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

VHLC

Cul-2

HIFproteasome

PHD

HIF

HRE

VHL targets HIFVHL targets HIF for ubiquitination for ubiquitination PHDs hydroxylate HIFPHDs hydroxylate HIF

O2O2

O2

O2O2

HIF :activates genes involved in O:activates genes involved in O2 2 homeostasishomeostasis

Regulation of Hypoxia Inducible Genes

O2O2

O2

O2O2

B

VHLC

Cul-2

HIF

proteasome

PHD

HIF

HRE

ub-HIFub-HIF exported to cytoplasm for degradation exported to cytoplasm for degradation

Regulation of Hypoxia Inducible Genes

B

VHLC

Cul-2

proteasome

PHD

HIF

HRE

HIF

PHDs are inactivated in low oxygen tensionPHDs are inactivated in low oxygen tension HIFHIF evades recognition by VHL and binds HIF evades recognition by VHL and binds HIF

O2O2

O2

O2O2

Regulation of Hypoxia Inducible Genes

HIFHIF evades recognition by VHL and binds HIF evades recognition by VHL and binds HIF

B

VHLC

Cul-2

proteasome

PHD

HIF HIF

Glut-1VEGFMMPTGF

HRE

HIF heterodimers activate hypoxia inducible genesHIF heterodimers activate hypoxia inducible genes

O2

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 -2.1 -1.0 -0.5

TGF Promoter

ATG

TGF Proximal Promoter Analysis Reveals HRE and EBS

+ stable HIF-1

no activity -

+ stable HIF-2

high activity ++- -+ + - -

- -+ ++ +

- -- - - -

GFP

super-HIF1

super-HIF2

VHL+

RCC

LucTGF/Luc

4

3

2

1

+ stable HIF-2and Ets-1

high activity +++

+ stable HIF-2- Ets-1 (shRNA or DN)

no activity -

n=3

FGFP sHIF2

0.5

0

1

1.5

2

3

2.5

GFPEts-1Ets-1 DN

Rel

ativ

e F

old

Exp

ress

ion

Endogenous TGF Expression

sHIF1

TGF Proximal Promoter Analysis Reveals HRE and EBS

Ets-1 and HIF2 Physically Interact and Bind the TGF Promoter1 2

Ets-1

HIF2

HIF1

HIF

1

HIF

2

FL

AG

sH

IF1

Inp

ut

FG

FP

Inp

ut

Inp

ut

sH

IF2

sH

IF1

FG

FP

sH

IF2

WT7 Infected

Blo

t

IP

HIF-1

HIF-2

Ets-1

Identification of HIF2 Interacting Factors

FLAG sHIF-1 sHIF-2

RBM4

Infect cells with adenovirusto express stable variants of HIF1 or HIF2

FLAG control FLAG sHIF-1 FLAG sHIF-2

Immunoprecipitate FLAG

SDS-PAGE/Silver Stain

Isolate unique bandstryptic digest/mass spec

FLAG sHIF1 sHIF2

Elute protein complexes

HIF-2enhanced 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