix. With the use of this model, four potential areas for future directions for radiobiologic-clinical research are identified. The first area concerns the influence of human papillomavirus infection and p53 mutations on tumor biology, with particular reference to radiosensitivity and metastatic potential. Research in this area should be most fruitful. The second area focuses on the influence of hypoxia on clinical outcome in carcinoma of the cervix. The use of selective hypoxic cell toxins (e.g., tirapazamine) for phase II testing in hypoxic tumors is recommended. The third area concerns the development and clinical confirmation of assays for the prediction of intrinsic tumor radiosensitivity (e.g., surviving fraction after 2 Gy) and normal tissue radiosensitivity. The need exists for more rapid assays so that their results can be available prior to institution of therapy. The influence of the intrinsic radiosensitivity of normal tissues (especially in patients who are heterozygotes for ataxia-telangiectasia and patients with autoimmune disease) may permit identification of those at increased risk for complications so that alternative, less toxic treatment can be allocated. The fourth area for additional study concerns the influence of both intrinsic (c-myc amplification, matrix metalloproteinase levels) and extrinsic factors (fever, immunosuppression) on the development of distant metastases. Such investigations will permit identification of patients at high risk of developing distant metastases so that adjuvant treatments (e.g., chemotherapy or metalloproteinase inhibitors) can be explored. It is believed that future clarification of our proposed model will lead to other worthwhile areas for therapeutic intervention.

  View details for PubMedID 9023843

• MITOGEN-ACTIVATED PROTEIN-KINASE ACTS AS A NEGATIVE REGULATOR OF THE HEAT-SHOCK RESPONSE IN NIH3T3 CELLS CANCER RESEARCH Mivechi, N. F., Giaccia, A. J. 1995; 55 (23): 5512-5519

  Abstract

  Exposure of NIH3T3 cells to elevated temperatures induces the phosphorylation and activation of mitogen-activated protein (MAP) kinases [or extracellular signal-regulated kinases (ERKs)]. To investigate the significance of MAP kinase activation by heat shock, we examined the effect of inhibiting the activity of MAP kinase on heat shock protein 70 (hsp 70) expression. Overexpression of a dominant inhibitory mutant of ERK1, but not ERK2, in heat-shocked cells increased hsp70 reporter gene activity, suggesting that ERK1 acts as a repressor of hsp70 gene expression. Increases in ERK1 activity through treatment of cells with sodium vanadate (SV), an inhibitor of the dual-specificity MAP kinase phosphatase 1 (PAC1), resulted in increased phosphorylation of the heat shock transcription factor-1 (HSF-1) in unheated cells, delayed the activation of HSF-1 by heat shock, and inhibited the induction of hsp 70 by heat shock. Furthermore, the induction of thermotolerance was reduced significantly in cells that increased ERK1 activity by SV pretreatment. Immune complex kinase assays of heat shocked or SV-pretreated cells indicated that HSF-1 is a potential in vivo substrate for ERK1 phosphorylation. Taken together, these results suggest that agents that modulate MAP kinase act as negative regulators of the heat shock response in mammalian cells by modulating HSF-1 activity and hsp 70 expression.

  View details for Web of Science ID A1995TG21100007

  View details for PubMedID 7585624

• CHEMOTHERAPEUTIC TUMOR TARGETING USING CLOSTRIDIAL SPORES Meeting on Solventogenic Clostridia III Minton, N. P., MAUCHLINE, M. L., LEMMON, M. J., Brehm, J. K., Fox, M., Michael, N. P., Giaccia, A., Brown, J. M. ELSEVIER SCIENCE BV. 1995: 357–64

  Abstract

  The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.

  View details for Web of Science ID A1995TA64700015

  View details for PubMedID 7576773

• ATTENUATED RESPONSE OF P53 AND P21 IN PRIMARY CULTURES OF HUMAN PROSTATIC EPITHELIAL-CELLS EXPOSED TO DNA-DAMAGING AGENTS CANCER RESEARCH Girinsky, T., Koumenis, C., Graeber, T. G., Peehl, D. M., Giaccia, A. J. 1995; 55 (17): 3726-3731

  Abstract

  The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.

  View details for Web of Science ID A1995RR73900010

  View details for PubMedID 7543816

• Reduced p53 response to DNA damage in human prostate epithelial cells 10th International Congress of Radiation Research (10th ICRR) - Radiation Research 1895-1995 Girinsky, T., Koumenis, C., Graeber, T. G., Peehl, D. M., Giaccia, A. J. 10TH ICRR SOCIETY (INT CONGRESS RADIATION RES). 1995: 552–555

  View details for Web of Science ID A1995BH47W00115

• Genetic complementation of the radiosensitive: SCID phenotype: A role for DNA-dependent protein kinase in DNA double-strand break repair 10th International Congress of Radiation Research (10th ICRR) - Radiation Research 1895-1995 Kirchgessner, C. U., Fried, L. M., Koumenis, C., Patil, C. K., Cuomo, C. A., Evans, J. W., VANZIJL, P., Oettinger, M. A., Giaccia, A. J., Brown, J. M. 10TH ICRR SOCIETY (INT CONGRESS RADIATION RES). 1995: 408–411

  View details for Web of Science ID A1995BH47W00082

• HYPOXIC ACTIVATION OF NUCLEAR FACTOR-KAPPA-B IS MEDIATED BY A RAS AND RAF SIGNALING PATHWAY AND DOES NOT INVOLVE MAP KINASE (ERK1 OR ERK2) CANCER RESEARCH Koong, A. C., CHEN, E. Y., Mivechi, N. F., Denko, N. C., STANNBROOK, P., Giaccia, A. J. 1994; 54 (20): 5273-5279

  Abstract

  We have previously shown that hypoxia causes the activation of nuclear factor-kappa B (NF-kappa B), and the phosphorylation of its inhibitory subunit, I kappa B alpha, on tyrosine residues. With the use of dominant negative mutants of Ha-Ras and Raf-1, we investigated some of the early signaling events leading to the activation of NF-kappa B by hypoxia. Both dominant negative alleles of Ha-Ras and Raf-1 inhibited NF-kappa B induction by hypoxia, suggesting that the hypoxia-induced pathway of NF-kappa B induction is dependent on Ras and Raf-1 kinase activity. Furthermore, although conditions of low oxygen can also activate mitogen-activated protein kinases (ERK1 and ERK2), these kinases do not appear to be involved in regulating NF-kappa B by low oxygen conditions, as dominant negative mutants of mitogen-activated protein kinase do not inhibit NF-kappa B activation by hypoxia. Since Ras and Raf-1 have been previously shown to work downstream from membrane-associated tyrosine kinases such as Src, we determined if the Src membrane-associated kinase was also activated by low oxygen conditions. We detected an increase in Src proto-oncogene activity within 15-30 min of cellular exposure to hypoxia. We postulate that Src activation by hypoxia may be one of the earliest events that precedes Ras activation in the signaling cascade which ultimately leads to the phosphorylation and dissociation of the inhibitory subunit of NF-kappa B, I kappa B alpha.

  View details for Web of Science ID A1994PL55800007

  View details for PubMedID 7923153

• MODULATION OF POTASSIUM CHANNELS BY PROTEIN-TYROSINE KINASE INHIBITORS JOURNAL OF CELLULAR PHYSIOLOGY Saad, A. H., Kuo, S. S., Koong, A. C., Hahn, G. M., Giaccia, A. J. 1994; 161 (1): 142-148

  Abstract

  Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.

  View details for Web of Science ID A1994PK53000016

  View details for PubMedID 7929599

• HYPOXIA INDUCES ACCUMULATION OF P53 PROTEIN, BUT ACTIVATION OF A G(1)-PHASE CHECKPOINT BY LOW-OXYGEN CONDITIONS IS INDEPENDENT OF P53 STATUS MOLECULAR AND CELLULAR BIOLOGY Graeber, T. G., Peterson, J. F., Tsai, M., MONICA, K., Fornace, A. J., Giaccia, A. J. 1994; 14 (9): 6264-6277

  Abstract

  It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.

  View details for Web of Science ID A1994PC75000068

  View details for PubMedID 8065358

• THE HUMAN HA-RAS ONCOGENE INDUCES GENOMIC INSTABILITY IN MURINE FIBROBLASTS WITHIN ONE-CELL CYCLE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Denko, N. C., Giaccia, A. J., Stringer, J. R., Stambrook, P. J. 1994; 91 (11): 5124-5128

  Abstract

  Many human tumors contain an activating mutation in one of the ras protooncogenes. Additionally, these tumor cells are often heteroploid and characterized by chromosome breaks and rearrangements that are consequences of the genomic instability that is thought to contribute to tumor progression. The concurrence of ras mutations and genomic instability in tumors prompted us to ask whether selective induction of an activated Ha-ras gene could render a genome unstable. The NIH 3T3 cells used in this study contained mutant p53 genes and carried a selectively inducible activated (EJ) Ha-ras transgene under the control of bacterial lactose regulatory elements. When stably transfected cells were induced to express activated Ha-ras by isopropyl beta-D-thiogalactoside administration, there was a marked increase in the number of gross chromosomal aberrations including acentric fragments, multicentric chromosomes, and double minutes, which occurred within the time frame of a single cell cycle from the time of induction. To confirm that these aberrations occurred within the first cell cycle after mutant Ha-ras induction, the cells were arrested in G1 phase by serum depletion and, subsequently, released by administration of isopropyl beta-D-thiogalactoside or serum. The mitoses from cells released with isopropyl beta-D-thiogalactoside contained a 3-fold elevation in the fraction of chromosomes containing aberrations compared to mitoses from parallel cell cultures that were released with serum. Thus, the induction of activated Ha-ras gene expression in these cells results in genomic instability that can be detected as aberrant chromosomes at the next mitosis.

  View details for Web of Science ID A1994NN21400109

  View details for PubMedID 8197195

• ACTIVATORS OF PROTEIN-KINASE-C SELECTIVELY MEDIATE CELLULAR CYTOTOXICITY TO HYPOXIC CELLS AND NOT AEROBIC CELLS 8th International Conference on Chemical Modifiers of Cancer Treatment Koong, A. C., CHEN, E. Y., Kim, C. Y., Giaccia, A. J. PERGAMON-ELSEVIER SCIENCE LTD. 1994: 259–65

  Abstract

  By understanding the signal transduction pathways through which a cell responds to changes in environmental oxygen levels, we may be able to therapeutically exploit this response by manipulating these pathways.The human adenocarcinoma cell line A549 was exposed to varying durations of hypoxia alone and then plated for survival, or treated with PKC activating agents for 1 h before plating for survival. Western blots were used to determine the kinetics of PKC epsilon and phospholipase C induction.The level of hypoxic killing was directly related to the time of exposure and inversely related to the level of oxygen in the environment. Exposure of the cells to protein kinase C (PKC) activators for 1 h after chronic hypoxic exposure increased cell killing by at least an additional three logs beyond that found for hypoxia alone. Treatment of cells with an inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (PDA) resulted in no increase in hypoxic cell killing, even at the highest concentrations of PDA which produced no detectable toxicity under normal aerobic conditions. Using inhibitors of phospholipases A2 and C, we were able to completely inhibit the additional hypoxic cell killing induced by TPA, but not the uninduced hypoxic cell killing.These studies suggest that accumulation of phospholipid breakdown products may be responsible for TPA induced cell killing, and that hypoxic cells differ from aerobic cells in their ability to tolerate these products.

  View details for Web of Science ID A1994NN51200007

  View details for PubMedID 8195017

• ANALYSIS OF HSF-1 PHOSPHORYLATION IN A549 CELLS TREATED WITH A VARIETY OF STRESSES INTERNATIONAL JOURNAL OF HYPERTHERMIA Mivechi, N. F., Koong, A. C., Giaccia, A. J., Hahn, G. M. 1994; 10 (3): 371-379

  Abstract

  In the absence of stress, heat shock transcription factor-1 (HSF-1) exists as a monomer. After the treatment of cells with variety of stresses, HSF-1 forms a trimer and binds to the heat shock element (HSE), a motif consisting of three consecutive NGAAN sequences located in an inverted orientation upstream of the heat shock genes. HSF-1 is then phosphorylated causing transactivation of heat shock mRNAs. Treatment of cells with some of the stresses has been shown to increase HSF binding to HSE without detectably increasing the synthesis of heat shock mRNAs. Here we used antibody specific to HSF-1 to detect its phosphorylation status following exposure of A549, a human lung carcinoma cell line to a variety of stresses in order to correlate HSF-1 phosphorylation with its transactivation ability. Our studies show that HSF-1 is phosphorylated following heat shock (43 degrees C for 1 h), hypoxia (5 h exposure to 0.02% oxygen), 8% ethanol (1 h exposure at 37 degrees C), or 200 microM sodium arsenite (1 h exposure at 37 degrees C). All such stresses have previously been shown to increase the synthesis of heat shock proteins (hsps). However, there are no detectable increases in HSF-1 phosphorylation after the treatment of cells with X-irradiation (2-8 Gy) or 100 microM canavanine, an amino acid analogue (1 h exposure at 37 degrees C). Treatment of cells with X-irradiation increases HSF binding to HSE without increasing the synthesis of hsps, while treatment of cells with canavanine has been shown to increase the synthesis of hsps.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1994NP47600009

  View details for PubMedID 7930803

• THE REGULATION OF GRP78 AND MESSENGER-RNA LEVELS BY HYPOXIA IS MODULATED BY PROTEIN-KINASE-C ACTIVATORS AND INHIBITORS Workshop on Molecular, Cellular, and Genetic Basis of Radiosensitivity at Low Doses: A Case of Induced Repair Koong, A. C., AUGER, E. A., CHEN, E. Y., Giaccia, A. J. RADIATION RESEARCH SOC. 1994: S60–S63

  Abstract

  In this study, we have shown that steady-state levels of glucose-regulated 78 kDa (GRP78) protein and messenger RNA increase during a 5-h exposure to 0.02% oxygen. This increase in GRP78 protein and mRNA induced by hypoxia can be abolished by a 1-h pretreatment of cells before hypoxia with the protein kinase C (PKC) inhibitors staurosporine and H7 at concentrations at which the drugs themselves do not cause cytotoxicity. Although all studies using protein kinase inhibitors must be interpreted with caution, staurosporine and H7 have been shown to be potent inhibitors of PKC activity, suggesting a role for PKC in mediating the transcriptional regulation of GRP78 by hypoxia. Further support for PKC in regulating GRP78 gene expression by hypoxia stems from gel-mobility shift studies in mixtures of nuclear extracts from aerobic or hypoxic cells with a 36 bp region of the GRP78 promoter (-170 to -135). Binding of this factor could be inhibited by pretreating cells with the PKC inhibitor staurosporine before hypoxia or activated by treating cells with the PKC-activating phorbol ester TPA. These data suggest that activation of this hypoxia-responsive factor is sensitive to oxygen levels and seems to be mediated through a PKC signal transduction pathway.

  View details for Web of Science ID A1994NF37900016

  View details for PubMedID 8146329

• HYPOXIA CAUSES THE ACTIVATION OF NUCLEAR FACTOR KAPPA-B THROUGH THE PHOSPHORYLATION OF I-KAPPA-B-ALPHA ON TYROSINE RESIDUES CANCER RESEARCH Koong, A. C., CHEN, E. Y., Giaccia, A. J. 1994; 54 (6): 1425-1430

  Abstract

  The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B.

  View details for Web of Science ID A1994NA88700009

  View details for PubMedID 8137243

• INCREASED CYTOTOXICITY OF CHRONIC HYPOXIC CELLS BY MOLECULAR INHIBITION OF GRP78 INDUCTION INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Koong, A. C., CHEN, E. Y., Lee, A. S., Brown, J. M., Giaccia, A. J. 1994; 28 (3): 661-666

  Abstract

  To develop a molecular strategy of increasing cytotoxicity of chronically hypoxic cells by inhibiting Glucose Regulated Protein 78 kDal (GRP78) induction.A mutant nonGRP78 inducing cell line (78WO) was developed from its parent (DG44) by overexpressing antisense GRP78 mRNA. Following exposure to varying durations of hypoxia, Northern and Western blot analysis were used to characterize the amount of GRP78 expression both at the RNA and protein level. Hypoxia was achieved by placing cells in specially designed hypoxic chambers which were subjected to successive rounds of evacuation and flushing with 95% CO2/N2 to reduce the oxygen in the environment to 0.02% oxygen. After treatment with hypoxia, cells were assayed for colony forming ability.GRP78 mRNA and protein induction following exposure to hypoxia was 3-4 fold lower in the 78WO cell line than in the parental DG44 cell line. Furthermore, it was observed that there was no difference in the cytotoxicity of 78WO and DG44 cells after 10 h of hypoxia. However, after 15 h of hypoxia, the survival of 78WO cells decreased by 1 log and after 20 h of hypoxia, the survival of 78WO decreased by another log.These results show that stress protein induction is important for cellular survival to chronic hypoxia and that inhibition of GRP78 induction may represent a novel therapeutic strategy by selectively sensitizing chronically hypoxic cells within solid tumors.

  View details for Web of Science ID A1994NA83600012

  View details for PubMedID 8113109

• TUMOR HYPOXIA - THE PICTURE HAS CHANGED IN THE 1990S Radiation Research 93 - Annual Meeting of the Association-for-Radiation-Research Brown, J. M., Giaccia, A. J. TAYLOR & FRANCIS LTD. 1994: 95–102

  Abstract

  Since the 1950s, the presence of hypoxic cells in human tumours has been widely regarded as a problem, and a variety of strategies have been developed and tested, both in experimental and clinical studies, to overcome this perceived problem. One of these strategies was the development of bioreductive cytotoxins--drugs which in themselves were relatively innocuous, but when metabolized under hypoxic conditions, became highly cytotoxic, thereby preferentially killing the hypoxic cells. Modelling studies and experimental data with newly developed hypoxic cytotoxins, such as SR 4233 (tirapazamine) and RSU 1069, have led to the realization not only that it is better to kill hypoxic cells in tumours than to radiosensitize or oxygenate them, but also that with these bioreductive cytotoxins hypoxic cells in tumours can be an advantage in cancer therapy. However, to realize the advantage of adding the drug with each radiation dose, the tumour must undergo a process analogous to reoxygenation, which we have termed 'rehypoxiation', by which hypoxic cells are regenerated after each dose of the hypoxic cytotoxin. In addition, we also discuss the fact that hypoxia is a cellular stress which activates many new genes. The activation of these genes will be a major focus for research in coming years and will undoubtedly lead to new approaches in cancer detection and treatment. In summary, the 1990s are bringing a fundamental change in our perception of tumour hypoxia, from a position of being a problem to that of being a solution in cancer treatment.

  View details for Web of Science ID A1994NE75800013

  View details for PubMedID 7905916

• CHROMOSOMAL RADIOSENSITIVITY AT INTRACHROMOSOMAL TELOMERIC SITES GENES CHROMOSOMES & CANCER ALVAREZ, L., Evans, J. W., Wilks, R., Lucas, J. N., Brown, J. M., Giaccia, A. J. 1993; 8 (1): 8-14

  Abstract

  Telomeric DNA is composed of highly conserved sequences which are present at the termini of chromosomes as well as at intrachromosomal locations. Here, we studied a Chinese hamster ovary (CHO) cell line, BL-10, with highly stable amplified telomeric DNA at the termini as well as at intrachromosomal locations. We show that intrachromosomal or interstitial telomeric sites in this cell line and in another CHO cell line, HA-I, are radiosensitive in that they are more prone to breakage than would be expected based on the percentage of the genome composed of telomeric sequences. The frequency of breakage at interstitial telomeric sites is 4.3 to 8.3 times higher than that in the CHO genome overall. These conclusions are reached by both conventional cytogenetic analysis of two CHO cell lines which have the same survival rates after exposure to ionizing radiation, and by use of double fluorescence in situ hybridization (FISH) with a pan-telomere-specific probe and a CHO chromosome-specific library in the same metaphase cells after irradiation.

  View details for Web of Science ID A1993LU38000002

  View details for PubMedID 7691162

• ACTIVATION OF POTASSIUM CHANNELS BY HYPOXIA AND REOXYGENATION IN THE HUMAN LUNG ADENOCARCINOMA CELL-LINE A549 JOURNAL OF CELLULAR PHYSIOLOGY Koong, A. C., Giaccia, A. J., Hahn, G. M., Saad, A. H. 1993; 156 (2): 341-347

  Abstract

  Active oxygen species are generated in cells during pathophysiologic conditions such as inflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We investigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents.

  View details for Web of Science ID A1993LP82200016

  View details for PubMedID 8344990

• HEAT SENSITIVITY OF BLEOMYCIN-SENSITIVE CHO DERIVATIVES IS NOT DUE TO IMPROPER INITIALIZATION OF HEAT-SHOCK RESPONSE INTERNATIONAL JOURNAL OF HYPERTHERMIA AUGER, E. A., Giaccia, A. J., Hahn, G. M. 1993; 9 (2): 275-284

  Abstract

  We have investigated whether differences in the heat shock response exist between CHO and three bleomycin-sensitive, heat-sensitive CHO derivatives. The binding of heat shock factor (HSF) in response to heat and varying concentrations of bleomycin in the four cell lines was examined using a gel shift assay and a synthetic heat shock element (HSE). Heat (45 degrees C, 10 min) and exposure to 1 micrograms/ml bleomycin for 1 h at 37 degrees C induced similar levels of HSF binding in all four cell lines. We also examined if bleomycin dose and the length of recovery from bleomycin treatment affected the induction of HSF binding. The level of activated HSF binding to HSE was higher in cells treated with low doses (1 ng/ml) of bleomycin than in cells treated with 1 or 25 micrograms/ml bleomycin. The amount of activated HSF was directly proportional to the time elapsed since bleomycin treatment. Our results therefore indicate no difference between CHO and its bleomycin-sensitive derivatives in the ability to initiate the heat shock response as determined by the production of activated HSF in response to either heat or bleomycin. We conclude that the intrinsic thermosensitivity of these cell lines is not related to the early response to heat shock, but either occurs later in the pathway or is unrelated to events after heating.

  View details for Web of Science ID A1993KR12400008

  View details for PubMedID 7682247

• POTASSIUM-CHANNEL ACTIVATION IN RESPONSE TO LOW-DOSES OF GAMMA-IRRADIATION INVOLVES REACTIVE OXYGEN INTERMEDIATES IN NONEXCITATORY CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuo, S. S., Saad, A. H., Koong, A. C., Hahn, G. M., Giaccia, A. J. 1993; 90 (3): 908-912

  Abstract

  Active oxygen species are generated during pathophysiologic conditions such as inflammation and ionizing radiation exposure. We tested the hypothesis that an early cellular event in response to these species involves regulation of ion channels. We exposed cells to gamma-irradiation or treated them with hydrogen peroxide, xanthine/xanthine oxidase, or [3H]thymidine and then monitored channel activity by the technique of whole-cell voltage clamping. Recordings showed that both normal and tumor cells exhibit an increase in K+ currents after treatment with radiation, H2O2, and xanthine/xanthine oxidase but not with high specific activity [3H]thymidine, suggesting that the signal for K+ channel activation originates at the cell membrane. A single noncytotoxic dose of 10 cGy induced measurable levels of K+ currents, suggesting that the induction of currents regulates biochemical changes in response to stress. To test whether channel activity is sensitive to active oxygen species, we pretreated cells with N-acetyl-L-cysteine (NAC) to increase cellular pools of free radical scavengers before radiation. In NAC-pretreated cells, K+ channel activation by gamma-irradiation was abolished. It has previously been shown that protein kinase C (PKC) is activated by ionizing radiation and can regulate K+ channels in some cells. However, the effect of radiation on induction of K+ channel activity was independent of PKC, since cells chronically exposed to phorbol esters still produced K+ currents after radiation. These results suggest that an early cellular response to oxidative stress is the activation of K+ channels.

  View details for Web of Science ID A1993KK81300028

  View details for PubMedID 8430104

• ANALYSIS OF RESTRICTION ENZYME-INDUCED CHROMOSOMAL-ABERRATIONS BY FLUORESCENCE IN-SITU HYBRIDIZATION ENVIRONMENTAL AND MOLECULAR MUTAGENESIS COLUMNA, E. A., Giaccia, A. J., Evans, J. W., Yates, B. L., Morgan, W. F. 1993; 22 (1): 26-33

  Abstract

  Fluorescence in situ hybridization and Giemsa staining of metaphase chromosomes were used to determine the relative frequencies of symmetric exchange aberrations (translocations) and asymmetric exchange aberrations (rings, dicentrics, and polycentrics) after exposure of human lymphoblastoid cells to restriction enzymes or X-rays. The yield of symmetric exchanges was determined with the use of chromosome-specific probes for human chromosomes 2 or 4, which were hybridized to metaphase chromosomes from cells exposed to the enzymes PvuII, SacI, or XbaI or 3 or 5 Gy of X-rays. The yield of asymmetric exchanges was determined in Giemsa-stained metaphase chromosomes from the same enzyme-treated or irradiated cell population. About 1.5- to 3-fold more symmetric than asymmetric exchanges were induced after restriction enzyme treatment. However, after X-ray treatment the yield of dicentrics relative to the yield of reciprocal translocations was close to the expected 1:1 ratio.

  View details for Web of Science ID A1993LP83100004

  View details for PubMedID 8393403

• DIFFERENTIAL EXPRESSION OF PROTEIN-KINASE-C EPSILON-PROTEIN IN LUNG-CANCER CELL-LINES BY IONIZING-RADIATION BRITISH JOURNAL OF CANCER Kim, C. Y., Giaccia, A. J., Strulovici, B., Brown, J. M. 1992; 66 (5): 844-849

  Abstract

  The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.

  View details for Web of Science ID A1992JW71300014

  View details for PubMedID 1329908

• THE USE OF ASYMMETRIC-FIELD INVERSION GEL-ELECTROPHORESIS TO PREDICT TUMOR-CELL RADIOSENSITIVITY RADIOTHERAPY AND ONCOLOGY Giaccia, A. J., SCHWARTZ, J., Shieh, J., Brown, J. M. 1992; 24 (4): 231-238

  Abstract

  The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radiosensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique [21], we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60-80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1992JN42500004

  View details for PubMedID 1410578

• WHAT ROLE DO GLUTATHIONE S-TRANSFERASES PLAY IN THE CELLULAR-RESPONSE TO IONIZING-RADIATION 7TH INTERNATIONAL CONF ON CHEMICAL MODIFIERS OF CANCER TREATMENT CHOLON, A., Giaccia, A. J., Lewis, A. D., Hickson, I., Brown, J. M. ELSEVIER SCIENCE INC. 1992: 759–63

  Abstract

  The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.

  View details for Web of Science ID A1992HJ37700028

  View details for PubMedID 1544849

• ACTIVATION OF THE HEAT-SHOCK TRANSCRIPTION FACTOR BY HYPOXIA IN NORMAL AND TUMOR-CELL LINES INVIVO AND INVITRO 33RD MEETING OF THE AMERICAN SOC FOR THERAPEUTIC RADIOLOGY AND ONCOLOGY Giaccia, A. J., AUGER, E. A., Koong, A., Terris, D. J., Minchinton, A. I., Hahn, G. M., Brown, J. M. PERGAMON-ELSEVIER SCIENCE LTD. 1992: 891–97

  Abstract

  Cells exposed to hypoxia increase their synthesis of a specific set of proteins called oxygen regulated proteins. Recently, three of these proteins have been identified as hemoxygenase, Glucose Regulated Protein 78 kilodaltons and Glucose Regulated Protein 94 kilodaltons. In contrast, reoxygenation from hypoxic conditions increases the synthesis of the heat shock proteins. Although the molecular signals required for regulation of both sets of proteins by hypoxia and reoxygenation are still under investigation, it is known that their expression is regulated at the transcriptional level. This finding suggests that these stresses work either singularly or together to control the activation of nuclear transcription factors which bind distinct regulatory sequences in the promoter region of these genes. One possible nuclear transcription factor which could act as a transcriptional regulator for both hypoxia and reoxygenation gene transcription is the heat shock transcription factor. In this report, we focused on the kinetics of HSF activation by hypoxia in normal and tumor cell lines of murine and human origins. In cell culture, both the normal diploid cell line AG1522 and the tumor cell line JSQ-3 possess the same kinetics of HSF activation (binding to the heat shock element) by hypoxia, with maximal induction at or after 3 hr. We have also shown that the activation of HSF occurs in the SCCVII tumor in vivo without clamping, but not in SCCVII cells grown in monolayers. When SCCVII tumors are dissociated and allowed to reoxygenate in cell culture, HSF binding decreased in 5 hr, and was undetectable after 18 hr. Furthermore, one human tumor biopsy tested for the presence of hypoxia by both the pO2 histograph (Eppendorf, Germany) and HSF binding showed good agreement for both techniques. These results suggest that HSF binding may be a useful marker for monitoring the tumor hypoxia.

  View details for Web of Science ID A1992JD82800027

  View details for PubMedID 1618682

• CHARACTERIZATION OF A CHO CELL-LINE RESISTANT TO KILLING BY THE HYPOXIC CELL CYTOTOXIN SR-4233 7TH INTERNATIONAL CONF ON CHEMICAL MODIFIERS OF CANCER TREATMENT Giaccia, A. J., Biedermann, K. A., TOSTO, L. M., Minchinton, A. I., Kovacs, M. S., Brown, J. M. ELSEVIER SCIENCE INC. 1992: 681–84

  Abstract

  One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7-fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of D0's. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/10(6) cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity.

  View details for Web of Science ID A1992HJ37700011

  View details for PubMedID 1544836

• THE HYPERSENSITIVITY OF THE CHINESE-HAMSTER OVARY VARIANT BL-10 TO BLEOMYCIN KILLING IS DUE TO A LACK OF GLUTATHIONE S-TRANSFERASE-ALPHA ACTIVITY CANCER RESEARCH Giaccia, A. J., Lewis, A. D., Denko, N. C., CHOLON, A., Evans, J. W., Waldren, C. A., Stamato, T. D., Brown, J. M. 1991; 51 (16): 4463-4469

  Abstract

  As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T.D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588-1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) alpha also resides on chromosome 6. Measurements of selenium-independent peroxidase (alpha-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium-dependent peroxidase activity, using H2O2 as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little alpha-GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the alpha-GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for alpha-GST in the resistance of cells to bleomycin.

  View details for Web of Science ID A1991GA54300054

  View details for PubMedID 1714344

• BIOCHEMICAL-EVIDENCE FOR 2 DIFFERENT MECHANISMS FOR BLEOMYCIN-INDUCED CELL KILLING MUTATION RESEARCH Giaccia, A. J., Shieh, J., CHOLON, A., Brown, J. M. 1991; 263 (2): 69-75

  Abstract

  Using pulsed-field gel electrophoresis, we have measured the ability of two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not cross-sensitive to other DNA breaking agents. A 1-h treatment of bleomycin induces a similar number of DNA double-strand breaks in XR-1, BL-10 and CHO cells. However, XR-1 is unable to repair bleomycin-induced DNA double-strand breaks, whereas BL-10 possesses the same kinetics of repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is DNA DSB-dependent, and the other seems to be DNA DSB-independent.

  View details for Web of Science ID A1991FT58100002

  View details for PubMedID 1710776

• LOSS OF THE INTRINSIC HEAT-RESISTANCE OF HUMAN-CELLS AND CHANGES IN MR 70,000 HEAT-SHOCK PROTEIN EXPRESSION IN HUMAN X HAMSTER HYBRIDS CANCER RESEARCH Anderson, R. L., Fong, K. J., Gabriele, T., Lavagnini, P., Hahn, G. M., Evans, J. W., Waldren, C. A., Stamato, T. D., Giaccia, A. J. 1991; 51 (10): 2636-2641

  Abstract

  Since mammalian cells vary widely in their intrinsic thermoresistance, we have investigated the genetic basis underlying this phenomenon in human and rodent cell lines. Typically, human cells are considerably more resistant to killing by heat than rodent cell lines. To determine whether the heat-resistant phenotype is dominant or recessive and to locate the chromosome(s) bearing determinants for heat resistance, we have prepared hybrids of heat-resistant human HT1080 cells and heat-sensitive Chinese hamster ovary (CHO) cells to test their response to heat. For both mass hybrid cultures and individual clones, the heat response of the hybrids was similar to that of the CHO parent. Analysis by in situ hybridization revealed the presence of five to 20 human chromosomes per cell in the mass hybrids and four to eight intact chromosomes plus some fragments in individual clones isolated from the hybrid cell population. A similar result was obtained using a different human cell line, AG1522. These data suggest that heat resistance is a recessive trait. Consistent with this conclusion are the results from a study of a fusion of HT1080 to a CHO mutant, BL-10, which was found to be hypersensitive to heat-induced killing. These hybrids had a normal CHO heat response and not the more heat-resistant phenotype of HT1080 cells. Two hybrid clones, H2 and H4, from the HT1080/BL-10 fusion were studied in more detail. Both clones possess similar amounts of Mr 70,000 heat shock protein (HSP70), despite the fact that H4 contains three human chromosomes (Nos. 6, 14, and 21) which carry HSP70 genes while H2 contains only one (chromosome 6). Both hybrid cell lines have the same response to heat. Although we found a wide range of sensitivities to heat, all cell lines contained a similar amount of constitutive HSP70, suggesting that HSP70 levels per se are not the critical determinant of intrinsic heat resistance.

  View details for Web of Science ID A1991FK91000022

  View details for PubMedID 2021941

• THE USE OF FLUORESCENCE INSITU HYBRIDIZATION COMBINED WITH PREMATURE CHROMOSOME CONDENSATION FOR THE IDENTIFICATION OF CHROMOSOME-DAMAGE BRITISH JOURNAL OF CANCER Evans, J. W., Chang, J. A., Giaccia, A. J., Pinkel, D., Brown, J. M. 1991; 63 (4): 517-521

  Abstract

  The technique of fusing mitotic cells to interphase cells, thereby producing condensation of the chromosomes of the interphase cell (so-called 'premature chromosome condensation' or PCC), has allowed detection of the initial number of chromosome breaks and their repair following ionising radiation. However, the difficulty and tedium of scoring all the chromosome fragments, as well as the inability to readily detect exchange aberrations, has limited the use of PCC. We describe here the use of the recently developed technique of fluorescence in situ hybridisation with whole chromosome libraries to stain individual human chromosomes (also called 'chromosome painting') with the PCC's and show that this overcomes most of the limitations with the analysis of PCC's. First, by focusing on a single chromosome, scoring of breaks in the target chromosome is easy and rapid and greatly expands the radiation dose range over which the PCC technique can be used. Second, it allows the easy recognition of exchange type aberrations. A number of new applications of this technology, such as predicting the radiosensitivity of human tumours in situ, are feasible.

  View details for Web of Science ID A1991FH59600011

  View details for PubMedID 2021536

• SCID MUTATION IN MICE CONFERS HYPERSENSITIVITY TO IONIZING-RADIATION AND A DEFICIENCY IN DNA DOUBLE-STRAND BREAK REPAIR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Biedermann, K. A., Sun, J. R., Giaccia, A. J., TOSTO, L. M., Brown, J. M. 1991; 88 (4): 1394-1397

  Abstract

  C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

  View details for Web of Science ID A1991EY61700064

  View details for PubMedID 1996340

• HUMAN CHROMOSOME-5 COMPLEMENTS THE DNA DOUBLE-STRAND BREAK-REPAIR DEFICIENCY AND GAMMA-RAY SENSITIVITY OF THE XR-1 HAMSTER VARIANT AMERICAN JOURNAL OF HUMAN GENETICS Giaccia, A. J., Denko, N., MacLaren, R., Mirman, D., Waldren, C., Hart, I., Stamato, T. D. 1990; 47 (3): 459-469

  Abstract

  XR-1 is a Chinese hamster ovary (CHO) cell mutant which is unusually sensitive to killing by gamma rays in the G1 portion of the cell cycle but has nearly normal resistance to gamma-ray damage in late S phase. The cell-cycle sensitivity correlates with the mutant's inability to repair DNA double-strand breaks (DSBs) produced by ionizing radiation and restriction enzymes. We have previously shown in somatic cell hybrids of XR-1 cells and human fibroblasts that the XR-1 mutation is a recessive mutation. In this study, using somatic cell hybrids formed between XR-1 and human fibroblasts, we map the human complementing gene to chromosome 5 by chromosome-segregation analysis. This gene biochemically restores the hamster defect to wild-type levels of gamma-ray and bleomycin resistance as well as restoring its proficiency to repair DNA DSBs, suggesting that a single gene is responsible for the XR-1 phenotype. We have tentatively assigned the name XRCC4 (X-ray-complementing Chinese hamster gene 4) to this human gene until its biochemical function in repair is discovered.

  View details for Web of Science ID A1990DX22300011

  View details for PubMedID 1697445

• USE OF FLUORESCENT INSITU HYBRIDIZATION TO DETECT CHROMOSOMAL REARRANGEMENTS IN SOMATIC-CELL HYBRIDS GENES CHROMOSOMES & CANCER Giaccia, A. J., Evans, J. W., Brown, J. M. 1990; 2 (3): 248-251

  Abstract

  In situ hybridization of hamster/human hybrids with biotinylated human genomic DNA has revealed that human chromosomal DNA can integrate into the hamster genome and is not always cytologically detectable. This finding helps to explain why discordancy can arise in gene mapping by failing to recognize small pieces of foreign DNA in the rodent genome. Fluorescent in situ hybridization allows one to locate these fragments in rodent chromosomes visually and possibly to identify their chromosome of origin.

  View details for Web of Science ID A1990ED57700013

  View details for PubMedID 2078516

• INCREASED SENSITIVITY TO KILLING BY RESTRICTION ENZYMES IN THE XR-1 DNA DOUBLE-STRAND BREAK REPAIR-DEFICIENT MUTANT MUTATION RESEARCH Giaccia, A. J., MACLAREN, R. A., Denko, N., Nicolaou, D., Stamato, T. D. 1990; 236 (1): 67-76

  Abstract

  Repair or misrepair of DNA double-strand breaks (DSBs) is critical in determining cellular survival after gamma-irradiation. In this report, we focus on the cellular and biochemical consequences of restriction enzyme induced DSBs in wild-type Chinese hamster ovary (CHO) cells and the DNA DSB repair-defective mutant XR-1. We find that XR-1 possesses reduced cellular survival after the introduction of restriction enzymes that produce either cohesive or blunt ends. XR-1's sensitivity to killing by restriction enzymes strongly mimics its response to gamma-rays. Using pulsed field electrophoresis, we find that for each enzyme, similar numbers of DNA DSBs are being introduced in both cell lines. The simplest explanation for the increased sensitivity to restriction enzymes in the mutant is that the biochemical defect in XR-1 is not confined to the repair of ionizing radiation induced ends, but extends to DSBs that possess ligatable 3'-hydroxyl and 5'-phosphate ends as well.

  View details for Web of Science ID A1990DQ16900009

  View details for PubMedID 2164147

• MEASUREMENT OF THE RELATIVE PROPORTION OF SYMMETRICAL AND ASYMMETRICAL CHROMOSOME-TYPE INTERCHANGES INDUCED BY GAMMA-RADIATION IN HUMAN HAMSTER HYBRID-CELLS RADIATION RESEARCH BAHARI, I. B., Bedford, J. S., Giaccia, A. J., Stamato, T. D. 1990; 123 (1): 105-107

  Abstract

  Using hamster-human hybrid cells and methods for differential staining of the hamster and human chromosomes, the relative proportion of induced chromosome-type symmetrical and asymmetrical interchange aberrations was measured after a 6.5-Gy dose of gamma radiation. The ratio of these aberration types was not significantly different from 1:1, i.e., the proportion of such interchanges that are symmetrical is very near 0.5, in agreement with the conclusion of others (J.A. Heddle, Genetics 52, 1329-1334, 1965; M. Holmberg and J. Jonasson, Hereditas 74, 57-68, 1973; J.R.K. Savage and D.G. Papworth, Mutat. Res. 95, 7-18, 1983). This proportion is important because virtually all estimates of radiation-induced chromosome-type exchange aberrations are based on measurement of the easily observed but unstable and lethal asymmetrical types, while some of the biological effects of concern from the point of view of oncogenesis and mutagenesis are thought to result from production of stable and nonlethal symmetrical types.

  View details for Web of Science ID A1990DN66500016

  View details for PubMedID 2371375

• GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF THE CHO-UV-1 MUTANT DEFECTIVE IN POSTREPLICATION RECOVERY OF DNA CANCER RESEARCH Hentosh, P., COLLINS, A. R., Correll, L., Fornace, A. J., Giaccia, A., Waldren, C. A. 1990; 50 (8): 2356-2362

  Abstract

  The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.

  View details for Web of Science ID A1990CZ34900025

  View details for PubMedID 2317821

• MOLECULAR-CLONING OF THE BREAKPOINT REGION ON CHROMOSOME-6 IN CUTANEOUS MALIGNANT-MELANOMA - EVIDENCE FOR DELETION IN THE C-MYB LOCUS AND TRANSLOCATION OF A SEGMENT OF CHROMOSOME-12 ONCOGENE Dasgupta, P., Linnenbach, A. J., Giaccia, A. J., Stamato, T. D., Reddy, E. P. 1989; 4 (10): 1201-1205

  Abstract

  Nonrandom involvement of chromosome 6 in cutaneous malignant melanoma have been noted by several investigators. Recently an alteration in the c-myb locus (6q22-23) has been identified by Southern analysis in the WM983A cell line which was derived from a primary melanoma of the vertical growth phase. In the present study, the nature of this rearrangement in the WM983A cell line has been further characterized by molecular cloning and nucleotide sequence analysis of the break-point region in the c-myb locus. The results of this investigation demonstrate that the rearrangement in the 6q22-23 region results in deletion of the 3'-end of the c-myb locus with the concomitant translocation of a portion of chromosome 12 to chromosome 6.

  View details for Web of Science ID A1989AU50800006

  View details for PubMedID 2677917

• ISOLATION AND CHARACTERIZATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE-DEFICIENT CHINESE-HAMSTER CELLS DERIVED FROM PURE MUTANT COLONIES MUTAGENESIS Stamato, T. D., Richardson, E., IANACONE, J., MACLAREN, R. A., Denko, N., Giaccia, A. 1989; 4 (4): 259-264

  Abstract

  Ten pure glucose-6-phosphate dehydrogenase (G6PD)-deficient mutants were isolated from colonies composed entirely of cells which lacked G6PD staining activity. These mutants were analyzed for G6PD enzyme activity and the presence of immunologically cross-reactive proteins using immunoblotting techniques and antiserum directed against bovine G6PD. Four mutants had no detectable enzyme activity and did not contain protein which produces a detectable cross-reaction with G6PD antibody. One mutant had residual enzyme activity and altered electrophoretic mobility but did not have detectable immunological cross-reactivity. These results could be explained by either a DNA deletion or point mutational mechanism. On the other hand, five of the 10 mutants analyzed had characteristics consistent with a point mutation in the G6PD gene. All contain a protein which cross-reacts with the G6PD antibody and have the same subunit molecular weight as the parent cell's G6PD enzyme. Four of the five mutants had residual G6PD enzyme activity. Thus, the mechanism for the formation of pure mutants is not simply DNA deletion but is probably a more complex process involving the transfer of altered genetic information from one DNA strand to the other.

  View details for Web of Science ID A1989AG75300003

  View details for PubMedID 2674604

• AN ASYMMETRIC FIELD INVERSION GEL-ELECTROPHORESIS METHOD FOR THE SEPARATION OF LARGE DNA-MOLECULES ANALYTICAL BIOCHEMISTRY Denko, N., Giaccia, A., Peters, B., Stamato, T. D. 1989; 178 (1): 172-176

  Abstract

  An electrophoretic method for separating large DNA molecules which uses periodically inverted electric fields of different magnitude in the two directions is described. Net DNA migration is either in the high field direction or in the low field direction, depending on the relative duration of the pulses. With this approach, molecules of up to 1.6 million base pairs can be separated in parallel lanes after a single run under fixed timing conditions. An inexpensive switching unit is the only device needed in addition to the conventional gel box.

  View details for Web of Science ID A1989T871000030

  View details for PubMedID 2729570

• GENETIC-ANALYSIS OF XR-1 MUTATION IN HAMSTER AND HUMAN HYBRIDS SOMATIC CELL AND MOLECULAR GENETICS Giaccia, A. J., Richardson, E., Denko, N., Stamato, T. D. 1989; 15 (1): 71-77

  Abstract

  We investigated the dominant/recessive nature of the XR-1 mutant locus in intraspecies Chinese hamster ovary (CHO) hybrids and interspecies hybrids with human cell lines that possess different radioresistances. The XR-1 cell is abnormally sensitive to killing by gamma rays in the G1 phase of the cell cycle, while late-S-phase cells have wild-type resistance. [3H]Thymidine selection was used to eliminate the resistant S-phase population. In both intraspecies and interspecies hybrids, the XR-1 mutation is recessive to the wild-type cell and is not influenced by differences in chromosome ploidy. Analysis of hybrids between human ataxia telangiectasia fibroblasts AT5BI and XR-1 cells revealed that they possess different genetic defects as they complemented each other in three of four hybrids tested. These data suggest that the XR-1 locus is evolutionarily conserved between hamster and human cells.

  View details for Web of Science ID A1989T192900008

  View details for PubMedID 2916163

• CELL-CYCLE-DEPENDENT REPAIR OF POTENTIALLY LETHAL DAMAGE IN THE XR-1 GAMMA-RAY-SENSITIVE CHINESE-HAMSTER OVARY CELL RADIATION RESEARCH Stamato, T. D., DIPATRI, A., Giaccia, A. 1988; 115 (2): 325-333

  Abstract

  Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.

  View details for Web of Science ID A1988P773800010

  View details for PubMedID 3406371

• ISOLATION AND CHARACTERIZATION OF BLEOMYCIN-SENSITIVE CHINESE-HAMSTER OVARY CELLS CANCER RESEARCH Stamato, T. D., Peters, B., Patil, P., Denko, N., Weinstein, R., Giaccia, A. 1987; 47 (6): 1588-1592

  Abstract

  Nineteen bleomycin-sensitive Chinese hamster ovary cell mutants have been isolated using a replica plating and photography approach. As judged by the dose which reduces cell survival to 37% of the untreated control, these mutants are from 2.5- to 32-fold more sensitive to a 16-h bleomycin treatment than the parental cell, while for chronic bleomycin exposure, the increase in sensitivity was 5 to 58 times that of the parental cell. Four bleomycin-sensitive mutants had increased sensitivities to killing by gamma-rays (2- to 3-fold), mitomycin C (2-fold), and ethyl methane sulfonate (4- to 5-fold), while six other mutants were resistant to these agents. Nine other bleomycin-sensitive mutants displayed a variable pattern of cross-sensitivities to these agents. Using the technique of alkaline elution, the relative frequency of single-strand DNA breaks introduced by varying concentrations of bleomycin was examined in one mutant and its parent cell. The elution profiles of both cells were similar, suggesting that the bleomycin sensitivity of this mutant is not due to a greater frequency of single-strand breaks introduced by bleomycin.

  View details for Web of Science ID A1987G548300020

  View details for PubMedID 2434221

• DELAYED MUTATION IN CHINESE-HAMSTER CELLS SOMATIC CELL AND MOLECULAR GENETICS Stamato, T., Weinstein, R., Peters, B., Hu, J., Doherty, B., Giaccia, A. 1987; 13 (1): 57-66

  Abstract

  The possibility was examined that mutational events can be delayed for more than one or two cell divisions following treatment of Chinese hamster cells with the DNA alkylating agent ethyl methane sulfonate. If mutations in mammalian cells are delayed, the proportion of mutant cells in colonies grown from single mutagen-treated cells will reflect the cell division at which the mutation is genetically fixed, i.e., a first division mutation yields a 1/2 mutant colony, a fifth division mutation produces a 1/32 mutant colony, etc. In the present study, replating of cells from single colonies grown for six to seven days after mutagen treatment resulted in the discrete ratios of glucose-6-phosphate dehydrogenase (G6PD)-deficient mutant to wild-type colonies expected for a delayed mutational process which produces mutations over at least 8-10 cell generations. Further, when cells from 7- to 10-day colonies, grown from ethyl methane sulfonate (EMS)-treated cells were replated into selective medium containing 6-thioguanine (6TG), the number of 6TG-resistant colonies obtained per flask was distributed over a very wide range, consistent with a mutational delay process. These results could not be explained by differences in the number of cells per colony or plating efficiency in selective medium. Assuming that the relative number of 6TG-resistant colonies per flask reflects the time of mutation, EMS treatment produced two groups of mutational events: one which occurred within the first five cell generations and another uniformly distributed over at least the next eight to nine divisions. These results support the conclusion that EMS induces mutants for at least 10-14 cell generations after treatment and raise the possibility that current methods to assess the mutagenic potential of an agent might lead to significant underestimation. The role of delayed mutation in the phenomenon of "mutation expression time" is also discussed.

  View details for Web of Science ID A1987G091300006

  View details for PubMedID 3468633

• CELL CYCLE-DEPENDENT REPAIR OF DOUBLE-STRAND DNA BREAKS IN A GAMMA-RAY-SENSITIVE CHINESE-HAMSTER CELL SOMATIC CELL AND MOLECULAR GENETICS Giaccia, A., Weinstein, R., Hu, J., Stamato, T. D. 1985; 11 (5): 485-491

  Abstract

  A Chinese hamster cell mutant has been isolated which is extremely sensitive to killing by gamma-irradiation in the G1 and early S phases of the cell cycle (LD50 of 20 vs. 250 rads for parent), but which has nearly normal resistance in late S. The mutant cell is able to repair single-stranded DNA breaks introduced by gamma-radiation. However, in comparison to its parental cell, the mutant is deficient in the repair of double-stranded DNA breaks produced by gamma-irradiation during the sensitive G1-early S period, while in the resistant late S period, the repair is nearly the same for both cell types. This correlation between gamma-ray sensitivity and repair strongly suggests that an inability to repair double-strand DNA breaks in G1 is the basis for the hypersensitivity of the mutant to killing by gamma-rays in this phase of the cell cycle. It also provides direct evidence in mammalian cells that the ability to repair double-strand DNA breaks induced by ionizing radiation is an important biochemical function in cell survival and supports the hypothesis that unrepaired double-strand breaks are a major lethal lesion in mammalian cells. A plausible explanation for the appearance of the cell cycle phenotype of the mutant is that in normal cells there are at least two pathways for the repair of double-strand breaks, one of which functions primarily in late S phase, and the other, either throughout the cell cycle or only in the G1 and early S phases.

  View details for Web of Science ID A1985ARK8400008

  View details for PubMedID 3862244

• TIMING OF MUTATION FIXATION EVENTS IN ETHYL METHANE SULFONATE TREATED CHINESE-HAMSTER CELLS SOMATIC CELL AND MOLECULAR GENETICS Stamato, T. D., Weinstein, R., Giaccia, A. 1984; 10 (4): 429-434

  Abstract

  Exposure of single Chinese hamster ovary (CHO) cells to the mutagen, ethyl methane sulfonate, produces two types of mutant colonies lacking glucose-6-phosphate dehydrogenase activity: colonies uniformly deficient in enzyme activity, and mosaic colonies containing both mutant and nonmutant cell phenotypes in various relative proportions and sectored patterns (1/8, 1/4, 1/2). We find that the relative size of the mutant sector in these mosaic colonies primarily reflects the cell division at which the mutation was genetically fixed. Thus, the mutation-fixation event occurs before the first cell division in 1/2 sector and pure mutant colonies, between the first and second divisions for 1/4 sectors, and between the second and third divisions for 1/8 sectors. Delay in the formation of mutations could also explain the phenomenon of "mutation expression time" which is observed when drug resistance is used to select for mutants. Colony sectoring offers for the first time in mammalian cells the opportunity to observe an agent's effect on the timing of the mutational process.

  View details for Web of Science ID A1984TC41700012

  View details for PubMedID 6589795

• ISOLATION OF CELL CYCLE-DEPENDENT GAMMA-RAY-SENSITIVE CHINESE-HAMSTER OVARY CELL SOMATIC CELL GENETICS Stamato, T. D., Weinstein, R., Giaccia, A., Mackenzie, L. 1983; 9 (2): 165-173

  Abstract

  A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.

  View details for Web of Science ID A1983QJ46700002

  View details for PubMedID 6836453