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

slide4

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

slide5

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

slide6

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

slide7

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
slide9

TGFa Proximal Promoter Analysis Reveals HRE and EBS

ATG

kb

-2.1

-1.0

-0.5

TGFa Promoter

Luciferase

slide10

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 -

slide11

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
slide14

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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