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The Hypoxic Tumour Microenvironment: Ets-1 Promotes Hypoxia Inducible Factor- a Target Specificity

The Hypoxic Tumour Microenvironment: Ets-1 Promotes Hypoxia Inducible Factor- a Target Specificity. Chet Holterman, PhD Dr. Stephen Lee. The Hallmarks of Cancer. Self-sufficiency in growth signals. Current genomic and proteomic studies have revealed a broad spectrum of cancer “genotypes”

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The Hypoxic Tumour Microenvironment: Ets-1 Promotes Hypoxia Inducible Factor- a Target Specificity

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

  2. The Hallmarks of Cancer Self-sufficiency in growth signals • 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 • 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 Evading apoptosis Insensitivity to anti-growth signals HIF Tissue invasion & metastasis Sustained angiogenesis Limitless potential Modified from; Hanahan and Weinberg, (2000) Cell pg 58

  3. O2 O2 O2 VHL HIFb O2 O2 :activates genes involved in O2 homeostasis HIFa Regulation of Hypoxia Inducible Genes PHD Cul-2 B C HIFa proteasome HRE VHL targets HIFa for ubiquitination PHDs hydroxylate HIFa

  4. O2 O2 O2 VHL HIFb O2 O2 Regulation of Hypoxia Inducible Genes PHD Cul-2 B C HIFa proteasome HRE ub-HIFa exported to cytoplasm for degradation

  5. VHL HIFb Regulation of Hypoxia Inducible Genes O2 O2 PHD Cul-2 B O2 C proteasome HIFa O2 HRE O2 PHDs are inactivated in low oxygen tension HIFa evades recognition by VHL and binds HIFb

  6. VHL HIFa HIFb Regulation of Hypoxia Inducible Genes O2 PHD Cul-2 B C Glut-1 VEGF MMP TGFa proteasome HRE HIFa evades recognition by VHL and binds HIFb HIF heterodimers activate hypoxia inducible genes

  7. HIF2a is the Oncogenic Variant in Renal Clear Cell Carcinoma • Two HIFaisoforms are expressed in RCC • HIF-1aand HIF-2a • activate unique target genes • HIF-2a is the critical oncogenic isoform: • 1)Stabilization of HIF-2a but not HIF-1ais sufficient to drive tumourigenesis • 2) Silencing of HIF-2a abolishes tumourigenesis in vivo, silencing HIF-1adoes not VHL HIF-2 TGF EGFR Growth Autonomy TUMORIGENESIS Pathway demonstrated in several human cancer cell lines

  8. Understanding HIF2a Oncogenic Activity • How is isoform specificity achieved? (TGFa/EGFR pathway) • interaction with specific co-factors • promoter analysis • co-immunoprecipitation • The role of HIF-2a in post-transcriptional regulation • EGFR and other receptor tyrosine kinases • What are the role of HIFs in the generation/maintenance of tumour initiating cells

  9. TGFa Proximal Promoter Analysis Reveals HRE and EBS ATG kb -2.1 -1.0 -0.5 TGFa Promoter Luciferase

  10. TGFa Proximal Promoter Analysis Reveals HRE and EBS + stable HIF-1a no activity - Endogenous TGFa Expression + stable HIF-2a GFP 3 high activity ++ Ets-1 2.5 Ets-1 DN 2 1.5 Relative Fold Expression + stable HIF-2a and Ets-1 1 0.5 high activity +++ 0 sHIF1 FGFP sHIF2 n=3 + stable HIF-2a - Ets-1 (shRNA or DN) no activity -

  11. Ets-1 and HIF2a Physically Interact and Bind the TGFa Promoter 2 1 HIF-1a Ets-1 HIF-2a ChIP IP 786-0 U87MG In -ve +ve Ets1 HIF2 In -ve +ve Ets1 HIF2 HIF2a HIF1a FLAG Input Input Input GAPDH HIF1a TGFa F1/R1 HIF2a Blot TGFa F2/R2 Ets-1 FGFP FGFP sHIF2 sHIF1 sHIF2 sHIF1 WT7 Infected

  12. Identification of HIF2a Interacting Factors Infect cells with adenovirus to express stable variants of HIF1a or HIF2a FLAG sHIF-1a sHIF-2a RBM4 HIF-2a RBM4 FLAG control FLAG sHIF-1a FLAG sHIF-2a Immunoprecipitate FLAG Elute protein complexes SDS-PAGE/Silver Stain FLAG sHIF1a sHIF2a Isolate unique bands tryptic digest/mass spec enhanced translation EGFR mRNA

  13. Summary • HIF-2a 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-2a • Rbm4:HIF-2a = post-transcriptional regulation of EGFR

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

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