Estrogen receptor directly regulates the hypoxia inducible factor

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

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

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

ERα and HIF-1α 6 We have previously shown that HIF-1α and ERα can coordinate expression of genes, such as lysine-specific demethylase 4 B/Jumonji domain-containing 2 B (KDM 4 B/JMJD 2 B), an H 3 K 9 me 3/me 2 histone demethylase, which is targeted by both ERα and HIF-1α and epigenetically regulates cell cycle progression. The genomic locus of KDM 4 B 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

ERα signaling regulates hypoxia/HIF-1α pathway 8 We have previously shown that knockdown of ERα significantly down-regulated histone demethylase KDM 4 B expression, a HIF-1α transcriptional target. J Biol Chem 283(52): 36542– 36552

ERα signaling regulates hypoxia/HIF-1α pathway 9 ER antagonist MCF 7 N C H ICI 24 hr C ICI (ICI 182780 : 1μM) Extracted RNA Microarray Global gene-expression profile analysis Dually responsive gene

ERα signaling regulates hypoxia/HIF-1α pathway 10 Library of Integrated Network-based Cellular Signatures l 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

Ch. IP Chromatin Immunoprecipitation DNA-protein Cross-linking 11 Cell lysis Sonication or enzyme digestion Protei n Fragmented chromatin Immunoprecipitation with specific antibody DNA purification Analysis of bound DNA PCR q. PCR Sequencing Microarray

ERα signaling regulates hypoxia/HIF-1α pathway 12 Library of Integrated Network-based Cellular Signatures l 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 13 High-resolution genome-wide mapping of HIF-binding sites by Ch. IP-seq Blood 117(23): e 207–e 217 A CTCF-independent role for cohesin in tissue-specific transcription Genome Res 20(5): 578– 588 Kyoto Encyclopedia of Genes and Genomes l 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 14 Hypoxia mimetic Deferoxifine MCF 7 N C E 2 DFO H 500μm 1%O 2 C E 2 (Estrogen : 100 nm) 6 hr Western blot l Conclusion : E 2 greatly enhanced HIF-1α expression in hypoxia

ERα signaling regulates HIF-1α expression 15 Condition Hypoxia(1%O 2) 1μm ICI 182780 24 hr Breast cancer cell ERα (+) ERα(-) l Conclusion : ERα signaling regulates HIF-1α expression

ERα signaling regulates HIF-1α expression 16 Hypoxia mimetic Dimethyloxalylglycine si. ERα Transfect into MCF 7 Proteasome inhibitor 200 μm DMOG 10 μm MG 132 1%O 2 Hypoxia 16 hr l Conclusion : ERα signaling pathway regulates HIF-1α expression Western blot

ERα signaling regulates HIF-1α gene expression 17 Cell Culture (Normoxia) +E 2 / +drug 100 n. M 1μM 24 hr RNA extraction RT-PCR l 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/+E 2/+Tam (100 n. M) 24 hr 18 Ch. IP C/+E 2/+Tam ERα ERα ｜ ｜ ｜ NB 1 ERE NB 2 RT-PCR l 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 19 Wild type ERE Mutant type ERE clone into p. GL 3 -Luciferase reporter Transfect Luciferase assay l Conclusion : The luciferase activity was significantly high, but the ERE mutant abrogated the activity in MCF 7 cells

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

Machanisms of Tamoxifen and ICI 182780 21 Histone deacetylase Cell Culture (Normoxia) C/+Tam/+ICI (1000 n. M) 48 hr Ch. IP C/+Tam/+ICI Ig. G HDAC ERα ｜ ｜ ｜ ERE ERE RT-PCR l Conclusion : These data indicate that the two compounds inhibit ERα function through different mechanisms

Tamoxifen-bound ERα inhibits HIF-1α expression 22 Resistant to Tamoxifen Condition Tam-MCF 7 BT 474 100 n. M Tamoxifen l Result : When ERα was depleted in tamoxifen-resistant cell, HIF-1α expression was up-regulated

Tamoxifen-bound ERα inhibits HIF-1α expression 23 Short Exposure ERα expression plasmid Transfection Long Exposure (Normoxia) 48 hr Western Blot l Result : Longer exposure of the film showed that overexpression of ERα enhanced HIF-1α in parental cells l Result : Overexpression of ERα in Tam. R-MCF 7 cells significantly reduced HIF-1α expression

The working model between E 2 ERα and HIF-1α The working model between Tamoxifen and HIF-1α 24 l Conclusion : E 2 -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 c. DNA Retroviral vector Transfection 25 18 d 4 wk western blot l 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 26 Tumorsphere Formation Assay Inhibits HIF-1α expression Induces apoptosis of MCF 7 l Result : HIF-1α conferred significant resistance to tamoxifen and ICI 182780 compared with the parental control

High HIF-1α gene expression show a poor response to 27 Tamoxifen treatment in ERα+ breast cancer Relapse free survival Tamoxifen treatment l 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 Overall survival Tamoxifen treatment with chemotherapy l Result : When chemotherapy was included for those patients who received tamoxifen, HIF-1α is also associated with poor overall survival 28

HIF-1α Overexpression Confers Advantage of Tumor 29 Growth and Resistance to Tamoxifen Treatment NSG mice C C Tam HIF-1 C Tam (Tamoxifen : 5 mg) l Result : Tamoxifen treatmen only modestly delayed tumor growth with HIF-1α overexpression , similar to the in vitro data l Conclusion : HIF-1α is able to confer tamoxifen resistance

30 Discussion

The working model between ERα and HIF-1 pathway 31

HIF-1 might not be required for ERα activity but synergizes with ERα 32 Previous studies also show that some genes, such as KDM 4 B, STC 2, 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 KDM 4 B expression in hypoxia, whereas depletion of ERα nearly abrogated KDM 4 B expression. Cancer Res 70(16): 6456– 6466 HIF-1 might not be required for ERα activity but synergizes with ERα.

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