Derek Richard Clements

All Publications

• Alveolar macrophages and epithelial cells: The art of living together. The Journal of experimental medicine Clements, D., Idoyaga, J. 2021; 218 (10)

  Abstract

  In this issue of JEM, Gschwend et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210745) reveal the indispensable role of alveolar epithelial cells type 2 in controlling the density of alveolar macrophages. This study highlights the intricate crosstalk that lung stroma and macrophages undergo to maintain homeostasis.

  View details for DOI 10.1084/jem.20211583

  View details for PubMedID 34491265

• Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy. Journal for immunotherapy of cancer Prokopi, A., Tripp, C. H., Tummers, B., Hornsteiner, F., Spoeck, S., Crawford, J. C., Clements, D. R., Efremova, M., Hutter, K., Bellmann, L., Cappellano, G., Cadilha, B. L., Kobold, S., Boon, L., Ortner, D., Trajanoski, Z., Chen, S., de Gruijl, T. D., Idoyaga, J., Green, D. R., Stoitzner, P. 2021; 9 (1)

  Abstract

  BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described.METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells.RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ andCD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon gamma (IFNgamma)/tumor necrosis factor alpha (TNFalpha). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth.CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

  View details for DOI 10.1136/jitc-2020-000832

  View details for PubMedID 33408092

• Trial Watch: oncolytic viro-immunotherapy of hematologic and solid tumors Oncolmmunology Pol, J. G., Levesque, S., Workenhe, S. T., Gujar, S., Le Boeuf, F., Clements, D. R., Fahrner, J., Fend, L., Bell, J. C., Mossman, K. L., Fucikova, J., Spisek, R., Zitvogel, L., Kroemer, G., Galluzi, L. 2018
• Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages. Journal of proteome research Clements, D. R., Murphy, J. P., Sterea, A. n., Kennedy, B. E., Kim, Y. n., Helson, E. n., Almasi, S. n., Holay, N. n., Konda, P. n., Paulo, J. A., Sharif, T. n., Lee, P. W., Weekes, M. P., Gygi, S. P., Gujar, S. n. 2017; 16 (9): 3391–3406

  Abstract

  Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host-virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G-, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G-, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral-host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.

  View details for DOI 10.1021/acs.jproteome.7b00425

  View details for PubMedID 28768414

  View details for PubMedCentralID PMC5648240

• All that glitters is not gold: the need to consider desirable and undesirable immune aspects of oncolytic virus therapy ONCOIMMUNOLOGY Clements, D. R., Kim, Y., Gujar, S. A., Lee, P. W. 2016; 5 (1)

  Abstract

  Oncolytic viruses (OVs), a novel class of anticancer therapeutic agents, can overturn cancer-mediated immunosuppression and initiate antitumor immunity. Contrary to this paradigm, our recent study illustrates that oncolytic reovirus transiently augments cancer-associated immunosuppression immediately following its therapeutic administration. To achieve the optimum efficacy for OV-based anticancer therapies, the pathophysiological as well as clinical implications of this phenomenon need to be considered.

  View details for DOI 10.1080/2162402X.2015.1057674

  View details for Web of Science ID 000373383600002

  View details for PubMedID 26942069

  View details for PubMedCentralID PMC4760287

• Dendritic Cells in Oncolytic Virus-Based Anti-Cancer Therapy VIRUSES-BASEL Kim, Y., Clements, D. R., Sterea, A. M., Jang, H. W., Gujar, S. A., Lee, P. W. 2015; 7 (12): 6506-6525

  Abstract

  Dendritic cells (DCs) are specialized antigen-presenting cells that have a notable role in the initiation and regulation of innate and adaptive immune responses. In the context of cancer, appropriately activated DCs can induce anti-tumor immunity by activating innate immune cells and tumor-specific lymphocytes that target cancer cells. However, the tumor microenvironment (TME) imposes different mechanisms that facilitate the impairment of DC functions, such as inefficient antigen presentation or polarization into immunosuppressive DCs. These tumor-associated DCs thus fail to initiate tumor-specific immunity, and indirectly support tumor progression. Hence, there is increasing interest in identifying interventions that can overturn DC impairment within the TME. Many reports thus far have studied oncolytic viruses (OVs), viruses that preferentially target and kill cancer cells, for their capacity to enhance DC-mediated anti-tumor effects. Herein, we describe the general characteristics of DCs, focusing on their role in innate and adaptive immunity in the context of the TME. We also examine how DC-OV interaction affects DC recruitment, OV delivery, and anti-tumor immunity activation. Understanding these roles of DCs in the TME and OV infection is critical in devising strategies to further harness the anti-tumor effects of both DCs and OVs, ultimately enhancing the efficacy of OV-based oncotherapy.

  View details for DOI 10.3390/v7122953

  View details for Web of Science ID 000367536700027

  View details for PubMedID 26690204

  View details for PubMedCentralID PMC4690876

• Newly Recruited CD11b(+), GR-1(+), Ly6C(high) Myeloid Cells Augment Tumor-Associated Immunosuppression Immediately following the Therapeutic Administration of Oncolytic Reovirus JOURNAL OF IMMUNOLOGY Clements, D. R., Sterea, A. M., Kim, Y., Helson, E., Dean, C. A., Nunokawa, A., Coyle, K. M., Sharif, T., Marcato, P., Gujar, S. A., Lee, P. W. 2015; 194 (9): 4397-4412

  Abstract

  Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.

  View details for DOI 10.4049/jimmunol.1402132

  View details for Web of Science ID 000353727400038

  View details for PubMedID 25825443

• Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms BRITISH JOURNAL OF CANCER Gujar, S. A., CLEMENTS, D., Dielschneider, R., Helson, E., Marcato, P., Lee, P. W. 2014; 110 (1): 83-93

  Abstract

  Reovirus preferentially infects and kills cancer cells and is currently undergoing clinical trials internationally. While oncolysis is the primary mode of tumour elimination, increasing evidence illustrates that reovirus additionally stimulates anti-tumour immunity with a capacity to target existing and possibly relapsing cancer cells. These virus-induced anti-tumour immune activities largely determine the efficacy of oncotherapy. On the other hand, anti-viral immune responses can negatively affect oncotherapy. Hence, the strategic management of anti-tumour and anti-viral immune responses through complementary therapeutics is crucial to achieve the maximum anti-cancer benefits of oncotherapy.Intra-peritoneal injection of mouse ovarian surface epithelial cells (ID8 cells) into wild-type C57BL/6 mice was treated with a therapeutic regimen of reovirus and/or gemcitabine and then analysed for prolonged survival, disease pathology, and various immunological parameters. Furthermore, in vitro analyses were conducted to assess apoptosis, viral spread, and viral production during reovirus and/or gemcitabine treatment.We demonstrate that reovirus and gemcitabine combination treatment postpones peritoneal carcinomatosis development and prolongs the survival of cancer-bearing hosts. Importantly, these anti-cancer benefits are generated through various immunological mechanisms, including: (1) inhibition of myeloid-derived suppressor cells recruitment to the tumour microenvironment, (2) downmodulation of pro-MDSC factors, and (3) accelerated development of anti-tumour T-cell responses.The complementation of reovirus with gemcitabine further potentiates virus-initiated anti-cancer immunity and enhances the efficacy of oncotherapy. In the context of ongoing clinical trials, our findings represent clinically relevant information capable of enhancing cancer outcomes.

  View details for DOI 10.1038/bjc.2013.695

  View details for Web of Science ID 000329493700013

  View details for PubMedID 24281006

  View details for PubMedCentralID PMC3887295

• Two is better than one Complementing oncolytic virotherapy with gemcitabine to potentiate antitumor immune responses ONCOIMMUNOLOGY Gujar, S. A., Clements, D., Lee, P. W. 2014; 3 (1)

  Abstract

  Oncolytic viruses (OVs) preferentially infect and kill cancer cells. Additionally, OV-induced immune responses subvert cancer-associated immunosuppression and promote antitumor immunity. We have recently demonstrated that the complementation of oncolytic virotherapy with gemcitabine accentuates its immunostimulatory effects, hence exerting superior antineoplastic activity.

  View details for DOI 10.4161/onci.27622

  View details for Web of Science ID 000339952700045

  View details for PubMedID 24804161

  View details for PubMedCentralID PMC4010537

• Reovirus in cancer therapy: an evidence-based review. Oncolytic virotherapy Clements, D., Helson, E., Gujar, S. A., Lee, P. W. 2014; 3: 69-82

  Abstract

  Reovirus, a double-stranded ribonucleic acid virus and benign human pathogen, preferentially infects and kills cancer cells in its unmodified form, and is one of the leading oncolytic viruses currently undergoing clinical trials internationally. With 32 clinical trials completed or ongoing thus far, reovirus has demonstrated clinical therapeutic applicability against a multitude of cancers, including but not limited to breast cancer, prostate cancer, pancreatic cancer, malignant gliomas, advanced head and neck cancers, and metastatic ovarian cancers. Phase I trials have demonstrated that reovirus is safe to use via both intralesional/intratumoral and systemic routes of administration, with the most common adverse reactions being grade I/II toxicities, such as flu-like illness (fatigue, nausea, vomiting, headache, fever/chills, dizziness), diarrhea, and lymphopenia. In subsequent Phase II trials, reovirus administration was demonstrated to successfully decrease tumor size and promote tumor necrosis, thereby complementing compelling preclinical evidence of tumor destruction by the virus. Importantly, reovirus has been shown to be effective as a monotherapy, as well as in combination with other anticancer options, including radiation and chemotherapeutic agents, such as gemcitabine, docetaxel, paclitaxel, and carboplatin. Of note, the first Phase III clinical trial using reovirus in combination with paclitaxel and carboplatin for the treatment of head and neck cancers is under way. Based on the evidence from clinical trials, we comprehensively review the use of reovirus as an anticancer agent, acknowledge key obstacles, and suggest future directions to ultimately potentiate the efficacy of reovirus oncotherapy.

  View details for DOI 10.2147/OV.S51321

  View details for PubMedID 27512664

  View details for PubMedCentralID PMC4918368

• Quantitative Proteome Responses to Oncolytic Reovirus in GM-CSF- and M-CSF-Differentiated Bone Marrow-Derived Cells. Journal of proteome research Giacomantonio, M. A., Sterea, A. M., Kim, Y., Paulo, J. A., Clements, D. R., Kennedy, B. E., Bydoun, M. J., Shi, G., Waisman, D. M., Gygi, S. P., Giacomantonio, C. A., Murphy, J. P., Gujar, S. 2020; 19 (2): 708-718

  Abstract

  The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.

  View details for DOI 10.1021/acs.jproteome.9b00583

  View details for PubMedID 31884793

  View details for PubMedCentralID PMC7294930

• Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo JOURNAL OF VIROLOGY Mohamed, A., Clements, D. R., Gujar, S. A., Lee, P. W., Smiley, J. R., Shmulevitz, M. 2020; 94 (4)

  Abstract

  Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

  View details for DOI 10.1128/JVI.01688-19

  View details for Web of Science ID 000510867500018

  View details for PubMedID 31748391

  View details for PubMedCentralID PMC6997766

• Inhibition of Pyruvate Dehydrogenase Kinase Enhances the Antitumor Efficacy of Oncolytic Reovirus. Cancer research Kennedy, B. E., Murphy, J. P., Clements, D. R., Konda, P., Holay, N., Kim, Y., Pathak, G. P., Giacomantonio, M. A., Hiani, Y. E., Gujar, S. 2019; 79 (15): 3824-3836

  Abstract

  Oncolytic viruses (OV) such as reovirus preferentially infect and kill cancer cells. Thus, the mechanisms that dictate the susceptibility of cancer cells to OV-induced cytotoxicity hold the key to their success in clinics. Here, we investigated whether cancer cell metabolism defines its susceptibility to OV and if OV-induced metabolic perturbations can be therapeutically targeted. Using mass spectrometry-based metabolomics and extracellular flux analysis on a panel of cancer cell lines with varying degrees of susceptibility to reovirus, we found that OV-induced changes in central energy metabolism, pyruvate metabolism, and oxidative stress correlate with their susceptibility to reovirus. In particular, reovirus infection accentuated Warburg-like metabolic perturbations in cell lines relatively resistant to oncolysis. These metabolic changes were facilitated by oxidative stress-induced inhibitory phosphorylation of pyruvate dehydrogenase (PDH) that impaired the routing of pyruvate into the tricarboxylic acid cycle and established a metabolic state unsupportive of OV replication. From the therapeutic perspective, reactivation of PDH in cancer cells that were weakly sensitive for reovirus, either through PDH kinase (PDK) inhibitors dichloroacetate and AZD7545 or short hairpin RNA-specific depletion of PDK1, enhanced the efficacy of reovirus-induced oncolysis in vitro and in vivo. These findings identify targeted metabolic reprogramming as a possible combination strategy to enhance the antitumor effects of OV in clinics. SIGNIFICANCE: This study proposes targeted metabolic reprogramming as a valid combinatorial strategy to enhance the translational efficacy of oncolytic virus-based cancer therapies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/15/3824/F1.large.jpg.

  View details for DOI 10.1158/0008-5472.CAN-18-2414

  View details for PubMedID 31088833

• Therapy-Induced MHC I Ligands Shape Neo-Antitumor CD8 T Cell Responses during Oncolytic Virus-Based Cancer Immunotherapy JOURNAL OF PROTEOME RESEARCH Murphy, J., Kim, Y., Clements, D. R., Konda, P., Schuster, H., Kowalewski, D. J., Paulo, J. A., Cohen, A. M., Stevanovic, S., Gygi, S. P., Gujar, S. 2019; 18 (6): 2666-2675

  Abstract

  Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.

  View details for DOI 10.1021/acs.jproteome.9b00173

  View details for Web of Science ID 000471212200029

  View details for PubMedID 31095916

  View details for PubMedCentralID PMC7294931

• The lysosomal TRPML1 channel regulates triple negative breast cancer development by promoting mTORC1 and purinergic signaling pathways CELL CALCIUM Xu, M., Almasi, S., Yang, Y., Yan, C., Sterea, A., Syeda, A., Shen, B., Derek, C., Huang, P., Gujar, S., Wang, J., Zong, W., Trebak, M., El Hiani, Y., Dong, X. 2019; 79: 80-88

  Abstract

  The triple-negative breast cancer (TNBC) that comprises approximately 10%-20% of breast cancers is an aggressive subtype lacking effective therapeutics. Among various signaling pathways, mTORC1 and purinergic signals have emerged as potentially fruitful targets for clinical therapy of TNBC. Unfortunately, drugs targeting these signaling pathways do not successfully inhibit the progression of TNBC, partially due to the fact that these signaling pathways are essential for the function of all types of cells. In this study, we report that TRPML1 is specifically upregulated in TNBCs and that its genetic downregulation and pharmacological inhibition suppress the growth of TNBC. Mechanistically, we demonstrate that TRPML1 regulates TNBC development, at least partially, through controlling mTORC1 activity and the release of lysosomal ATP. Because TRPML1 is specifically activated by cellular stresses found in tumor microenvironments, antagonists of TRPML1 could represent anticancer drugs with enhanced specificity and potency. Our findings are expected to have a major impact on drug targeting of TNBCs.

  View details for DOI 10.1016/j.ceca.2019.02.010

  View details for Web of Science ID 000463866200010

  View details for PubMedID 30889511

  View details for PubMedCentralID PMC6698368

• Multiplexed Relative Quantitation with Isobaric Tagging Mass Spectrometry Reveals Class I Major Histocompatibility Complex Ligand Dynamics in Response to Doxorubicin ANALYTICAL CHEMISTRY Murphy, J., Yu, Q., Konda, P., Paulo, J. A., Jedrychowski, M. P., Kowalewski, D. J., Schuster, H., Kim, Y., Clements, D., Jain, A., Stevanovic, S., Gygi, S. P., Mancias, J. D., Gujar, S. 2019; 91 (8): 5106-5115

  Abstract

  MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.

  View details for DOI 10.1021/acs.analchem.8b05616

  View details for Web of Science ID 000465189600028

  View details for PubMedID 30779550

  View details for PubMedCentralID PMC7302430

• TRPM2 ion channel promotes gastric cancer migration, invasion and tumor growth through the AKT signaling pathway. Scientific reports Almasi, S., Sterea, A. M., Fernando, W., Clements, D. R., Marcato, P., Hoskin, D. W., Gujar, S., El Hiani, Y. 2019; 9 (1): 4182

  Abstract

  Transient Receptor Potential Melastatin-2 (TRPM2) ion channel is emerging as a great therapeutic target in many types of cancer, including gastric cancer - a major health threat of cancer related-death worldwide. Our previous study demonstrated the critical role of TRPM2 in gastric cancer cells bioenergetics and survival; however, its role in gastric cancer metastasis, the major cause of patient death, remains unknown. Here, using molecular and functional assays, we demonstrate that TRPM2 downregulation significantly inhibits the migration and invasion abilities of gastric cancer cells, with a significant reversion in the expression level of metastatic markers. These effects were concomitant with decreased Akt and increased PTEN activities. Finally, TRPM2 silencing resulted in deregulation of metastatic markers and abolished the tumor growth ability of AGS gastric cancer cells in NOD/SCID mice. Taken together, our results provide compelling evidence on the important function of TRPM2 in the modulation of gastric cancer cell invasion likely through controlling the PTEN/Akt pathway.

  View details for PubMedID 30862883

• TRPM2 Silencing Causes G2/M Arrest and Apoptosis in Lung Cancer Cells via Increasing Intracellular ROS and RNS Levels and Activating the JNK Pathway. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology Almasi, S., Long, C. Y., Sterea, A., Clements, D. R., Gujar, S., El Hiani, Y. 2019; 52 (4): 742–57

  Abstract

  BACKGROUND/AIMS: The oxidative stress sensor transient receptor potential melastatin-2 (TRPM2) ion channel has recently gained attention in many types of cancer. The lung tissue is highly susceptible to oxidative stress-mediated injury and diseases; therefore, we aimed to determine whether TRPM2 plays an essential role in protecting lung cancer cells from oxidative damage while promoting cancer cell survival and metastasis.METHODS: We used two non-small cell lung (NSCLC) cell lines A549 and H1299 as a lung cancer model. We investigated the functional expression of TRPM2 using electrophysiology, qRT-PCR and Western blots. CFSE and flow cytometry were used to study TRPM2 role in proliferation, cell cycle and apoptosis. Gap closure chambers and Three-Tiered Chemotaxis Chamber were used to study the role of TRPM2 in metastasis. SCID mice were used to study the role of TRPM2 in lung tumor growth and metastasis.RESULTS: we demonstrate that TRPM2 is functionally expressed in NSCLC cells and that its downregulation significantly inhibits cell proliferation and promotes apoptosis. These results were concomitant with an induction in DNA damage and G2/M cell cycle arrest. TRPM2 silencing inhibits also lung cancer cells invasion ability and alters EMT processes. Mechanistically, TRPM2 downregulation causes an increase in the intracellular levels of reactive oxygen (ROS) and nitrogen (RNS) species, which in turn causes DNA damage and JNK activation leading to G2/M arrest, and an ultimate cell death. Finally, TRPM2 downregulation suppresses the growth of human lung tumour xenograft in SCID mice and TRPM2 depleted tumours exhibited a significant reduction in the mRNA expression level of EMT markers compared to the control tumors.CONCLUSION: Our data provide new insights on the functional expression of TRPM2 in lung cancer, its essential role in tumour growth and metastasis through the control of JNK signaling pathway, and that TRPM2 could be exploited for targeted lung cancer therapies.

  View details for PubMedID 30933439

• RTN4 Knockdown Dysregulates the AKT Pathway, Destabilizes the Cytoskeleton, and Enhances Paclitaxel-Induced Cytotoxicity in Cancers MOLECULAR THERAPY Pathak, G. P., Shah, R., Kennedy, B. E., Murphy, J., Clements, D., Konda, P., Giacomantonio, M., Xu, Z., Schlaepfer, I. R., Gujar, S. 2018; 26 (8): 2019–33

  Abstract

  Reticulon-4 (RTN4), commonly known as a neurite outgrowth inhibitor (Nogo), is emerging as an important player in human cancers. Clinically, we found lower RTN4 expression in patient-derived tumors was associated with significantly better survival in lung, breast, cervical, and renal cancer patients. To identify the role of RTN4 in cancer biology, we performed mass spectrometry-based quantitative proteomic analysis on cancer cells following RTN4 knockdown and found its link with pro-survival as well as cytoskeleton-related processes. Subsequent mechanistic investigations revealed that RTN4 regulates lipid homeostasis, AKT signaling, and cytoskeleton modulation. In particular, downregulation of RTN4 reduced sphingomyelin synthesis and impaired plasma membrane localization of AKT, wherein AKT phosphorylation, involved in many cancers, was significantly reduced without any comparable effect on AKT-related upstream kinases, in a sphingolipid-dependent manner. Furthermore, knockdown of RTN4 retarded proliferation of cancer cells in vitro as well as tumor xenografts in mice. Finally, RTN4 knockdown affected tubulin stability and promoted higher cytotoxic effects with chemotherapeutic paclitaxel in cancer cells both in vitro and in vivo. In summary, RTN4 is involved in carcinogenesis and represents a molecular candidate that may be targeted to achieve desired antitumor effects in clinics.

  View details for DOI 10.1016/j.ymthe.2018.05.026

  View details for Web of Science ID 000441547500019

  View details for PubMedID 30078441

  View details for PubMedCentralID PMC6094397

• Epigenetic Silencing of TAP1 in Aldefluor+ Breast Cancer Stem Cells Contributes to Their Enhanced Immune Evasion. Stem cells (Dayton, Ohio) Sultan, M., Vidovic, D., Paine, A. S., Huynh, T. T., Coyle, K. M., Thomas, M. L., Cruickshank, B. M., Dean, C. A., Clements, D. R., Kim, Y., Lee, K., Gujar, S. A., Weaver, I. C., Marcato, P. 2018; 36 (5): 641-654

  Abstract

  Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.

  View details for DOI 10.1002/stem.2780

  View details for PubMedID 29341428

• Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis. Acta neuropathologica communications Warford, J. R., Lamport, A. C., Clements, D. R., Malone, A., Kennedy, B. E., Kim, Y., Gujar, S. A., Hoskin, D. W., Easton, A. S. 2018; 6 (1): 4

  Abstract

  Proteoglycans are promising therapeutic targets in Multiple Sclerosis (MS), because they regulate many aspects of the immune response. This was studied using surfen, an agent that binds both heparan sulphate proteoglycans (HSPGs) and chondroitin sulphate proteoglycans (CSPGs). Initial cell culture work on bone marrow derived macrophages (BMDMs) found that surfen reduced concentrations of the chemokines CCL2, CCL4 and CCL5, with reduced messenger (m)RNA expression for Tumor Necrosis Factor, IL-6, IL-1β and inducible nitric oxide synthase. These data were further explored using Experimental Autoimmune Encephalomyelitis (EAE) in mice. Surfen reduced clinical signs during EAE when administered from disease onset, and reduced infiltration by CD4 positive T cells and macrophages into the central nervous system. These mice also showed reduced mRNA expression for the chemokines CCL3 and CCL5, with reduced concentrations of CCL2, CCL3 and CCL5. During EAE, surfen treatment induced a persistent increase in Interleukin (IL)-4 concentrations which may enhance T helper 2 responses. During EAE, surfen treatment reduced mRNA expression for HSPGs (NDST1, agrin, syndecan-4, perlecan, serglycin, syndecan-1) and the CSPG versican. By contrast, surfen increased mRNA expression for the CSPG aggrecan, with no effect on neurocan. During EAE, significant positive correlations were found between mRNA expression and clinical score for syndecan-4, serglycin and syndecan-1 and a significant negative correlation for aggrecan. These correlations were absent in surfen treated mice. Repair in the later stages of MS involves remyelination, which was modeled by injecting lysolecithin (lysophosphatidylcholine, LPC) into mouse corpus callosum to create regions of demyelination. When surfen was injected 2 days after LPC, it delayed remyelination of the lesions, but had no effect when injected 7 days after LPC. The delayed remyelination was associated with local increases in CSPG expression. Therefore surfen suppresses inflammation but inhibits remyelination in these models. A mechanism in common may be increased CSPG expression.

  View details for DOI 10.1186/s40478-017-0506-9

  View details for PubMedID 29301568

  View details for PubMedCentralID PMC5755315

• The NAD+ Salvage Pathway Supports PHGDH-Driven Serine Biosynthesis Cell Reports Murphy, J. P., Giacomantonio, M., Paulo, J. A., Everley, R. A., Kennedy, B. E., Pathak, G. P., Clements, D. R., Kim, Y., Dai, C., Sharif, T., Gygi, S. P., Gujar, S. 2018

  View details for DOI 10.1016/j.celrep.2018.07.086

• MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies JOURNAL OF PROTEOME RESEARCH Murphy, J. P., Konda, P., Kowalewski, D. J., Schuster, H., Clements, D., Kim, Y., Cohen, A. M., Sharif, T., Nielsen, M., Stevanovic, S., Lee, P. W., Gujar, S. 2017; 16 (4): 1806-1816

  Abstract

  Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.

  View details for DOI 10.1021/acs.jproteome.6b00971

  View details for Web of Science ID 000398985700038

  View details for PubMedID 28244318

• Autophagic homeostasis is required for the pluripotency of cancer stem cells AUTOPHAGY Sharif, T., Martell, E., Dai, C., Kennedy, B. E., Murphy, P., Clements, D. R., Kim, Y., Lee, P. W., Gujar, S. A. 2017; 13 (2): 264-284

  Abstract

  Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate-limiting enzyme in the NAD+ synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (KD) of NAMPT or the KD of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin β 3 class III), CSN2 (casein β), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT KD promoted while POU5F1 KD inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 KD), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD+ biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.

  View details for DOI 10.1080/15548627.2016.1260808

  View details for Web of Science ID 000395125200005

  View details for PubMedID 27929731

  View details for PubMedCentralID PMC5324853

• Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1 ONCOTARGET Coyle, K. M., Murphy, J. P., Vidovic, D., Vaghar-Kashani, A., Dean, C. A., Sultan, M., Clements, D., Wallace, M., Thomas, M. L., Hundert, A., Giacomantonio, C. A., Helyer, L., Gujar, S. A., Lee, P. W., Weaver, I. C., Marcato, P. 2016; 7 (28): 44096-44112

  Abstract

  Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

  View details for DOI 10.18632/oncotarget.9858

  View details for Web of Science ID 000385395700092

  View details for PubMedID 27286452

  View details for PubMedCentralID PMC5190082

• The NAD(+) salvage pathway modulates cancer cell viability via p73 CELL DEATH AND DIFFERENTIATION Sharif, T., Ahn, D., Liu, R., Pringle, E., Martell, E., Dai, C., NUNOKAWA, A., Kwak, M., CLEMENTS, D., Murphy, J. P., Dean, C., Marcato, P., McCormick, C., Godbout, R., Gujar, S. A., Lee, P. W. 2016; 23 (4): 669-680

  Abstract

  The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

  View details for DOI 10.1038/cdd.2015.134

  View details for Web of Science ID 000374126800012

  View details for PubMedID 26586573

  View details for PubMedCentralID PMC4986639

• Aldehyde dehydrogenase 1A3 influences breast cancer progression via differential retinoic acid signaling. Molecular oncology Marcato, P., Dean, C. A., Liu, R., Coyle, K. M., Bydoun, M., Wallace, M., Clements, D., Turner, C., Mathenge, E. G., Gujar, S. A., Giacomantonio, C. A., Mackey, J. R., Godbout, R., Lee, P. W. 2015; 9 (1): 17-31

  Abstract

  Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

  View details for DOI 10.1016/j.molonc.2014.07.010

  View details for PubMedID 25106087

  View details for PubMedCentralID PMC5528683

• Core Needle Biopsy of Breast Cancer Tumors Increases Distant Metastases in a Mouse Model NEOPLASIA Mathenge, E. G., Dean, C. A., Clements, D., Vaghar-Kashani, A., Photopoulos, S., Coyle, K. M., Giacomantonio, M., Malueth, B., Nunokawa, A., Jordan, J., Lewis, J. D., Gujar, S. A., Marcato, P., Lee, P. W., Giacomantonio, C. A. 2014; 16 (11): 950-960

  Abstract

  Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome.To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice.Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4 + T cells, CD8 + T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group.CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.

  View details for DOI 10.1016/j.neo.2014.09.004

  View details for Web of Science ID 000345516900007

  View details for PubMedID 25425969

  View details for PubMedCentralID PMC4240917

• The NAD(+) synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target CELL CYCLE Pan, L., Ahn, D., Sharif, T., Clements, D., Gujar, S. A., Lee, P. W. 2014; 13 (6): 1041-1048

  Abstract

  NAD(+) metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD(+) metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD(+) biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD(+) synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD(+) levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD(+) synthesis is congruent with p53's emerging role as a key regulator of metabolism and related cell fate.

  View details for DOI 10.4161/cc.28128

  View details for Web of Science ID 000336659200024

  View details for PubMedID 24552824

  View details for PubMedCentralID PMC3984302

• Gemcitabine-mediated tumour regression and p53-dependent gene expression: implications for colon and pancreatic cancer therapy CELL DEATH & DISEASE Hill, R., Rabb, M., Madureira, P. A., CLEMENTS, D., Gujar, S. A., Waisman, D. M., Giacomantonio, C. A., Lee, P. W. 2013; 4

  Abstract

  Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g.with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine's mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3σ protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3σ and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.

  View details for DOI 10.1038/cddis.2013.307

  View details for Web of Science ID 000325370300008

  View details for PubMedID 24008735

  View details for PubMedCentralID PMC3789167

• Multifaceted Therapeutic Targeting of Ovarian Peritoneal Carcinomatosis Through Virus-induced Immunomodulation MOLECULAR THERAPY Gujar, S., Dielschneider, R., Clements, D., Helson, E., Shmulevitz, M., Marcato, P., Pan, D., Pan, L., Ahn, D., Alawadhi, A., Lee, P. W. 2013; 21 (2): 338-347

  Abstract

  Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, β2-microglobulin (β2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.

  View details for DOI 10.1038/mt.2012.228

  View details for Web of Science ID 000314434600011

  View details for PubMedID 23299799

  View details for PubMedCentralID PMC3594021