The hypoxic tumour microenvironment ets 1 promotes hypoxia inducible factor a target specificity
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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

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

Chet Holterman, PhD

Dr. Stephen Lee


The hallmarks of cancer

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


Regulation of hypoxia inducible genes

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


Chet holterman phd dr stephen lee

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


Chet holterman phd dr stephen lee

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


Chet holterman phd dr stephen lee

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


Chet holterman phd dr stephen lee

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


Understanding hif2 a oncogenic activity

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


  • Chet holterman phd dr stephen lee

    TGFa Proximal Promoter Analysis Reveals HRE and EBS

    ATG

    kb

    -2.1

    -1.0

    -0.5

    TGFa Promoter

    Luciferase


    Chet holterman phd dr stephen lee

    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 -


    Chet holterman phd dr stephen lee

    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


    Identification of hif2 a interacting factors

    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


    Summary

    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


  • Chet holterman phd dr stephen lee

    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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