The Hypoxic Tumour Microenvironment: Ets-1 Promotes Hypoxia Inducible Factor-  Target Specificity Chet Holterman, PhD Dr. Stephen Lee

The Hallmarks of Cancer Modified from; Hanahan and Weinberg, (2000) Cell pg 58 Self-sufficiency in growth signals Insensitivity to anti-growth signals Evading apoptosis Sustained angiogenesis Tissue invasion & metastasis Limitless potential HIF RTK – EGFR Cell cycle regulation – c-myc/cyclin D Death (p53) vs. Survival (IGF1/IGF-1R) Angiogenesis – VEGF/VEGFR ECM interaction/degradation – Integrins/MMP “Stemness” – Oct4/Nanog/ABCG2 Current genomic and proteomic studies have revealed a broad spectrum of cancer “genotypes” no unifying aberrant genetic theme Despite their genetic diversity cancers share several hallmark traits required for tumourigenesis

B VHL C Cul-2 HIF  proteasome PHD HIF  HRE VHL targets HIF  for ubiquitination VHL targets HIF  for ubiquitination PHDs hydroxylate HIF  PHDs hydroxylate HIF  O2O2 O2O2 O2O2 O2O2 O2O2 HIF  :activates genes involved in O 2 homeostasis :activates genes involved in O 2 homeostasis Regulation of Hypoxia Inducible Genes

O2O2 O2O2 O2O2 O2O2 O2O2 B VHL C Cul-2 HIF  proteasome PHD HIF  HRE ub-HIF  exported to cytoplasm for degradation Regulation of Hypoxia Inducible Genes

B VHL C Cul-2 proteasome PHD HIF  HRE HIF  PHDs are inactivated in low oxygen tension PHDs are inactivated in low oxygen tension HIF  evades recognition by VHL and binds HIF  HIF  evades recognition by VHL and binds HIF  O2O2 O2O2 O2O2 O2O2 O2O2 Regulation of Hypoxia Inducible Genes

HIF  evades recognition by VHL and binds HIF  HIF  evades recognition by VHL and binds HIF  B VHL C Cul-2 proteasome PHD HIF  HIF  Glut-1 VEGF MMP TGF  HRE HIF heterodimers activate hypoxia inducible genes HIF heterodimers activate hypoxia inducible genes O2O2 Regulation of Hypoxia Inducible Genes

Two HIF  isoforms are expressed in RCC –HIF-1  and HIF-2  –activate unique target genes HIF-2  is the critical oncogenic isoform: 1) Stabilization of HIF-2  but not HIF-1  is sufficient to drive tumourigenesis 2) Silencing of HIF-2  abolishes tumourigenesis in vivo, silencing HIF-1  does not HIF2  is the Oncogenic Variant in Renal Clear Cell Carcinoma VHL HIF-2  TGF  EGFR Growth Autonomy TUMORIGENESIS Pathway demonstrated in several human cancer cell lines

Understanding HIF2  Oncogenic Activity How is isoform specificity achieved? (TGF  /EGFR pathway) interaction with specific co-factors »promoter analysis »co-immunoprecipitation The role of HIF-2  in post-transcriptional regulation EGFR and other receptor tyrosine kinases What are the role of HIFs in the generation/maintenance of tumour initiating cells

Luciferase kb TGF  Promoter ATG TGF  Proximal Promoter Analysis Reveals HRE and EBS

+ stable HIF-1  no activity - + stable HIF-2  high activity ++ + stable HIF-2  and Ets-1 high activity stable HIF-2  - Ets-1 (shRNA or DN) no activity - n=3 FGFPsHIF GFP Ets-1 Ets-1 DN Relative Fold Expression Endogenous TGF  Expression sHIF1 TGF  Proximal Promoter Analysis Reveals HRE and EBS

Ets-1 and HIF2  Physically Interact and Bind the TGF  Promoter 1 2 Ets-1 HIF2  HIF1  HIF2  FLAG sHIF1 Input FGFP Input sHIF2sHIF1 FGFP sHIF2 WT7 Infected Blot IP ChIP In GAPDH TGF  F2/R2 TGF  F1/R1 -ve+veEts1HIF2In-ve+veEts1HIF U87MG HIF-1  HIF-2  Ets-1

Identification of HIF2  Interacting Factors FLAG sHIF-1  sHIF-2  RBM4 Infect cells with adenovirus to express stable variants of HIF1  or HIF2  FLAG control FLAG sHIF-1  FLAG sHIF-2  Immunoprecipitate FLAG SDS-PAGE/Silver Stain Isolate unique bands tryptic digest/mass spec FLAG sHIF1  sHIF2  Elute protein complexes HIF-2  RBM4 enhanced translation EGFR mRNA

Summary HIF-2  activates a unique repertoire of target genes achieved through interaction with other transcription factors –Ets-1 –???? Interactions with protein co-factors may explain non-canonical functions of HIF-2  Rbm4:HIF-2  = post-transcriptional regulation of EGFR

Acknowledgments FundingS. Lee Lab Members Tim Audas, PhD Josianne Payette Aleksandra Franovic, PhD Mireille Khacho, PhD Stephanie Langlois, PhD Gabriel Lachance James Uniacke, PhD Canadian Institute of Health Research National Cancer Institute of Canada Camille Fransisco Mathieu Jacob Thank you to NOSM and NHRC organizers