Haiyu Zhang

All Publications

• The highs and lows of cyclic thrombocytopenia. British journal of haematology Zhang, H., Villar-Prados, A., Bussel, J. B., Zehnder, J. L. 2023

  Abstract

  Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.

  View details for DOI 10.1111/bjh.19239

  View details for PubMedID 38083878

• The role of CD8+ T cell clones in immune thrombocytopenia. Blood Malik, A., Sayed, A. A., Han, P., Tan, M. M., Watt, E., Constantinescu-Bercu, A., Cocker, A. T., Khoder, A., Saputil, R. C., Thorley, E. V., Teklemichael, A., Ding, Y., Hart, A. C., Zhang, H., Mitchell, W. A., Imami, N., Crawley, J. T., Salles-Crawley, I. I., Bussel, J. B., Zehnder, J. L., Adams, S. P., Zhang, B. M., Cooper, N. 2023

  Abstract

  Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

  View details for DOI 10.1182/blood.2022018380

  View details for PubMedID 36749920

• SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19. Journal of neuropathology and experimental neurology Serrano, G. E., Walker, J. E., Tremblay, C., Piras, I. S., Huentelman, M. J., Belden, C. M., Goldfarb, D., Shprecher, D., Atri, A., Adler, C. H., Shill, H. A., Driver-Dunckley, E., Mehta, S. H., Caselli, R., Woodruff, B. K., Haarer, C. F., Ruhlen, T., Torres, M., Nguyen, S., Schmitt, D., Rapscak, S. Z., Bime, C., Peters, J. L., Alevritis, E., Arce, R. A., Glass, M. J., Vargas, D., Sue, L. I., Intorcia, A. J., Nelson, C. M., Oliver, J., Russell, A., Suszczewicz, K. E., Borja, C. I., Cline, M. P., Hemmingsen, S. J., Qiji, S., Hobgood, H. M., Mizgerd, J. P., Sahoo, M. K., Zhang, H., Solis, D., Montine, T. J., Berry, G. J., Reiman, E. M., Roltgen, K., Boyd, S. D., Pinsky, B. A., Zehnder, J. L., Talbot, P., Desforges, M., DeTure, M., Dickson, D. W., Beach, T. G. 2022

  Abstract

  Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

  View details for DOI 10.1093/jnen/nlac056

  View details for PubMedID 35818336

• Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3, and MPL mutations. Blood advances Zhang, H., Chien, M., Hou, Y., Shomali, W., Brar, R., Ho, C., Han, P., Xu, D., Zhang, B. M., Guo, X., Tolentino, L., Wu, N. C., Tsai, A. G., Jin, J., Witteles, W. H., Chen, Z., Abidi, P., Jangam, D., Krieger, M. S., Craig, M., Bussel, J. B., Gotlib, J. R., Zehnder, J. L. 2022

  Abstract

  Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to two patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

  View details for DOI 10.1182/bloodadvances.2021006701

  View details for PubMedID 35381066

• A Blueprint for Identifying Phenotypes and Drug Targets in Complex Disorders with Empirical Dynamics. Patterns (New York, N.Y.) Krieger, M. S., Moreau, J. M., Zhang, H., Chien, M., Zehnder, J. L., Craig, M. 2020; 1 (9): 100138

  Abstract

  A central challenge in medicine is translating from observational understanding to mechanistic understanding, where some observations are recognized as causes for the others. This can lead not only to new treatments and understanding, but also to recognition of novel phenotypes. Here, we apply a collection of mathematical techniques (empirical dynamics), which infer mechanistic networks in a model-free manner from longitudinal data, to hematopoiesis. Our study consists of three subjects with markers for cyclic thrombocytopenia, in which multiple cells and proteins undergo abnormal oscillations. One subject has atypical markers and may represent a rare phenotype. Our analyses support this contention, and also lend new evidence to a theory for the cause of this disorder. Simulations of an intervention yield encouraging results, even when applied to patient data outside our three subjects. These successes suggest that this blueprint has broader applicability in understanding and treating complex disorders.

  View details for DOI 10.1016/j.patter.2020.100138

  View details for PubMedID 33336196

• Investigating circulating tumor cells anddistant metastases in patient-derivedorthotopic xenograft models of triple-negative breast cancer Breast Cancer Research Ramani, V., Lemaire, C., Triboulet, M., Casey, K., Heirich, K., Renier, C., Vilches-Moure, J., Gupta, R., Razmara, A., Zhang, H., Sledge, G., Sollier, E., Jeffrey, S. 2019; 21

  View details for DOI 10.1186/s13058-019-1182-4

• Blood transcriptome and clonal T cell correlates of response and nonresponse to eltrombopag therapy in a cohort of patients with chronic immune thrombocytopenia. Haematologica Zhang, H., Zhang, B., Guo, X., Xu, L., You, X., West, R., Bussel, J., Zehnder, J. 2019

  View details for DOI 10.3324/haematol.2019.226688

• Identification of a Novel MPL Loss of Function Mutation in a Patient with Cyclic Thrombocytopenia and Characterization of This Syndrome Zhang, H., Hou, Y., Brar, R. S., Zhang, B., Chen, Z., Abidi, P., Jin, J., Gotlib, J. R., Zehnder, J. L. AMER SOC HEMATOLOGY. 2016

  View details for Web of Science ID 000394452300114

• Gender and duration of disease differentiate responses to rituximab-dexamethasone therapy in adults with immune thrombocytopenia AMERICAN JOURNAL OF HEMATOLOGY Chapin, J., Lee, C. S., Zhang, H., Zehnder, J. L., Bussel, J. B. 2016; 91 (9): 907–11

  Abstract

  Adults often develop chronic immune thrombocytopenia (ITP) for which treatment order is uncertain. Rituximab and three cycles of dexamethasone (4R + 3Dex) improve treatment responses and short-term disease control but long-term outcome is not known. In adults with ITP treated with 4R + 3D, we sought long-term outcome and associated prognostic variables. Forty-nine adults treated at Weill-Cornell received 4R + 3Dex. Their clinical characteristics were reviewed. Duration was median time to treatment failure; Kaplan-Meier estimates were developed. Vbeta Tcell receptor (VBTCR) repertoire was obtained after treatment in 36 patients. Patients were adults with ITP 18-64 years old, median age 37. The 27 females were twice as likely to have an ongoing response to 4R + 3Dex (44.1%) as males (19.6%; P = 0.009). For ITP duration <12 months, 52.7% of patients had continuing responses to 4R + 3Dex compared to 15.3% of patients with diagnosis >12 months (P = 0.02). Females with ITP duration of <12 months had continuing responses in 78.6%, compared to males with <12 months duration of ITP (21.2%). For patients with disease duration <12 months, 67% of females had continuing responses, compared to 31% of males (P = 0.004). Post-treatment polyclonal VBTCR was seen in 9/10 continuing responders (six female, three male) but only 13/26 relapsers/nonresponders (P = 0.068). Durable remissions after treatment with 4R + 3Dex were more frequent in female patients with <12 months of ITP duration and those with polyclonal VBTCR after treatment, emphasizing the roles of duration of disease, gender and T cells in chronic ITP. Differences in pathophysiology of ITP by gender and by duration of ITP require further study. Am. J. Hematol. 91:907-911, 2016. © 2016 Wiley Periodicals, Inc.

  View details for PubMedID 27220625

• Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms. Blood Hinds, D. A., Barnholt, K. E., Mesa, R. A., Kiefer, A. K., Do, C. B., Eriksson, N., Mountain, J. L., Francke, U., Tung, J. Y., Nguyen, H. M., Zhang, H., Gojenola, L., Zehnder, J. L., Gotlib, J. 2016; 128 (8): 1121-1128

  Abstract

  We conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10(-89)), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10(-32)), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10(-14)), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10(-9)). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm.

  View details for DOI 10.1182/blood-2015-06-652941

  View details for PubMedID 27365426

• Effects of Thrombopoietin Mimetics on Patients with Chronic ITP: Perspectives from Blood Transcriptome Profiling Analysis Zhang, H., Zhang, B., Guo, X., Haq, N., West, R. B., Bussel, J. B., Zehnder, J. L. AMER SOC HEMATOLOGY. 2014

  View details for Web of Science ID 000349233800107

• Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow BMC CANCER Deng, G., Krishnakumar, S., Powell, A. A., Zhang, H., Mindrinos, M. N., Telli, M. L., Davis, R. W., Jeffrey, S. S. 2014; 14

  Abstract

  Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes.We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for known hotspot mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations.Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture.Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

  View details for DOI 10.1186/1471-2407-14-456

  View details for Web of Science ID 000338965100001

• Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition BREAST CANCER RESEARCH Zhang, H., Cohen, A. L., Krishnakumar, S., Wapnir, I. L., Veeriah, S., Deng, G., Coram, M. A., Piskun, C. M., Longacre, T. A., Herrler, M., Frimannsson, D. O., Telli, M. L., Dirbas, F. M., Matin, A. C., Dairkee, S. H., Larijani, B., Glinsky, G. V., Bild, A. H., Jeffrey, S. S. 2014; 16 (2)

  View details for DOI 10.1186/bcr3640

  View details for Web of Science ID 000338990900021

• CD137 Ligand Is Expressed in Primary and Secondary Lymphoid Follicles and in B-cell Lymphomas Diagnostic and Therapeutic Implications AMERICAN JOURNAL OF SURGICAL PATHOLOGY Zhao, S., Zhang, H., Xing, Y., Natkunam, Y. 2013; 37 (2): 250-258

  Abstract

  CD137 ligand (4-1BB ligand, TNFSF9, CD137L) is a member of the tumor necrosis factor family whose binding to its receptor, CD137 (4-1BB, TNFRSF9), mediates costimulatory and prosurvival signals necessary for T-cell activation and regulation of humoral immune responses. Recent studies have shown that anti-CD137 immunotherapy has promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. Here, we define the tissue expression profile of CD137L, which has not been previously explored. We characterized the expression of CD137L in normal and neoplastic human hematopoietic and nonhematopoietic tissue and found that CD137L is preferentially expressed in B cells of the primary follicles, mantle zones of the secondary follicles, germinal centers, and in normal endothelial cells. Double immunofluorescence labeling in tissue sections and flow cytometry analysis further showed that CD137L is a potential new marker of memory B cells. Evaluation of over 700 human hematopoietic tumors revealed that the majority of B-cell lymphomas expressed CD137L, which include mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. In contrast, CD137L expression was lacking in Hodgkin lymphoma and T-cell lymphoma. Our findings suggest that CD137L is a novel diagnostic marker of subtypes of non-Hodgkin B-cell lymphomas and raise the possibility that its expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and other malignancies.

  View details for DOI 10.1097/PAS.0b013e318268c6ea

  View details for Web of Science ID 000313576200012

  View details for PubMedID 23095505

• Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines PLOS ONE Powell, A. A., Talasaz, A. H., Zhang, H., Coram, M. A., Reddy, A., Deng, G., Telli, M. L., Advani, R. H., Carlson, R. W., Mollick, J. A., Sheth, S., Kurian, A. W., Ford, J. M., Stockdale, F. E., Quake, S. R., Pease, R. F., Mindrinos, M. N., Bhanot, G., Dairkee, S. H., Davis, R. W., Jeffrey, S. S. 2012; 7 (5)

  Abstract

  To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.

  View details for DOI 10.1371/journal.pone.0033788

  View details for PubMedID 22586443

• A pharmacogenomic method for individualized prediction of drug sensitivity MOLECULAR SYSTEMS BIOLOGY Cohen, A. L., Soldi, R., Zhang, H., Gustafson, A. M., Wilcox, R., Welm, B. E., Chang, J. T., Johnson, E., Spira, A., Jeffrey, S. S., Bild, A. H. 2011; 7

  Abstract

  Identifying the best drug for each cancer patient requires an efficient individualized strategy. We present MATCH (Merging genomic and pharmacologic Analyses for Therapy CHoice), an approach using public genomic resources and drug testing of fresh tumor samples to link drugs to patients. Valproic acid (VPA) is highlighted as a proof-of-principle. In order to predict specific tumor types with high probability of drug sensitivity, we create drug response signatures using publically available gene expression data and assess sensitivity in a data set of >40 cancer types. Next, we evaluate drug sensitivity in matched tumor and normal tissue and exclude cancer types that are no more sensitive than normal tissue. From these analyses, breast tumors are predicted to be sensitive to VPA. A meta-analysis across breast cancer data sets shows that aggressive subtypes are most likely to be sensitive to VPA, but all subtypes have sensitive tumors. MATCH predictions correlate significantly with growth inhibition in cancer cell lines and three-dimensional cultures of fresh tumor samples. MATCH accurately predicts reduction in tumor growth rate following VPA treatment in patient tumor xenografts. MATCH uses genomic analysis with in vitro testing of patient tumors to select optimal drug regimens before clinical trial initiation.

  View details for DOI 10.1038/msb.2011.47

  View details for Web of Science ID 000293351900012

  View details for PubMedID 21772261

  View details for PubMedCentralID PMC3159972

• Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype BRITISH JOURNAL OF CANCER Ameri, K., Luong, R., Zhang, H., Powell, A. A., Montgomery, K. D., Espinosa, I., Bouley, D. M., Harris, A. L., Jeffrey, S. S. 2010; 102 (3): 561-569

  Abstract

  Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs.We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared.MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts.CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.

  View details for DOI 10.1038/sj.bjc.6605491

  View details for Web of Science ID 000274194700015

  View details for PubMedID 20051957

  View details for PubMedCentralID PMC2805847

• RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies BMC MOLECULAR BIOLOGY Ribeiro-Silva, A., Zhang, H., Jeffrey, S. S. 2007; 8

  Abstract

  The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp.Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86-100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp.All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

  View details for DOI 10.1186/1471-2199-8-118

  View details for Web of Science ID 000253110900001

  View details for PubMedID 18154675

  View details for PubMedCentralID PMC2233637

• Polyphosphate kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum with a role in cytokinesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, H., Gomez-Garcia, M. R., Shi, X., Rao, N. N., Kornberg, A. 2007; 104 (42): 16486-16491

  Abstract

  Polyphosphate kinase 1 (PPK1), the principal enzyme responsible for reversible synthesis of polyphosphate (poly P) from the terminal phosphate of ATP, is highly conserved in bacteria and archaea. Dictyostelium discoideum, a social slime mold, is one of a few eukaryotes known to possess a PPK1 homolog (DdPPK1). Compared with PPK1 of Escherichia coli, DdPPK1 contains the conserved residues for ATP binding and autophosphorylation, but has an N-terminal extension of 370 aa, lacking homology with any known protein. Polyphosphate or ATP promote oligomerization of the enzyme in vitro. The DdPPK1 products are heterogeneous in chain length and shorter than those of E. coli. The unique DdPPK1 N-terminal domain was shown to be necessary for its enzymatic activity, cellular localization, and physiological functions. Mutants of DdPPK1, as previously reported, are defective in development, sporulation, and predation, and as shown here, in late stages of cytokinesis and cell division.

  View details for DOI 10.1073/pnas.0706847104

  View details for Web of Science ID 000250373400020

  View details for PubMedID 17940044

  View details for PubMedCentralID PMC2034253

• Inorganic polyphosphate in the social life of Myxococcus xanthus: Motility, development, and predation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, H. Y., Rao, N. N., Shiba, T., Kornberg, A. 2005; 102 (38): 13416-13420

  Abstract

  Inorganic polyphosphate (poly P), a polymer of tens or hundreds of phosphate residues linked by high-energy, ATP-like bonds, is found in all organisms and performs a wide variety of functions. Myxococcus xanthus, a social bacterium that feeds on other bacteria and forms fruiting bodies and spores, depends on poly P for motility, development, and nutritional predation. Two poly P metabolizing enzymes were studied in M. xanthus: poly P kinase 1, which synthesizes poly P reversibly from ATP, and poly P:AMP phosphotransferase, which uses poly P as a donor to also reversibly convert AMP to ADP. The null mutant of ppk1 is defective in social motility, overproduces pilin protein on the cell surface, is delayed in fruiting body formation, produces fewer spores, is delayed in germination, and forms far smaller plaques on a lawn of Klebsiella aerogenes. The pap mutant is also impaired in social motility, but shows only slightly reduced abilities in development and predation.

  View details for DOI 10.1073/pnas.0506520102

  View details for Web of Science ID 000232115100013

  View details for PubMedID 16174737

  View details for PubMedCentralID PMC1224657

• Inorganic polyphosphate in Dictyostelium discoideum: Influence on development, sporulation, and predation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, H. Y., Gomez-Garcia, M. R., Brown, M. R., Kornberg, A. 2005; 102 (8): 2731-2735

  Abstract

  Dictyostelium discoideum, a social slime mold that forms fruiting bodies with spores, depends on inorganic polyphosphate (poly P) for its cycles of development and for nutritional predation on bacteria. The synthesis of poly P, a polymer of tens or hundreds of phosphate residues linked by high energy, ATP-like bonds, is catalyzed in most bacteria by poly P kinase (PPK1). The eukaryote D. discoideum possesses a homolog of PPK1. We report here that mutants of D. discoideum PPK1 (DdPPK1) have reduced levels of poly P and are deficient in development. Fruiting bodies are smaller and produce fewer spores, which appear to germinate like the wild type (WT). The DdPPK1 mutant formed smaller plaques on bacterial lawns compared with those of the WT. Predation by D. discoideum, assessed by uptake and digestion of Klebsiella aerogenes, showed that fewer bacteria were taken up by the DdPPK1 mutant compared with the WT and were killed less rapidly, indicating a role of poly P and/or DdPPK1 in phagocytosis. On Pseudomonas aeruginosa lawns, cleared plaques were observed with the bacterial PPK1 mutant but not with the WT P. aeruginosa. Thus, poly P is important in predation both for the predator and prey.

  View details for DOI 10.1073/pnas.0500023102

  View details for Web of Science ID 000227232400014

  View details for PubMedID 15701689

  View details for PubMedCentralID PMC549442

• Polyphosphate is essential for Pseudomonas aeruginosa functions. International Pseudomonas Meeting Zhang, H., Ishige, K., Rashid, M., Kornberg, A. 2003
• A polyphosphate kinase (PPK2) widely conserved in bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, H. Y., Ishige, K., Kornberg, A. 2002; 99 (26): 16678-16683

  Abstract

  Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli. PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa. In a null mutant of P. aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP. The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity. The 5'-end is 177 bp upstream of the annotated genome sequence of a "conserved hypothetical protein"; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa. Sequences homologous to PPK2 are present in two other proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species. Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report.

  View details for DOI 10.1073/pnas.262655199

  View details for Web of Science ID 000180101600033

  View details for PubMedID 12486232

  View details for PubMedCentralID PMC139203

• Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ishige, K., Zhang, H. Y., Arthur, K. 2002; 99 (26): 16684-16688

  Abstract

  An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5. Poly P chains of 30-50 residues are optimal; those of 15-700 residues can also serve. GDP is preferred over ADP among nucleoside diphosphate acceptors. This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report. The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features. Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis. PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa.

  View details for DOI 10.1073/pnas.262655299

  View details for Web of Science ID 000180101600034

  View details for PubMedID 12482933

  View details for PubMedCentralID PMC139204

• The cloning and functional analysis of Sinorhizobium fredii 042BS regulatory nodulation genes. Acta Microbiologica Sinica Zhang, H., Zhang, H., Li, X., Wen, S., Yang, S. 2002; 42: 33-39
• Sinorhizobium fredii strain that effectively nodulate Medicago sativa. Acta Microbiologica Sinica Zhang, H., Zhang, H., Li, X., Yang, S. 2001; 41: 127-132
• Cloning of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B Acta Microbiologica sinica Chen , X., Zhang, H., Gao, W., Zhu, K., Kan, F., Yang, S. 1999; 39: 14-19
• The cloning and physical mapping of Sinorhizobium meliloti 042B DNA fragment related to common nodulation genes nodABC J. Agricultural Biotechnology Yang , X., Zhang, H., Yang, S. 1998; 6: 319-326