Biology of Human Tumors VEGF / VEGFR-2 Upregulates EZH 2 Expression in Lung Adenocarcinoma Cells and EZH 2 Depletion Enhances the Response to Platinum-Based and VEGFR-2 – Targeted Therapy

Purpose: To investigate the mechanisms of regulation and role associated with enhancer of zeste homolog 2 (EZH2) expression in lung cancer cells. Experimental Design:We investigated the mechanisms of EZH2 expression associated with the VEGF/ VEGFR-2 pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. In addition, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients’ clinical characteristics. Results: In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 andhypoxia-inducible factor-1a (HIF1a), and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. In addition, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patientswhodidnot receive this therapy. Furthermore,wedemonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti– VEGFR-2 drug AZD2171. Conclusion: Our results suggest that the VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF1a, and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy. Clin Cancer Res; 20(14); 3849–61. 2014 AACR.

[1]  John D. Minna,et al.  Human Lung Epithelial Cells Progressed to Malignancy through Specific Oncogenic Manipulations , 2013, Molecular Cancer Research.

[2]  Yoon-Jae Cho,et al.  The VEGF pathway in cancer and disease: responses, resistance, and the path forward. , 2012, Cold Spring Harbor perspectives in medicine.

[3]  M. Nishimura,et al.  Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells. , 2012, Lung cancer.

[4]  Huqun,et al.  Enhancer of zeste homolog 2 is a novel prognostic biomarker in nonsmall cell lung cancer , 2012, Cancer.

[5]  Qiang Yu,et al.  Polycomb protein EZH2 regulates cancer cell fate decision in response to DNA damage , 2011, Cell Death and Differentiation.

[6]  F. Liu,et al.  Polycomb Repressor Complex-2 Is a Novel Target for Mesothelioma Therapy , 2011, Clinical Cancer Research.

[7]  A. Ochiai,et al.  MEK–ERK pathway regulates EZH2 overexpression in association with aggressive breast cancer subtypes , 2011, Oncogene.

[8]  K. Coombes,et al.  Increased VEGFR-2 gene copy is associated with chemoresistance and shorter survival in patients with non-small-cell lung carcinoma who receive adjuvant chemotherapy. , 2011, Cancer research.

[9]  P. Tsichlis,et al.  FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. , 2011, Molecular cell.

[10]  Donglei Liu,et al.  MicroRNA-101 Exerts Tumor-Suppressive Functions in Non-small Cell Lung Cancer through Directly Targeting Enhancer of Zeste Homolog 2 , 2011, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[11]  T. Wurdinger,et al.  Down-Regulation of miR-101 in Endothelial Cells Promotes Blood Vessel Formation through Reduced Repression of EZH2 , 2011, PloS one.

[12]  W. Woodward,et al.  EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-β-catenin signaling. , 2011, Cancer cell.

[13]  Lesley A. Mathews,et al.  Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer , 2011, Molecular Cancer.

[14]  Laura A. Sullivan,et al.  Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. , 2010, Cancer research.

[15]  B. Tannous,et al.  miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis , 2010, Oncotarget.

[16]  G. Calin,et al.  Regulation of tumor angiogenesis by EZH2. , 2010, Cancer cell.

[17]  A. Sood,et al.  Collateral damage: toxic effects of targeted antiangiogenic therapies in ovarian cancer. , 2010, The Lancet. Oncology.

[18]  Qiang Yu,et al.  Polycomb protein EZH2 regulates E2F1-dependent apoptosis through epigenetically modulating Bim expression , 2010, Cell Death and Differentiation.

[19]  D. Small,et al.  Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML. , 2010, Blood.

[20]  A. Harris,et al.  Assessment of tumour hypoxia for prediction of response to therapy and cancer prognosis , 2009, Journal of cellular and molecular medicine.

[21]  Liz Y. Han,et al.  Functional significance of VEGFR‐2 on ovarian cancer cells , 2009, International journal of cancer.

[22]  S. Dhanasekaran,et al.  Repression of E-cadherin by the polycomb group protein EZH2 in cancer , 2008, Oncogene.

[23]  R. Weissleder,et al.  miR-296 regulates growth factor receptor overexpression in angiogenic endothelial cells. , 2008, Cancer cell.

[24]  A. Ochiai,et al.  Enhancer of zeste homolog 2 downregulates E‐cadherin by mediating histone H3 methylation in gastric cancer cells , 2008, Cancer science.

[25]  S. Dhanasekaran,et al.  Integrative genomics analysis reveals silencing of beta-adrenergic signaling by polycomb in prostate cancer. , 2007, Cancer cell.

[26]  Kristian Helin,et al.  The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. , 2007, Genes & development.

[27]  K. Uematsu,et al.  Prognostic value of expression of vascular endothelial growth factor and its flt-1 and KDR receptors in stage I non-small-cell lung cancer. , 2006, Lung cancer.

[28]  Elisabeth Brambilla,et al.  The 2004 World Health Organization classification of lung tumors. , 2005, Seminars in roentgenology.

[29]  H. Minn,et al.  [Tumor hypoxia:advantage or disadvantage?]. , 2005, Duodecim; laaketieteellinen aikakauskirja.

[30]  M. Tonato,et al.  Vascular endothelial growth factor, p53, Rb, Bcl-2 expression and response to chemotherapy in advanced non-small cell lung cancer. , 2004, Lung cancer.

[31]  T. Yazawa,et al.  Enhancement of pleural dissemination and lymph node metastasis of intrathoracic lung cancer cells by vascular endothelial growth factors (VEGFs). , 2004, Lung cancer.

[32]  Kristian Helin,et al.  EZH2 is downstream of the pRB‐E2F pathway, essential for proliferation and amplified in cancer , 2003, The EMBO journal.

[33]  G. Semenza.  Targeting HIF-1 for cancer therapy , 2003, Nature Reviews Cancer.

[34]  Debashis Ghosh,et al.  EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Wahlestedt,et al.  Small GTP-Binding Protein Rac Is an Essential Mediator of Vascular Endothelial Growth Factor-Induced Endothelial Fenestrations and Vascular Permeability , 2003, Circulation.

[36]  Weicheng Wu,et al.  Sphingosine Kinase Mediates Vascular Endothelial Growth Factor-Induced Activation of Ras and Mitogen-Activated Protein Kinases , 2002, Molecular and Cellular Biology.

[37]  S. Dhanasekaran,et al.  The polycomb group protein EZH2 is involved in progression of prostate cancer , 2002, Nature.

[38]  Hengbin Wang,et al.  Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing , 2002, Science.

[39]  C. Angeletti,et al.  A high vascular count and overexpression of vascular endothelial growth factor are associated with unfavourable prognosis in operated small cell lung carcinoma , 2002, British Journal of Cancer.

[40]  Adrian L. Harris,et al.  Hypoxia — a key regulatory factor in tumour growth , 2002, Nature Reviews Cancer.

[41]  Lena Claesson-Welsh,et al.  VEGF Receptor Signal Transduction , 2001, Science's STKE.

[42]  D. Hanahan,et al.  Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. , 1999, Science.

[43]  M. Shibuya,et al.  VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells , 1999, Oncogene.

[44]  C. Angeletti,et al.  Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas , 1999, British Journal of Cancer.

[45]  G. Semenza,et al.  Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 , 1996, Molecular and cellular biology.