Complex Formation and Function of Estrogen Receptor a in Transcription Requires RIP140

Published OnlineFirst August 21, 2014; DOI: 10.1158/0008-5472.CAN-13-3429

Cancer Molecular and Cellular Pathobiology Research

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. 2014 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 receptors. 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 2014 American Association for Cancer Research. ligand-binding pocket on the receptor as estrogen does, but

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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

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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 clinical 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 conducted ChIP-seq experiments using an antibody against ChIP, re-ChIP, and ChIP-seq endogenous RIP140 (Fig. 1). MCF7 cells were hormone- Chromatin immunoprecipitations (ChIP) were performed deprived for 3 days, after which the cells were treated for 3 as described before (29). For each ChIP, 10 mg of antibody was hours with estradiol (E2) or vehicle control, a time point used and 100 mL of Protein A magnetic beads (Invitrogen). The previously shown to be optimal for cofactor binding (32), as antibodies used were ERa (SC-543; Santa Cruz Biotechnology), compared with the earlier time point used for analysis of ERa RIP140 (6D7; ref. 5), total Histone 3 (generous gift from Fred binding (33). Importantly, chromatin binding of ERa and van Leeuwen, the Netherlands Cancer Institute, Amsterdam, cofactors after 3 hours of E2 treatment is still proximal to the Netherlands; ref. 30), H3K4me1 (ab8895; Abcam), H3K4me2 primary E2-responsive genes, including GREB1, XBP1, and (07-030; Millipore), and H3K4me3 (ab8580; Abcam). For the CCND1 (32). For each condition, two independent replicates sequential chromatin immunoprecipitation (re-ChIP) assay, were generated and only peaks found in both replicates were chromatin was eluted with dithiothreitol (10 mmol/L), as considered. As exemplified at the AREG locus, RIP140 chro- described before (31). All quantitative PCR reactions were matin binding was actively induced by estrogen treatment and carried out in duplicates from two independent biologic overlapped with ERa-binding sites (Fig. 1A). Despite the fact replicates, averaged, and expressed relative to the input signal. that RIP140 is an ERa-responsive gene, it is unlikely that the Primer sequences are shown in Supplementary Table S1. increased RIP140 chromatin binding after E2 exposure results

Figure 1. RIP140 genomic interactions in response to E2 treatment. A, genome browser snapshot of RIP140 chromatin interactions in MCF7 cells treated for 3 hours with vehicle control (green) or E2 (red). Data were compared with publicly available ERa ChIP-seq data of MCF7 cells treated for 3 hours with E2 (blue). B, Venn diagram of genomic binding sites, shared or unique for ERa (blue) or RIP140 (veh, green; E2, red). Number of chromatin-binding sites is depicted. C, heatmap visualization of the binding events shown in B. Data were centered on the top of the peak and visualized with a 5-kb window around the peak. Subclassiﬁcations of binding events represent subgroups shown in B. D, genomic distributions of binding sites shared or unique for ERa and RIP140 under E2 conditions. E, motif enrichment analysis of chromatin-binding sites that are shared or unique for ERa and RIP140 under E2 conditions. Numbers of binding sites assessed and P values (10log) are depicted. F, heatmap visualization of unique and shared ERa/RIP140 sites under E2 conditions. Data are sorted on z score of shared ERa/RIP140 sites, as indicated by the white–red color gradient. Top 20 motifs are shown. For all motif hits, see Supplementary Table S3.

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from elevated RIP140 protein levels, as Western blot analysis RIP140 is required for ERa transcriptional complex showed that RIP140 levels were not elevated after 3 hours of formation E2 treatment (Supplementary Fig. S1). In addition, exogenous We found a substantial overlap between RIP140 and ERa overexpression of RIP140 (for Western Blot analysis, see Sup- chromatin-binding patterns in breast cancer cells (Fig. 1). Next, plementary Fig. S1) did not increase RIP140 chromatin inter- we analyzed the binding relationship between ERa, RIP140, actions, both under vehicle and E2 conditions (Supplementary and other proteins known to be involved in the ERa transcrip- Fig. S2). In hormone-deprived conditions, 12,729 RIP140-bind- tion complex (Fig. 2). To this end, we compared ERa and ing events were found and following estrogen treatment this RIP140 with binding profiles of the p160 family members SRC1, increased to 21,934 RIP140-binding sites, of which 17,985 sites SRC2, and SRC3, p300 and CBP (32), as well as FOXA1 (33) and (82%) are shared with ERa (Fig. 1B). The hormone-indepen- GATA3 (36), all of which were previously mapped in our dent RIP140 chromatin-binding events are increased in inten- laboratories under similar experimental conditions. As ERa sity after hormonal treatment (Fig. 1C). In total, 19,210 (50%) is mainly found at enhancers, the enhancer-selective histone ERa-binding sites are not cobound by RIP140 and this is not modification H3K4me1 (37) was also analyzed. For all ChIP due to peak calling threshold issues (Fig. 1C, bottom). Note conditions, cross-comparisons were generated, and the per- that peaks unique for RIP140 in E2-treated cells (Fig. 1B) do centage of sites shared with any of the interaction partners was show low ERa signal, indicating that false negative peaks are determined. The vast majority of ERa and RIP140-binding sites observed. are shared with p300, CBP, and FOXA1. The overlap with the ERa is rarely found at promoters (5% of ERa binding p160 proteins, GATA3 and H3K4me1 was significantly lower. occurs at promoters; ref. 16) and the vast majority of ERa Importantly, the shared chromatin-binding events between chromatin interactions occurs at distal enhancers that are RIP140 and ERa are strongly associated with all factors, as mainly found within a 20-kb window from the transcription compared with sites bound by RIP140 or ERa alone. start site of responding genes (34). RIP140 has a comparable Next, we analyzed whether RIP140 and ERa are part of the genomic distribution as ERa (Fig. 1D); chromatin-binding same physical complex. Cells were hormone-deprived and events of RIP140 (regardless of whether they are cobound with treated with estrogen for 3 hours. ChIP was performed for ERa or not), are enriched at introns and distal intergenic ERa (Fig. 2B, top), after which a re-ChIP on the isolated regions, representing enhancer regions. Similar to ERa, chromatin was performed for RIP140. The reciprocal re-ChIP approximately 5% of RIP140-binding events are found at pro- was also conducted (Fig. 2B, bottom), and for all conditions, moters. To identify mechanisms of protein–DNA interactions, IgG was included as a control (Fig. 2B). Quantitative PCR motif enrichment analyses were performed for the different (qPCR) was performed for ten shared ERa/RIP140-binding categories of binding sites. The top 3 enriched motifs are shown sites adjacent to well-annotated estrogen-dependent genes, for each subset, with their corresponding P values included (Fig. including RARA, GREB1, and PGR (primer sequences are pro- 1E). In addition, the top 20 enriched motifs are depicted in a vided in Supplementary Table S1). For these well-defined heatmap visualization, ranked on z-score of the shared ERa/ chromatin-binding sites, enrichment was found over IgG con- RIP140 sites (Fig. 1F; entire list see Supplementary Table S3). trol, which was further enhanced by E2 treatment, indicating For all binding site subsets, ESR1 motifs were enriched. For the that RIP140 and ERa co-occupy the DNA together as an E2- RIP140 unique binding sites, weak ERa signal is still observed induced complex. (Fig. 1F), explaining the observed ESR1 motifs at these regions. As our findings suggest that RIP140 is an intrinsic part of the As expected, forkhead motifs were found, representing binding ERa transcriptional complex, we tested the effect of RIP140 sites for the ERa pioneer factor FOXA1 (16, 33, 35). FOXA1 knockdown on RNA Polymerase II recruitment (Fig. 2C). MCF- motifs were not observed for regions only bound by RIP140. 7 cells were transfected with siControl or siRNA targeting Instead, motifs underlying RIP140-binding events (either or not RIP140. The efficiency of the knockdown is shown on protein shared with ERa) were enriched for the alternative ERa pioneer and mRNA levels (Supplementary Fig. S3). ChIP-qPCR primers factor AP-2 (21) and RARA. Cumulatively, most RIP140 chro- were designed on the basis of chromatin-binding sites shared matin-binding sites are affected by estrogen treatment and by ERa and RIP140: enhancer regions for RARA, PGR, XBP1, shared with ERa in breast cancer cells, being enriched for and GREB1, representing ERa-responsive genes. For all these distinct transcription factor motifs at these sites. sites, RIP140 knockdown resulted in a significant decrease of

Figure 2. RIP140 as an intrinsic component of the ERa transcription complex in MCF7 cells. A, overlap of chromatin-binding sites, shared or unique for ERa or RIP140, with p300, CBP, SRC1, SRC2, SRC3, FOXA1, GATA3, or H3K4me1. Percentage of binding sites that were co-occupied with the indicated proteins was calculated, and data are shown in a heatmap visualization. B, re-ChIP analysis for ERa and RIP140. Hormone-deprived cells were treated with vehicle control or E2 for 3 hours, after which ChIP was performed for ERa (top) or RIP140 (bottom). Subsequently, isolated chromatin was split and reanalyzed using ChIP for RIP140, ERa, or IgG control. Data were normalized over input control. Error bars show SD values from technical replicates from two independent biologic replicate experiments. , enrichment over IgG control; #, enrichment over vehicle control. Student t test was performed; and #, P < 0.05. C, RIP140 is required for RNA Polymerase II recruitment to the ERa complex. MCF7 cells were transfected with siRIP140 or siCntrl and hormone-depleted for 3 days. Thereafter, cells were E2-treated for 3 hours, after which ChIP was performed for RNA Polymerase II. Chromatin-binding sites shared between ERa and RIP140 (RARA, PGR, XBP1, GREB1) were analyzed by qPCR. Error bars indicate SD from triplicate experiments. Student t test was performed; , P < 0.05. D, siRIP140 does not affect epigenetic modiﬁcations H3K4me1, H3K4me2, and H3K4me3 at ERa-binding sites. Experiments were performed analogous to C, but now ChIP-qPCR was performed for histone 3, H3K4me1, H3K4me2, and H3K4me3. Error bars indicate SD from triplicate experiments.

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Gene expression A B after siRIP140 Gene expression down High up

Low

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Downregulated Upregulated E2 response Downregulated 1.0 0 Upregulated Nonresponsive 73 0.5 243 262 147 Relative mRNA levels (a.u.) levels Relative mRNA

0.0 1 RIP140 Myc XBP1 GREB1 CCND1 FOS RARA Areg PR pS2 HES1 ESR1

E siRIP140-affected genes F RIP140 binding site<20K TSS Expression upon siRIP140 Expression up (382 proximal sites) down (141 proximal sites) ESR1 motif only ESR1 and AP2 motifs after E2 motif P (–10 log) motif P (–10 log) TFAP2A 154.37 ESR1 690.77 Upregulated Downregulated

ESR1 137.50 PAX5 368.36

SP1 128.78 FOS 357.64

G E2 response of RIP140-dependent genes ESR1 motif only ESR1 and AP2 motifs

Upregulated Downregulated Nonresponsive

Figure 3. RIP140 is required for ERa activity and ERa-responsive gene expression proﬁles. A, gene expression analysis in MCF7 cells treated for 6 hours with E2, transfected with siRIP140 or siCntrl. Six replicates were generated and differential gene expression (P < 0.01) was determined, as visualized in ared–blue heatmap (left). (Continued on the following page.)

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RNA Polymerase II recruitment in E2-treated cells, indicating genes, we determined what proportion of RIP140-affected that RIP140 contributes to ERa activity at these sites (Fig. 2C). genes are normally estrogen regulated (Fig. 3D). For this, we ChIP-qPCR for Histone 3 as well as epigenetically modified used a publicly available dataset of all differentially expressed H3K4me1, H3K4me2, and H3K4me3 did not show significant genes (P < 0.01) in hormone-deprived MCF7 cells following 6 alterations of these epigenetic histone marks after silencing of hours of E2 treatment (32), resulting in 1,812 downregulated RIP140 (Fig. 2D). genes and 1,821 upregulated genes in response to E2. In total, 506 of these genes were downregulated after RIP140 knock- RIP140 is required for estrogen receptor activity and down, of which, 262 (52%) are E2-upregulated genes. In con- downstream gene expression trast, of 220 genes upregulated after siRIP140, 147 (67%) are Knockdown of RIP140 abrogated E2-induced expression of downregulated by E2. Cumulatively, these data indicate that a selection of typical ERa-responsive genes, including CCND1, RIP140 enables ERa action both at genes that are upregulated AREG, and COX2 (Supplementary Fig. S3C). To assess the as well as those that are downregulated following E2 treatment. global role of RIP140 on E2-induced gene expression, hor- RIP140, just like ERa, has a bivalent function and knocking mone-deprived MCF7 cells were transfected with siControl down RIP140 results in both up- and downregulation of or siRIP140 and subsequently treated for 6 hours with E2 (Fig. responsive genes. To elucidate potential transcriptional reg- 3A). Six biologic replicates were generated for siControl and ulators that may underlie this divergent action of RIP140, siRIP140 and 743 probes were differentially expressed when RIP140 chromatin-binding sites were identified that were knocking down RIP140 (FDR < 0.01), which correspond to 726 proximal (<20 kb from the transcription start site) to E2- unique transcripts. Of these, 506 transcripts were downregu- responsive genes that were RIP140-dependent. Genes up- and lated and 220 transcripts were found to be upregulated after downregulated by E2 treatment were analyzed separately, re- knocking down RIP140. Ingenuity Pathway Analysis was per- sulting in 382 (for E2-upregulated genes) and 141 (for E2-down- formed, identifying b-estradiol (P ¼ 4.65E31) and ESR1 (P ¼ regulated genes) RIP140 chromatin-binding sites (Fig. 3E). 8.45E22) as the top upstream regulators, where signaling The RIP140 binding sites proximal to genes E2-downregulated cascades were predicted to be inhibited following RIP140 were most strongly enriched for ESR1 motifs, whereas an knockdown. The most enriched gene networks were centred additional enrichment of AP-2 and SP1 motifs was found for around ESR1, where direct ERa-responsive genes were found the RIP140 sites proximal to E2-upregulated genes. to be affected. These include classic ERa-upregulated genes, In line with these results, ESR1 motifs at RIP140-binding including GREB1 (Fig. 3B), CCND1, MYC, and XBP1, all of which sites were more frequently observed proximal to genes upre- were inhibited when RIP140 was silenced (Supplementary gulated by RIP140 knockdown (Fig. 3F) that were downregu- Table S4). Quantitative real-time PCR (qRT)-PCR was used to lated after E2 treatment (Fig. 3G). In contrast, genes with validate the effects of siRIP140 on expression of a selection of proximal ESR1/AP-2 motifs were mostly downregulated after ERa-upregulated genes: MYC, XBP1, GREB1, CCND1, FOS, RARA, RIP140 knockdown (Fig. 3F) and upregulated after E2 treat- AREG, and PgR (Fig. 3C). RIP140 was included as a control of the ment (Fig. 3G). knockdown. The selected genes were co-occupied by both ERa and RIP140, as identified through re-ChIP experiments (Fig. 2B) RIP140 is required for breast cancer cell proliferation and were all downregulated following siRIP140, indicating that and is indicative for breast cancer patient survival RIP140 functions as a coactivator for these genes. HES1 and RIP140 plays an essential role in ERa complex formation and TFF1 (pS2) were not differentially regulated by siRIP140 in the responsive gene expression. Next, we tested whether RIP140 microarray experiments and no differential mRNA expression expression is required for E2-induced cell proliferation (Fig. 4A– was found by qPCR following RIP140 silencing, indicating that C). Hormone-deprived MCF7 cells were transfected with effects of RIP140 depletion are not fully generalizable on a siRIP140 or siControl, treated with E2, and cellular confluence genome-wide scale. ERa downregulates its own expression was assessed. Knocking down RIP140 decreased hormone- levels in MCF7 cells (32) and was upregulated after knocking induced MCF7 cell proliferation (Fig. 4A). These data were down RIP140, indicating that RIP140 functions as a corepressor validated using an independent assay (Fig. 4B), where siRIP140- in this case (Supplementary Fig. S3C). or siCntrl-transfected cells were cultured for 1 week before As the vast majority of RIP140 chromatin-binding events analysis, in the presence of E2, tamoxifen, fulvestrant, or vehicle were shared with ERa binding, and RIP140 was required for control. A significant inhibition of cell proliferation was RNA Polymerase II recruitment to a subset of ERa-responsive observed for vehicle and E2-treated cells, again implicating the

(Continued.) Upregulated or downregulated genes after siRIP140 were clustered, and z scores (in green) were visualized in a heatmap. B, Ingenuity Pathway Analysis top enriched molecular network, revolving around ESR1. Colors indicate upregulated (red) or downregulated (green) transcripts after siRIP140. C, qRT-PCR validation of differentially expressed genes after siRIP140. Data were normalized over siCntrl, which was set at 1. Error bars indicate SD from triplicate analyses. D, differentially expressed genes after siRIP140 were separated into downregulated (left) and upregulated (right) after RIP140 knockdown. Thereafter, gene subsets were analyzed for responsiveness to E2 treatment, either downregulated (green) or upregulated (red) by 6-hour E2 treatment. E, motif analysis for RIP140 chromatin-binding events, proximal to E2 upregulated (left) or downregulated (right) genes. F, differential motif enrichment for RIP140-binding sites proximal to RIP140-dependent genes. Pie charts showing genes with proximal binding sites only containing ESR1 motifs (left) or sites containing both ESR1 and AP-2 motifs (right), either upregulated (red) or downregulated (green) following siRIP140. G, differential motif enrichment for RIP140-affected genes, based on response to E2 treatment. Pie charts were generated as in F, but now genes were scored on the basis of response to E2 treatment, being upregulated (red), downregulated (green), or not responsive to E2 treatment (white).

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ABCell proliferation MTT assay 10 3 SiCntrl SiRIP140 SiCntrl 8 SiRIP140 2 * 6

4 * 1

2 MTT cell proliferation (a.u.) Relative cell proliferation (a.u.) cell proliferation Relative 0 0 04080 120 veh E2 TAM ICI Time (hrs)

C Cell proliferation markers D siRIP140 gene classifier

SiCntrl SiRIP140

veh E2 veh E2 Cluster1 Cluster2 Gene expression High MCM4

Low PLK1

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Down HSP90

E Loi cohort F Wang cohort tamoxifen-treated patients non–endocrine-treated patients

P = 0.028 Recurrence-free survival P = 0.393 Distant metastasis-free survival HR = 1.84; 95% CI, 1.06–3.2 HR = 1.23; 95% CI, 0.77–1.96

Time (months) Time (months)

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involvement of RIP140 in cell proliferation. Knockdown of (cluster 2) correlate with active RIP140-responsive genes (RIP- RIP140 did not affect cell numbers under tamoxifen and fulves- 140 upregulated genes are increased, RIP140-downregulated trant conditions. As tamoxifen and fulvestrant block ERa- genes are decreased), whereas good outcome (cluster 1) dependent cell proliferation, these data indicate that RIP140 is patients generally represent cases with a less active RIP140- involved in cell proliferation rather than cell survival. These data responsive gene signature (RIP140-upregulated genes are were further confirmed by Western blot analysis of cell prolif- decreased, RIP140-downregulated genes are increased; Fisher eration markers MCM4, PLK1, and Ki67/MIB1 (Fig. 4C), with exact test, P ¼ 0.001; Supplementary Fig. S5). HSP90 as loading control (38, 39). Cells were transfected with On the basis of the hierarchical clustering analyses, these siCntrl or siRIP140 and hormone-deprived for 2 days. Subse- patient groups could be further segregated into smaller sub- quently, cells were treated with E2 or vehicle control for 4 days, sections, further identifying subgroups of patients with differ- after which cells were lysed and processed. As expected, E2 ential DMFS (Supplementary Fig. S6). These data were treatment increased levels of the cell proliferation markers validated using a second but smaller cohort of patients with in siCntrl-transfected cells, while this response to E2 was not tamoxifen-treated breast cancer (ref. 41; Supplementary Fig. observed after siRIP140. S7), and classification of patients with tamoxifen-treated To test for any clinical implications of RIP140 action, genes breast cancer over differential outcome was borderline signif- under RIP140 control were used to generate a gene classifier icant (P ¼ 0.056; HR ¼ 1.73; 95% CI, 0.98–3.06). No correlation that was tested in multiple cohorts of patients with breast with survival was found for a cohort of 209 patients with ERa- cancer. As RIP140 is an intrinsic part of the ERa complex and positive breast cancer who did not receive any adjuvant dictates E2 responsiveness, siRIP140-affected genes could also endocrine treatment (ref. 42; P ¼ 0.393; HR ¼ 1.23; 95% CI, be directly affected under tamoxifen-resistant conditions, sug- 0.77–1.96; Fig. 4F). gesting that differential expression of these direct target genes Cumulatively, we found that RIP140 is required for E2-driven in breast tumors could function as a hallmark for tamoxifen cell proliferation of the breast cancer cell line MCF7, and unresponsiveness. All genes differentially expressed following the RIP140-specific gene signature could selectively identify siRIP140 (Fig. 3A) were tested for correlation with distant patients with tamoxifen-treated breast cancer with a poor metastasis-free survival in a cohort of 263 tamoxifen-treated outcome. patients, diagnosed with primary ERa-positive breast cancer (40). Analyses of clinicopathologic parameters are provided in Discussion Supplementary Table S5. As visualized in a heatmap (Fig. 4D), Even though RIP140 was originally identified in the breast unsupervised clustering on the basis of our gene signature cancer cell line MCF7, its biologic role in breast cancer and its identified two distinct subgroups of patients. Directionality of effects on ERa function in this context has remained largely gene expression by siRIP140 in the MCF7 cell line (Fig. 3A) is elusive. Here, we show that RIP140 plays a crucial role in ERa highlighted in a red/blue color table on the left side of the transcription complex formation, being essential for RNA heatmap. ERa-mediated (32) and RIP140-affected (Fig. 3A) gene Polymerase II recruitment and ERa-mediated breast cancer expression involves both activation as well as inhibition of cell proliferation. Its physiologic role in ERa-mediated breast activity. In line with these data, no clear subgroups of directional tumours is further strengthened by the identification of a gene expression were identified on the basis of unsupervised RIP140-based gene expression classifier that could successfully hierarchical clustering. Functionally, the two largest gene clus- stratify patients with breast cancer on survival after adjuvant ters were enriched for networks involving ERa (geneset A) and tamoxifen treatment. NFkB (geneset B) related networks (Supplementary Fig. S4). RIP140 is critically involved in ERa function, and we showed Subsequently, distant metastasis-free survival (DMFS) after that RIP140/ERa complexes bind enhancer regions for the adjuvant tamoxifen treatment was determined (Fig. 4E), and RIP140 gene itself. While most ERa genomics analyses have fi signi cantly differential DMFS was found between the two been performed following 45 minutes of E2 treatment (33), we largest patient subgroups [P ¼ 0.028; HR ¼ 1.84; 95% confi- previously found coregulator/chromatin interactions, on a – dence interval (CI), 1.06 3.2]. Patients with a poor outcome genome-wide scale, to be more robust after 3 hours of E2

Figure 4. RIP140 is required for E2-driven cell proliferation and RIP140-responsive genes as classifier for breast cancer patient survival. A, hormone-deprived MCF7 cells were transfected with siRIP140 or siCntrl, and cell proliferation in the presence of E2 was determined by plate confluence and measured in time. Data were normalized over confluence at time point 0, which was set at 1. Error bars indicate SD from triplicate measurements. B, hormone-deprived cells were transfected with siRIP140 or siCntrl and cultured for 7 days in the presence of E2, tamoxifen, fulvestrant (ICI), or vehicle control. Subsequently, cells were processed for MTT analysis. Error bars indicate MTT signal from triplicate measurements. Between siRIP140 and siCntrl, Student t test was performed; , P < 0.05. C, Western blot analyses for cell proliferation markers in siRIP140 cells. Cells were transfected with siCntrl or siRIP140 and hormone depleted for 2 days. Thereafter, E2 or vehicle control was added and cells were incubated for an additional 4 days. Cell lysates were processed for Western blot analysis and stained for cell proliferation markers MCM4, PLK1, and MIB1/Ki67. Hsp90 was used as loading control. D, heatmap visualization of the RIP140-affected geneset, which identified two distinct subgroups from a publicly available dataset of 263 patients with tamoxifen-treated breast cancer (40), using unsupervised clustering. Colors indicate z score. Left look-up table shows expression genes of the corresponding genes after siRIP140 in MCF7 cells, shown in a second heatmap. E, Kaplan–Meier survival curve of 263 patients with ERa-positive breast cancer who received adjuvant tamoxifen treatment. Patient subgroups were used as generated in D. The x-axis shows time in months and the y-axis shows distant metastasis-free survival. F, Kaplan–Meier survival curve as in E, but now applying a publicly available dataset of 209 patients with ERa-positive breast cancer who did not receive adjuvant endocrine treatment (42).

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Rosell et al.

treatment (32). While E2 treatment does increase RIP140 levels, RIP140 was required for ERa activity and E2-driven cell no differential RIP140 protein levels were observed after 3 hours proliferation, and we therefore tested a possible correlation of of E2 exposure, nor did exogenous overexpression of RIP140 RIP140-action in ERa-dependent tumor cell proliferation. Being further increase chromatin interactions under vehicle or E2 a transcriptional regulator in ERa-action, antiestrogen treat- treatment. These data not only illustrate direct E2 dependence ment outcome may be correlated with expression of these of RIP140 chromatin interactions, but also suggest a higher downstream genes. Using two distinct cohorts, the gene expres- mode of regulation where RIP140 action provides a positive sion classifier successfully identified patients with breast cancer feedback loop in an E2-dependent fashion, further upregulating with a poor outcome after adjuvant tamoxifen treatment, its own activity at later time point of hormone exposure. whereas no correlation was observed in ERa-positive patients RIP140 is an atypical transcriptional regulator and depend- who did not receive adjuvant endocrine treatment. Future ing on the physiologic context (hormonal status, genomic research using randomized clinical trial samples is needed to environment, and transcription factor interaction repertoire) elucidate whether our classifier is merely a prognostic marker, it can function either as a repressor or as an activator of or that it also has a possible predictive component. transcription. In breast cancer, ERa is known to behave Cumulatively, we illustrate that RIP140 in breast tumor cells, similarly, downregulating as many genes as it upregulates analogous to its role in mammary gland development (7), after 6 hours of hormonal treatment (32). This bivalent action facilitates ERa activity and dictates the estrogen response in of ERa is facilitated by RIP140, as the response to E2 treat- breast cancer cells. As such, RIP140 is a pivotal player in ment (for both up and downregulated genes) was dampened endocrine treatment response in breast cancer and provides after RIP140 knockdown. Recently, we found that RIP140 has a gene expression classifier for breast cancer patient survival. a similar bivalent function in normal mammary gland development (7). Analogous to many other transcriptional core- Disclosure of Potential Conflicts of Interest gulators, RIP140 is shared by multiple transcription factors, No potential conflicts of interest were disclosed. including ERb (43) and E2F1 (12). RIP140 inhibits E2F1 function and is highly expressed in luminal breast cancer Authors' Contributions Conception and design: M. Rosell, J. Nautiyal, M.G. Parker, W. Zwart (12). As E2F1 is one of the major transcription factors Development of methodology: M. Rosell, A. Poliandri, W. Zwart mediating ERa action (44), the complexity of RIP140 func- Acquisition of data (provided animals, acquired and managed patients, tional behavior in breast cancer involves tight regulation and provided facilities, etc.): M. Rosell, S. Stelloo, J. Nautiyal, J.S. Carroll, M.G. Parker, W. Zwart interplay between multiple transcription factors, further Analysis and interpretation of data (e.g., statistical analysis, biostatistics, highlighting the complexity of the bivalent role of RIP140 in computational analysis): M. Rosell, E. Nevedomskaya, S. Stelloo, J.S. Carroll, M.G. Parker, W. Zwart transcriptional regulation. Writing, review, and/or revision of the manuscript: M. Rosell, S. Stelloo, This bivalent and complex role of RIP140 in transcriptional L.F.A. Wessels, M.G. Parker, W. Zwart regulation complicates interpretation of its role in clinical Administrative, technical, or material support (i.e., reporting or orga- fi nizing data, constructing databases): S. Stelloo, J.H. Steel outcome. RIP140-based gene expression classi ers cannot focus Study supervision: M. Rosell, L.F.A. Wessels, M.G. Parker, W. Zwart on directionality of gene expression, but should rather empha- size "physiologic RIP140-mediated gene expression directional- Acknowledgments ity"; a matter of attention also when generating expression The authors thank Gordon Brown and Suraj Menon from Cancer Research UK signatures based of ERa action. In addition, even though and University of Cambridge and Sander Canisius from NKI for bioinformatics support. The authors also thank James Hadfield and the entire genomic core ChIP-seq analyses do enable the identification of genesets facility at Cancer Research UK, University of Cambridge for help with Illumina directly responsive to ERa/RIP140 action, this complex is also sequencing. critically involved in the expression of other transcription factors, including Myc (45). Analyzing merely directly responsive Grant Support This work was supported by grants from the Dutch Cancer Society and the genes as determined through ChIP-seq represents an underes- Netherlands Organisation for Scientific Research (NWO). timation of the biological complexity of the system, and we The costs of publication of this article were defrayed in part by the payment of therefore studied the full spectrum of genes differentially page charges. This article must therefore be hereby marked advertisement in expressed after siRIP140. Yet, as the vast majority of RIP140 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chromatin-binding sites were involved in ERa complexes, Received December 2, 2013; revised July 1, 2014; accepted July 16, 2014; patient classification is likely biased towards ERa function. published OnlineFirst August 21, 2014.

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www.aacrjournals.org Cancer Res; 74(19) October 1, 2014 OF11

Complex Formation and Function of Estrogen Receptor α in Transcription Requires RIP140

Meritxell Rosell, Ekaterina Nevedomskaya, Suzan Stelloo, et al.

Cancer Res Published OnlineFirst August 21, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-3429

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