Download Normoxia

Estrogen receptor-α directly regulates the
hypoxia inducible factor 1 pathway associated
with antiestrogen response in breast cancer
PNAS 2015 112(49) 15172-15177
Speaker：Wu Po-Huei
Adviser : Jung-Yie Kao
Date：2016.11.01
2
Introduction
Estrogen receptor-α (ERα )
Estrogen
Estrogen receptor α
Estrogen receptor α dimer
(Transcription factor)
Estrogen response element
Mol Endocrinol 20(8):1707–1714
3
Tamoxifen (Tam)
CoR = Corepressor
HDAC = Histone deacetylase
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Mol Endocrinol 16(8) : 1778–1792
Hypoxia inducible factor 1(HIF-1α) 5
Normoxia
Proc Natl Acad Sci USA 100(11):6517–6522
ERα and HIF-1α
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We have previously shown that HIF-1α and ERα can coordinate
expression of genes, such as lysine-specific demethylase
4B/Jumonji domain-containing 2B (KDM4B/JMJD2B), an
H3K9me3/me2 histone demethylase, which is targeted by both
ERα and HIF-1α and epigenetically regulates cell cycle
progression. The genomic locus of KDM4B bears both HIF-1α
and ERα binding elements.
Cancer Res 70(16):6456–6466
Q : The role of ERα in the regulation of HIF-1 signaling
Q : How HIF-1 signaling is involved in
endocrine drug response
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Results
ERα signaling regulates hypoxia/HIF-1α pathway
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We have previously shown that knockdown of ERα
significantly down-regulated histone demethylase KDM4B
expression, a HIF-1α transcriptional target.
J Biol Chem 283(52):36542–36552
ERα signaling regulates hypoxia/HIF-1α pathway
ER antagonist
MCF7
N
C
H
ICI
24hr
C
ICI
(ICI182780 : 1μM)
Extracted RNA
Microarray
Global gene-expression
profile analysis
Dually responsive gene
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ERα signaling regulates hypoxia/HIF-1α pathway
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Library of Integrated
Network-based Cellular
Signatures
 Conclusion :
ERα
ERβ
HIF-1
These data
indicate that a
subgroup of
genes that are
targeted by
hypoxia/HIF-1α is
also regulated by
ERα signaling
DNA-protein
Cross-linking
ChIP
Chromatin Immunoprecipitation
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Cell lysis
Sonication or
enzyme digestion
Protein
Fragmented
chromatin
Immunoprecipitation
with specific antibody
DNA purification
Analysis of
bound DNA
PCR
qPCR
Sequencing
Microarray
ERα signaling regulates hypoxia/HIF-1α pathway
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Library of Integrated
Network-based Cellular
Signatures
 Conclusion :
ERα
ERβ
HIF-1
These data
indicate that a
subgroup of
genes that are
targeted by
hypoxia/HIF-1α is
also regulated by
ERα signaling
ERα and HIF-1α directly bind their response elements
in a subgroup of genes
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High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq
Blood 117(23):e207–e217
A CTCF-independent role for cohesin in tissue-specific transcription
Genome Res 20(5):578–588
Kyoto Encyclopedia of
Genes and Genomes
 Conclusion : We found that among the 356 genes bound by
HIF-1α, 202 (57%) of them were identified as the
common genes bound by ERα as well
Estrogen regulates HIF-1α expression
Hypoxia mimetic
Deferoxifine
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MCF7
N
C
E2
DFO
H
500μm
1%O2
C
E2
C
E2
(Estrogen : 100nm)
6hr
Western blot
 Conclusion : E2 greatly enhanced HIF-1α expression in hypoxia
ERα signaling regulates HIF-1α expression
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Condition
Hypoxia(1%O2)
1μm ICI182780
24hr
Breast cancer
cell
ERα (+)
Breast cancer
cell
ERα(-)
 Conclusion : ERα signaling regulates HIF-1α expression
ERα signaling regulates HIF-1α expression
Hypoxia mimetic
Dimethyloxalylglycine
siERα
Transfect into MCF7
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Proteasome inhibitor
200 μm DMOG
10 μm MG132
1%O2 Hypoxia
16hr
Western blot
 Conclusion : ERα signaling pathway regulates
HIF-1α expression
ERα signaling regulates HIF-1α gene expression
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Cell Culture
(Normoxia)
+E2 / +drug
100nM
1μM
24hr
RNA extraction
RT-PCR
 Conclusion : ERα signaling pathway directly regulates
HIF-1α gene expression
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
Cell Culture
(Normoxia)
C/+E2/+Tam
(100nM)
24hr
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ChIP
C/+E2/+Tam
ERα ERα ERα
｜ ｜ ｜
NB1 ERE NB2
RT-PCR
 Conclusion :
ERα directly binds EREs on the HIF-1α gene
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
Wt → Wild type
Mut → ERE mutant
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Wild type ERE
Mutant type ERE
clone into
pGL3-Luciferase reporter
Transfect
Luciferase assay
 Conclusion : The luciferase activity was significantly
high, but the ERE mutant abrogated the
activity in MCF7 cells
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription in hypoxia
Cell Culture
ChIP
N / H
N / H
48hr
IgG ERα
｜ ｜
ERE ERE
RT-PCR
 Conclusion : The results showed that ERα still bound at
the ERE of HIF-1α under hypoxia
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Machanisms of Tamoxifen and ICI182780
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Histone deacetylase
Cell Culture
(Normoxia)
C/+Tam/+ICI
(1000nM)
48hr
ChIP
C/+Tam/+ICI
IgG HDAC ERα
｜ ｜
｜
ERE ERE ERE
RT-PCR
 Conclusion : These data indicate that the two compounds
inhibit ERα function through different
mechanisms
Tamoxifen-bound ERα inhibits HIF-1α expression
Resistant to Tamoxifen
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Condition
Tam-MCF7
BT474
100 nM Tamoxifen
 Result : When ERα was depleted in tamoxifen-resistant
cell, HIF-1α expression was up-regulated
Tamoxifen-bound ERα inhibits HIF-1α expression
Short Exposure
ERα expression
plasmid
Transfection
(Normoxia)
Long Exposure
48hr
Western Blot
 Result : Longer exposure of the film showed that
overexpression of ERα enhanced HIF-1α in parental cells
 Result : Overexpression of ERα in TamR-MCF7 cells
significantly reduced HIF-1α expression
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The working model between E2 ERα and HIF-1α
The working model between Tamoxifen and HIF-1α24
 Conclusion : E2-bound ERα induces—but tamoxifen-bound
ERα suppresses—HIF-1α expression
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
Colony Formation assay
HIF-1α cDNA
Retroviral vector
Transfection
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18 d
4 wk
western blot
 Result : HIF-1α–expressing cells were at least twofold more
resistant in normoxia and long-term treatment
showed more remarkable effect
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
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Tumorsphere Formation Assay
Inhibits HIF-1α expression
Induces apoptosis of MCF7
 Result :
HIF-1α conferred significant
resistance to tamoxifen and ICI182780
compared with the parental control
High HIF-1α gene expression show a poor response to
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Tamoxifen treatment in ERα+ breast cancer
Relapse free survival
Tamoxifen treatment
 Result : Patients with high level of HIF-1α gene expression had a
poorer relapse-free survival to endocrine therapy or
tamoxifen treatment alone
High HIF-1α gene expression show a poor response to
Tamoxifen treatment in ERα+ breast cancer
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Overall survival
Tamoxifen treatment
with chemotherapy
 Result : When chemotherapy was included for those patients who
received tamoxifen, HIF-1α is also associated with poor
overall survival
HIF-1α Overexpression Confers Advantage of Tumor
Growth and Resistance to Tamoxifen Treatment 29
NSG mice
C
C
Tam
HIF-1
C
Tam
(Tamoxifen : 5mg)
 Result : Tamoxifen
treatment only modestly
delayed tumor growth with
HIF-1α overexpression ,
similar to the in vitro data
 Conclusion : HIF-1α is able to confer tamoxifen resistance
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Discussion
The working model between ERα and HIF-1 pathway
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HIF-1 might not be required for ERα activity
but synergizes with ERα
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Previous studies also show that some genes, such as KDM4B,
STC2, and VEGFA, bear both a hypoxia response element and
ERE.
Cancer Res 62(5):1289–1295
Exp Cell Res 316(3):466–476
We previously showed that depletion of HIF-1α only partially
affected KDM4B expression in hypoxia, whereas depletion of ERα
nearly abrogated KDM4B expression.
Cancer Res 70(16):6456–6466
HIF-1 might not be required for ERα
activity but synergizes with ERα.
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THE END