The Hypoxic Tumour Microenvironment:
Ets-1 Promotes Hypoxia Inducible Factora Target Specificity
Chet Holterman, PhD
Dr. Stephen Lee
The Hallmarks of Cancer
Self-sufficiency in
growth signals
•
• RTK – EGFR
Evading
Current genomic and proteomic
apoptosis
studies have revealed a broad
• “genotypes”
Cell cycle regulation – c-myc/cyclin D
spectrum of cancer
•
Insensitivity to
anti-growth signals
no unifying aberrant genetic
theme
• Death (p53) vs. Survival (IGF1/IGF-1R)
HIF
•
Despite their genetic
diversity
• Angiogenesis
– VEGF/VEGFR
cancers share several hallmark
traits required for tumourigenesis
Tissue invasion &
• ECM interaction/degradation
Sustained – Integrins/MMP
metastasis
angiogenesis
•
Limitless
“Stemness” – Oct4/Nanog/ABCG2
potential
Modified from; Hanahan and Weinberg, (2000) Cell pg 58
Regulation of Hypoxia Inducible Genes
O2
PHD
Cul-2
C
O2
B
VHL
O2
HIFa
proteasome
HIFb
O2
HRE
VHL
:activates
HIFa
genes
for
ubiquitination
involved in O2 homeostasis
HIFatargets
PHDs
hydroxylate
HIFa
O2
Regulation of Hypoxia Inducible Genes
O2
O2
PHD
Cul-2
O2
C
B
VHL
HIFa
proteasome
HIFb
O2
HRE
ub-HIFa exported to cytoplasm for degradation
O2
Regulation of Hypoxia Inducible Genes
O2
O2
PHD
Cul-2
O2
C
B
VHL
HIFa
proteasome
HIFb
O2
HRE
PHDsevades
HIFa
are inactivated
recognition
in low
by oxygen
VHL andtension
binds HIFb
O2
Regulation of Hypoxia Inducible Genes
O2
PHD
Cul-2
C
B
VHL
HIFa
HIFb
Glut-1
VEGF
MMP
TGFa
HRE
HIFaheterodimers
evades recognition
VHL and
binds HIFb
HIF
activateby
hypoxia
inducible
genes
proteasome
HIF2a is the Oncogenic Variant in Renal Clear Cell Carcinoma
•
Two HIFa isoforms are expressed
in RCC
–
–
•
HIF-1a and HIF-2a
activate unique target genes
HIF-2a is the critical oncogenic
isoform:
1) Stabilization of HIF-2a but not
HIF-1a is sufficient to drive
tumourigenesis
2) Silencing of HIF-2a abolishes
tumourigenesis in vivo, silencing
HIF-1a does not
VHL
HIF-2a
TGFa
EGFR
Growth Autonomy
TUMORIGENESIS
Pathway demonstrated in several
human cancer cell lines
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
TGFa Proximal Promoter Analysis Reveals HRE and EBS
kb -2.1
-1.0
TGFa Promoter
-0.5
ATG
Luciferase
TGFa Proximal Promoter Analysis Reveals HRE and EBS
+
VHL RCC
+ stable HIF-1a
4
no activity -
Luc
TGFa/Luc
3
2
+ stable HIF-2a
1
high activity ++
GFP
super-HIF1a
super-HIF2a
+ stable HIF-2a and Ets-1
+ + - - - - - + + - - - - - + +
Endogenous TGFa Expression
Relative Fold Expression
high activity +++
+ stable HIF-2a - Ets-1 (shRNA or DN)
no activity n=3
3
2.5
GFP
Ets-1
Ets-1 DN
2
1.5
1
0.5
0
FGFP
sHIF1
sHIF2
Ets-1 and HIF2a Physically Interact and Bind the TGFa Promoter
2
1
HIF-1a
Ets-1
HIF-2a
Input
Input
Input
FLAG
HIF1a
HIF2a
IP
ChIP
In
GAPDH
TGFa F1/R1
HIF2a
TGFa F2/R2
WT7 Infected
FGFP
sHIF1
sHIF2
FGFP
sHIF1
sHIF2
Blot
HIF1a
Ets-1
786-0
-ve +ve Ets1 HIF2
U87MG
In
-ve +ve Ets1 HIF2
Identification of HIF2a Interacting Factors
Infect cells with adenovirus
to express stable variants
of HIF1a or HIF2a
FLAG
sHIF-1a sHIF-2a
RBM4
FLAG control
FLAG sHIF-1a
FLAG sHIF-2a
Immunoprecipitate FLAG
Elute protein complexes
SDS-PAGE/Silver Stain
FLAG sHIF1a sHIF2a
RBM4
enhanced translation
HIF-2a
Isolate unique bands
tryptic digest/mass spec
EGFR mRNA
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
Acknowledgments
Thank you to NOSM and NHRC organizers
S. Lee Lab Members
Funding
Aleksandra Franovic, PhD
Canadian Institute of
Health Research
James Uniacke, PhD
Josianne Payette
Mireille Khacho, PhD
Stephanie Langlois, PhD
Tim Audas, PhD
Gabriel Lachance
Camille Fransisco
Mathieu Jacob
National Cancer Institute
of Canada