1. Estrogen receptor-α directly regulates the hypoxia inducible factor 1 pathway associated with antiestrogen response in breast cancer
PNAS (49)
Speaker：Wu Po-Huei Adviser : Jung-Yie Kao
Date：

2. Introduction

3. Estrogen receptor α dimer (Transcription factor)
Estrogen receptor-α (ERα )
Estrogen
Estrogen receptor α
Estrogen receptor α dimer
(Transcription factor)
Estrogen response element
Mol Endocrinol 20(8):1707–1714

4. Tamoxifen (Tam) CoR = Corepressor HDAC = Histone deacetylase
Mol Endocrinol 16(8) : 1778–1792
CoR = Corepressor
HDAC = Histone deacetylase

5. Hypoxia inducible factor 1(HIF-1α)
Normoxia
Proc Natl Acad Sci USA 100(11):6517–6522

6. ERα and HIF-1α
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

7. Results

8. ERα signaling regulates hypoxia/HIF-1α pathway
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

9. Global gene-expression profile analysis
ERα signaling regulates hypoxia/HIF-1α pathway
ER antagonist
Extracted RNA
Microarray
Global gene-expression profile analysis
MCF7 H
ICI C N
(ICI : 1μM)
24hr
Dually responsive gene

10. Library of Integrated Network-based Cellular Signatures
ERα signaling regulates hypoxia/HIF-1α pathway
Library of Integrated Network-based Cellular Signatures
Conclusion :
These data indicate that a subgroup of genes that are targeted by hypoxia/HIF-1α is also regulated by ERα signaling
ERα
ERβ
HIF-1

11. Chromatin Immunoprecipitation
ChIP
Chromatin Immunoprecipitation
DNA-protein
Cross-linking
Cell lysis
Sonication or
enzyme digestion
Protein
Fragmented
chromatin
Immunoprecipitation
with specific antibody
DNA purification
Analysis of
bound DNA
PCR
Sequencing
qPCR
Microarray

12. Library of Integrated Network-based Cellular Signatures
ERα signaling regulates hypoxia/HIF-1α pathway
Library of Integrated Network-based Cellular Signatures
Conclusion :
These data indicate that a subgroup of genes that are targeted by hypoxia/HIF-1α is also regulated by ERα signaling
ERα
ERβ
HIF-1

13. Kyoto Encyclopedia of Genes and Genomes
ERα and HIF-1α directly bind their response elements
in a subgroup of genes
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

14. Estrogen regulates HIF-1α expression
Western blot
MCF7 H E2 C
DFO N
500μm %O2
(Estrogen : 100nm)
6hr
Hypoxia mimetic
Deferoxifine
Conclusion : E2 greatly enhanced HIF-1α expression in hypoxia

15. ERα signaling regulates HIF-1α expression
Condition
Hypoxia(1%O2)
1μm ICI182780
24hr
Breast cancer cell
ERα (+)
Breast cancer cell
ERα(-)
Conclusion : ERα signaling regulates HIF-1α expression

16. Dimethyloxalylglycine
ERα signaling regulates HIF-1α expression
Hypoxia mimetic
Dimethyloxalylglycine
Proteasome inhibitor
siERα
Transfect into MCF7
200 μm DMOG
10 μm MG132
1%O2 Hypoxia
16hr
Western blot
Conclusion : ERα signaling pathway regulates
HIF-1α expression

17. ERα signaling regulates HIF-1α gene expression
Cell Culture
(Normoxia)
+E2 / +drug
100nM μM
24hr
RT-PCR
RNA extraction
Conclusion : ERα signaling pathway directly regulates
HIF-1α gene expression

18. ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
ChIP
C/+E2/+Tam
RT-PCR
Cell Culture
(Normoxia)
(100nM)
24hr
ERα ERα ERα
｜ ｜ ｜
NB1 ERE NB2
Conclusion :
ERα directly binds EREs on the HIF-1α gene

19. pGL3-Luciferase reporter
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
Wt → Wild type
Mut → ERE mutant
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

20. ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription in hypoxia
ChIP
N / H
RT-PCR
Cell Culture
48hr
IgG ERα
｜ ｜
ERE ERE
Conclusion : The results showed that ERα still bound at
the ERE of HIF-1α under hypoxia

21. Machanisms of Tamoxifen and ICI182780
Histone deacetylase
ChIP
C/+Tam/+ICI
RT-PCR
Cell Culture
(Normoxia)
(1000nM)
48hr
IgG HDAC ERα
｜ ｜ ｜
ERE ERE ERE
Conclusion : These data indicate that the two compounds
inhibit ERα function through different
mechanisms

22. Resistant to Tamoxifen
Tamoxifen-bound ERα inhibits HIF-1α expression
Resistant to Tamoxifen
Condition
Tam-MCF7
BT474
100 nM Tamoxifen
Result : When ERα was depleted in tamoxifen-resistant
cell, HIF-1α expression was up-regulated

23. ERα expression plasmid
Tamoxifen-bound ERα inhibits HIF-1α expression
ERα expression plasmid
Transfection
(Normoxia)
48hr
Western Blot
Short Exposure
Long Exposure
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

24. The working model between E2 ERα and HIF-1α
The working model between Tamoxifen and HIF-1α
Conclusion : E2-bound ERα induces—but tamoxifen-bound
ERα suppresses—HIF-1α expression

25. Colony Formation assay Transfection western blot
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
Colony Formation assay
18 d
4 wk
HIF-1α cDNA
Retroviral vector
Transfection western blot
Result : HIF-1α–expressing cells were at least twofold more
resistant in normoxia and long-term treatment
showed more remarkable effect

26. Tumorsphere Formation Assay
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
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

27. High HIF-1α gene expression show a poor response to
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

28. High HIF-1α gene expression show a poor response to
Tamoxifen treatment in ERα+ breast cancer
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

29. HIF-1α Overexpression Confers Advantage of Tumor
Growth and Resistance to Tamoxifen Treatment
NSG mice
HIF-1
Tam C
(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

30. Discussion

31. The working model between ERα and HIF-1 pathway

32. HIF-1 might not be required for ERα activity but synergizes with ERα.
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.

33. THE END