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