Published OnlineFirst August 21, 2014; DOI: 10.1158/0008-5472.CAN-13-3429 Cancer Molecular and Cellular Pathobiology Research Complex Formation and Function of Estrogen Receptor a in Transcription Requires RIP140 Meritxell Rosell1, Ekaterina Nevedomskaya2,3, Suzan Stelloo2, Jaya Nautiyal1, Ariel Poliandri1, Jennifer H. Steel1, Lodewyk F.A. Wessels3, Jason S. Carroll4, Malcolm G. Parker1, and Wilbert Zwart2 Abstract RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor a (ERa) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERa, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERa-complex formation, ERa-mediated gene expression, and ERa-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERa-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment. Cancer Res; 74(19); 1–11. Ó2014 AACR. Introduction RIP140 was first identified as a cofactor for ERa in breast RIP140, also known as nuclear receptor interacting protein 1 cancer cell lines (10, 11), but its putative function in ERa (Nrip1), is a transcriptional coregulator for a large number of biology remained elusive. Circumstantial evidence suggests transcription factors (reviewed in ref. 1). RIP140 is widely that RIP140 may have an important role in breast cancer, as fi expressed, playing crucial roles in metabolic control in adipose RIP140 expression is signi cantly decreased in basal-like breast tissues (2), skeletal (3), and cardiac muscle (4), as well as in liver cancers as compared with luminal tumors and RIP140 has been (5). RIP140 is also involved in circadian rhythm regulation (6) implicated in controlling cell proliferation, potentially by reg- and is essential for female fertility (6). Recently, we found ulating the activity of E2F transcription factors (12). In addi- RIP140 to be required for normal mammary gland develop- tion, RIP140 expression has also been found elevated in ductal ment, where it functions as an essential component in estrogen carcinomas in situ (13). ERa regulates the expression of many signaling (7). RIP140 has a dual role as a transcriptional components involved in ERa transcription complex formation, regulator, acting as both a corepressor for catabolic genes in including GATA3 (14), XBP1 (15), FOXA1 (15, 16), and GREB1 fi metabolic tissue (3, 8) and a coactivator for inflammatory (17). Analogous to these ndings, estrogen treatment of breast genes (9). RIP140 functions as a coactivator for ERa-responsive cancer cells directly induces RIP140 expression (18). genes in mammary gland formation (7). ERa belongs to the superfamily of nuclear hormone recep- tors. When activated by estrogen, ERa drives the expression of a large number of target genes, leading to breast tumor cell proliferation. Ligand binding results in receptor dimerization, 1Institute of Reproductive and Developmental Biology, Department of chromatin association, and the recruitment of a large set of Surgery and Cancer, Imperial College London, London, United Kingdom. coregulators required for transcription of target genes (19). For 2Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, the Netherlands. 3Division of Molecular Carcinogenesis, the ERa to associate with the chromatin, it requires the action of Netherlands Cancer Institute, Amsterdam, the Netherlands. 4Cancer so-called "pioneer factors" to enable chromatin accessibility. Research UK, Cambridge Institute, University of Cambridge, Cambridge, United Kingdom. Putative pioneer factors for ERa include FOXA1 (16), PBX1 (20), and AP-2g (21), all directly affecting ERa chromatin Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). binding and functionality in breast cancer (reviewed in ref. 22). ERa is expressed in 75% of all breast cancers, where it drives E. Nevedomskaya, S. Stelloo, and J. Nautiyal contributed equally to this article. cell proliferation and tumor growth. Because of the action of ERa, most treatment options for luminal breast cancer revolve Corresponding Author: Wilbert Zwart, Division of Molecular Pathology, The Netherlands Cancer Institute, plesmanlaan 121, 1066CX Amsterdam, around blocking the activity of this key transcription factor. the Netherlands. Phone: 312-0512-7920; Fax: 312-0512-2029; E-mail: This is achieved either by competitive inhibitors for estrogen [email protected] binding (such as tamoxifen) or by blocking estrogen synthesis doi: 10.1158/0008-5472.CAN-13-3429 (through aromatase inhibitors). Tamoxifen binds the same Ó2014 American Association for Cancer Research. ligand-binding pocket on the receptor as estrogen does, but www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 21, 2014; DOI: 10.1158/0008-5472.CAN-13-3429 Rosell et al. RIP140 RIP140 ERα ACveh E2 full medium Scale 10 kb chr4 75530000I 75540000I 75550000I 75560000I 80 ERα Shared 2 51 RIP140 RIP140 veh + veh RIP140 E2 RIP140 veh + 0 ERα 51 RIP140 E2 RIP140 E2 + ERα 2 AREG RIP140 veh B RIP140 E2 ERα RIP140 veh 594 19,210 1,489 8,981 1,665 9,004 2,087 RIP140 E2 ERα D αα 5 kb Motif enrichment E , F Top 20 motifs OF2 Cancer Res; 74(19) October 1, 2014 Cancer Research Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 21, 2014; DOI: 10.1158/0008-5472.CAN-13-3429 RIP140 in ERa Transcriptional Regulation alters receptor conformation to block coactivator binding and Solexa sequencing and bioinformatics is fully described in the consequently cell proliferation (23). Unfortunately, resistance Supplementary Methods. ChIP-seq read count and number of to endocrine treatment is common. ERa can acquire addi- aligned reads are shown in Supplementary Table S2. tional agonistic features through altered kinase activities (24– 26) or cofactor overexpression (27, 28). RNA extraction, qRT-PCR, and gene expression analyses In this study, we investigated the role of RIP140 in breast Total RNA was extracted with TRIzol. RNA for microarray cancer and its functional relationship with ERa. RIP140 was analysis was purified using QIAGEN RNeasy Mini columns. The shown to be a key component in the ERa transcription expression of target genes was determined using SYBR Green complex and required for estrogen-dependent transcriptional Reagent and gene-specific primers (Supplementary Table S1). regulation and breast cancer cell proliferation. In addition, a Relative expression levels were normalized to L13 ribosomal downstream gene signature for RIP140-regulated genes unit. Complete description of gene expression analyses is enabled the identification of breast cancer patients with a described in the Supplementary Methods. poor outcome after tamoxifen treatment, illustrating the clin- ical implications of our findings. Cell proliferation analysis MCF7 cells were seeded under hormone-depleted condi- Materials and Methods tions and transfected with siRIP140 or siControl. The next day, cells were trypsinized, reseeded in a clear-bottom 384-well Cell lines, transfections, antibodies, and tissue culture plate (IncuCyte) or 6-well plates (MTT assay), and hormones MCF7 cells were cultured in DMEM supplemented with 10% were added at the concentrations indicated. For MTT analyses, FBS and 1% antibiotics. For hormone depletion, cells were grown standard protocols were used (Millipore). For Incucyte anal- in phenol red-free DMEM with 5% charcoal-treated FBS and 1% yses, cell proliferation was determined by plate confluency and antibiotics for 3 days. For transient transfection of siRIP140, cells measured in time using an Incucyte Life Cell Imaging Device were transfected with Lipofectamine 2000 (Invitrogen) with 67 (Essen Bioscience). Four wells were measured per condition. nmol/L siRIP140 or siControl (NBS Biological), and assayed 48 hours after transfections, unless stated differently. RIP140 Data access overexpression experiments were performed using PEI (poly- All genomic data are deposited at ArrayExpress, with acces- ethylenimine; Polysciences, Inc.). For Western blotting, antibo- sion numbers E-MTAB-2576 (ChIP-seq) and E-MTAB-2577 dies were used for Ki67 (m7240; DAKO), HSP90 (sc-7947; Santa (microarray data). Cruz Biotechnology), MCM4 (559544; BD Biosciences), and PLK1 (sc-17783; Santa Cruz Biotechnology). Cell lines were obtained Results from the ATCC, authenticated through STR profiling, and used at RIP140 genome-wide chromatin-binding patterns in low passage after receipt from the vendor. Cells were cultured for MCF7 cells less than 6 months after validation. To identify the RIP140 genomic binding profile, we con- ducted ChIP-seq experiments using an antibody against ChIP, re-ChIP, and ChIP-seq endogenous RIP140