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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 4 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 6 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 ERα signaling regulates hypoxia/HIF-1α pathway 8 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 9 ERα signaling regulates hypoxia/HIF-1α pathway 10 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 11 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 12 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 13 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 14 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 15 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 16 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 17 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 18 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 19 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 20 Machanisms of Tamoxifen and ICI182780 21 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 22 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 23 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 25 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 26 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 27 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 28 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 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 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