Comparative Cistromics Reveals Genomic Cross-Talk Between FOXA1 and Era in Tamoxifen-Associated Endometrial Carcinomas

Published OnlineFirst May 6, 2016; DOI: 10.1158/0008-5472.CAN-14-1813 Cancer Molecular and Cellular Pathobiology Research

Comparative Cistromics Reveals Genomic Cross-talk between FOXA1 and ERa in Tamoxifen-Associated Endometrial Carcinomas Marjolein Droog1, Ekaterina Nevedomskaya1,2, Yongsoo Kim1,2, Tesa Severson1, Koen D. Flach1, Mark Opdam1, Karianne Schuurman1, Patrycja Gradowska3, Michael Hauptmann3, Gwen Dackus1, Harry Hollema4, Marian Mourits5, Petra Nederlof6, Hester van Boven6, Sabine C. Linn1,7, Lodewyk Wessels2,8, Flora E. van Leeuwen3, and Wilbert Zwart1

Abstract

Tamoxifen, a small-molecule antagonist of the transcription Multifactorial ChIP-seq data integration from the endometrial factor estrogen receptor alpha (ERa) used to treat breast cancer cell line Ishikawa illustrated a functional genomic cancer, increases risks of endometrial cancer. However, no network involving ERa and FOXA1 together with the enhancer- parallels of ERa transcriptional action in breast and endome- enriched transcriptional regulators p300, FOXM1, TEAD4, trial tumors have been found that might explain this effect. In FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of this study, we addressed this issue with a genome-wide assess- 230 primary endometrial tumor specimens showed that lack of ment of ERa-chromatin interactions in surgical specimens FOXA1 and ERa expression was associated with a longer obtained from patients with tamoxifen-associated endometrial interval between breast cancer and the emergence of endome- cancer. ERa was found at active enhancers in endometrial trial cancer, exclusively in tamoxifen-treated patients. Our cancer cells as marked by the presence of RNA polymerase II results deﬁne conserved sites for a genomic interplay between and the histone marker H3K27Ac. These ERa binding sites FOXA1 and ERa in breast cancer and tamoxifen-associated were highly conserved between breast and endometrial cancer endometrial cancer. In addition, FOXA1 and ERa are associated and enriched in binding motifs for the transcription factor with the interval time between breast cancer and endometrial FOXA1, which displayed substantial overlap with ERa binding cancer only in tamoxifen-treated breast cancer patients. Cancer sites proximal to genes involved in classical ERa target genes. Res; 76(13); 1–12. 2016 AACR.

Introduction lates ERa in certain other tissues, including the endometrium, specifically in a low estrogen environment, i.e., postmenopausal Of all breast tumors, roughly 75% depend on ERa for cell women (2–6). proliferation and tumor progression. Consequently, most breast Due to tissue-selective action of tamoxifen, postmenopausal cancer treatment modalities are aimed to inhibit ERa activity. breast cancer patients on this drug have an increased risk of Tamoxifen is the most widely applied hormonal therapy, which endometrial cancer development by 2- to 7-fold, depending on acts through competitive inhibition of ERa's natural ligand estro- the duration of use (4, 5, 7, 8). This increased ERa activity gen (1). Although tamoxifen inhibits ERa in breast cells, it stimu- was linked with an altered expression level of coactivator SRC1 (9) and PAX2 (10) in endometrial cancer cells. However, manip- 1Department of Molecular Pathology, Netherlands Cancer Institute, 2 ulating expression levels of either SRC1 or PAX2 in breast cancer Amsterdam, the Netherlands. Department of Molecular Carcinogen- fi esis, Netherlands Cancer Institute, Amsterdam, the Netherlands. cell line models did not support these ndings (11, 12), implying 3Department of Epidemiology, Netherlands Cancer Institute, Amster- that these proteins are not the sole drivers of agonistic features of 4 dam, the Netherlands. Department of Pathology, University Medical tamoxifen observed in endometrial tissue. In addition, ERa Center Groningen, Groningen, the Netherlands. 5Department of Gyne- cological Oncology, University Medical Center Groningen, Groningen, transcriptional regulation in endometrial cancer has exclusively the Netherlands. 6Department of Pathology, Netherlands Cancer Insti- been studied using cell lines (13) while ERa genomic behavior in tute, Amsterdam, the Netherlands. 7Department of Medical Oncology, 8 primary human endometrial tumors remains unexplored. Netherlands Cancer Institute, Amsterdam, the Netherlands. Faculty a of EEMCS, Delft University of Technology, Delft, the Netherlands. For ER to bind the chromatin in breast cancer cells, it requires FOXA1 (14–16). FOXA1 is a pioneer factor, regulating Note: Supplementary data for this article are available at Cancer Research a Online (http://cancerres.aacrjournals.org/). chromatin accessibility and thereby enables ER -dependent gene activation and proliferation of breast cancer cells (14). E. Nevedomskaya and Y. Kim contributed equally to this article. FOXA1 was previously identified as one of the luminal breast Corresponding Author: Wilbert Zwart, the Netherlands Cancer Institute, cancer–defining transcription factors (17, 18), and its expres- Department of Molecular Pathology, Plesmanlaan 121, 1066 CX Amsterdam, the sion correlates with a favorable outcome in breast cancer Netherlands. Phone: 31-20-512-2101; E-mail: [email protected] (19–21). In ERa-negative breast cancer cells, exogenous intro- doi: 10.1158/0008-5472.CAN-14-1813 duction of ERa and FOXA1 along with GATA3 was sufficient to 2016 American Association for Cancer Research. reprogram these cells toward hormone responsiveness (22).

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Cumulatively, these reports position FOXA1 as a crucial player dies (Seven Hills, WMAB-2F83 and WRAB-1200; Supplementary in ERa functionality in breast cancer. In endometrial cancer, the Fig. S1). Primer sequences used for ChIP-qPCR are listed pioneer factor for ERa is unknown, and the mechanisms that in Supplementary Table S3. All Ishikawa ChIP-seq and RNA-seq dictate ERa action in this tissue remain elusive. datasets were generated by the ENCODE consortium, and Sup- To study the genomic features of ERa in endometrial tumors, plementary Table S4 shows all accession numbers of the used we performed chromatin immunoprecipitation sequencing datasets that also includes breast cancer ERa ChIP-seq data from (ChIP-seq) in surgical samples from five endometrial tumors Jansen and colleagues (25). Patient characteristics of breast cancer of patients who were previously treated with tamoxifen. ERa tumors can be found in Supplementary Table S5. binding sites in endometrial tumors were enriched for RNA polymerase II as well as for H3K27Ac, the posttranslational Illumina sequencing and enrichment analysis histone modification that marks both active promoters and ChIP DNA was amplified as described (26). Sequences were active enhancers. Overlap of ERa chromatin binding sites was generated by the Illumina Hiseq 2000 genome analyzer (using observed between breast tumors and endometrial cancers. In 50-bp reads) and aligned to the Human Reference Genome (assem- addition, we identified a genomic functional network in the bly hg19, February 2009). Reads were filtered based on MAPQ endometrial cancer cell line Ishikawa, implicating FOXA1 and quality (reads with MAPQ>20) to eliminate reads from repetitive ERa as part of a large multiprotein transcriptional network that elements. Peak calling was performed over input, using Dfilter (27) enrolls FOXM1, TEAD4, and TCF12. The direct clinical implica- and MACS peak caller version 1.4 (28). Only peaks identified using tions of this coordinated action between ERa and FOXA1 in both algorithms were considered. MACS was run with the default endometrial carcinogenesis was assessed for 230 endometrial parameters, except P ¼ 10 7. DFilter was run using settings bs ¼ 50, cancer patients who were previously treated for breast cancer. In ks ¼ 30, refine, nonzero. Details on the number of reads obtained contrast to ERa, FOXA1 expression did not correlate with and the percentage of reads aligned, and number of peaks called can endometrial cancer patient survival. Interestingly, both ERa and be found in Supplementary Table S6. Details on bioinformatics FOXA1 expression did associate with short interval between analyses can be found in the Supplementary Methods. breast cancer treatment and endometrial cancer diagnosis, exclusively for the patients who received tamoxifen. RNA isolation and mRNA expression a With this, we present a genomic conservation of ER action RNA from tumors was isolated using the AllPrep DNA/RNA/ between breast and endometrial tumor specimens that highlights miRNA Universal Kit (Qiagen). cDNA was synthesized with Super- FOXA1 as a common player of hormonal response in both tissues. script III Reverse Transcriptase (Invitrogen), using random hexamer primers. RT-PCR was performed using SYBR Green (GC Biotech) on Materials and Methods a Roche Lightcycler, using 50 cycles of amplification for all genes Patient series tested. RT-PCR products were detected by agarose gel electropho- resis. Primer sequences are listed in Supplementary Table S7. Endometrial tumor tissue from 230 patients of the Tamoxifen Associated Malignancies: Aspects of RISK (TAMARISK) study was analyzed as described before (5, 7, 23). Clinicopathologic para- Immunohistochemistry and tissue microarray analyses meters from endometrial cancers can be found in Supplementary For immunohistochemical analysis, an antibody for FOXA1 Table S1. Tumor samples were analyzed anonymously, with was applied (2F83, Seven Hills Bioreagents). This antibody was fi coded leftover material that cannot be traced back to the patients previously reported to be highly speci c for FOXA1 (29). Anti- fi fi that these materials originated from. This study was performed in body speci city was con rmed by Western blot (Supplementary accordance with the Code of Conduct of the Federation of Fig. S1). Immunohistochemistry on the TAMARISK tissue micro- Medical Scientific Societies in the Netherlands (http://www. array (TMA) was performed using a Ventana Benchmark Ultra fmwv.nl). The study has been approved by the local medical system, applying standard protocols. A kappa of 0.78 was cal- a ethics committee of the Netherlands Cancer Institute. culated from scoring of two independent observers. ER staining was previously performed as described (7). Patients were categorized based on ERa and FOXA1 expression levels (at 10% ChIPs and analyses cutoff) and stratified over tamoxifen use. The mean time interval ChIPs were performed as described previously (24, 25) on between breast and endometrial cancer diagnosis was compared endometrioid andenocarcinoma tumors that are part of the between the groups and significance of the differences was TAMARISK study (5, 7, 23). These endometrial tumors were assessed using the t-test. For survival analysis, Kaplan–Meier collected from patients who were still on tamoxifen on the day survival plots were generated categorizing for ERa and FOXA1 of surgery or had stopped 1 to 2 months prior to surgery. expression levels and stratifying over tamoxifen use. Clinicopathologic parameters can be found in Supplementary Table S2. Tumor samples were cryosectioned, fixed in 1% form- Data access aldehyde for 20 minutes, and processed for sonication. For each All sequencing data can be found at GEO (GSE81213). ChIP, 10 mg of antibody, and 100 mL of Protein A (for ERa and H3K27ac) and Protein G (for FOXA1 and RNA polymerase II) magnetic beads (Invitrogen) were used. Antibodies raised to Results detect ERa (SC-543; Santa Cruz Biotechnology), RNA polymerase ERa is found at active enhancers in human endometrial tumor II (ab5408; Abcam), H3K27ac (39133; Active Motif), and FOXA1/ specimens 2 (SC-6554; Santa Cruz Biotechnology) were used. The specificity Tamoxifen exposure increases endometrial cancer risk (5). of SC-6554 antibody to detect FOXA1 was verified using immu- The genomic features of ERa in relation to tamoxifen in noprecipitation followed by Western blot using specific antibo- endometrial tumor specimens remain unexplored. To

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Figure 1. ERa binds to active enhancers in tamoxifen-associated endometrial tumor specimens. A, experimental overview. Tumor samples from five endometrial cancer patients were used to ChIP-seq ERa, RNA polymerase II (RNA Pol II), and H3K27ac. B, snapshots depicting ChIP-seq data for ERa (red), RNA polymerase II (purple) and H3K27Ac (blue) from five different endometrial tumor specimens. Genomic locations and read counts are shown. C, heatmap depicting raw reads of ChIP-seq data for ERa (red), RNA polymerase II (purple), and H3K27Ac (blue). ERa peaks are selected and sorted on intensity. Heatmaps are centered on the peak, showing reads within a 5 kb window around the peak. D, normalized average signal (in counts per million, CPM) of data shown in C. E, genomic distribution of ERa ChIP-seq peaks in five endometrial cancers. F, model for the genomic landscape of ERa in tamoxifen-associated endometrial tumors.

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investigate tamoxifen-associated endometrial cancer (5), five shared(Fig.2C).ERa sites shared between breast cancer and endometrial tumors were analyzed from postmenopausal endometrial cancer were robust as illustrated in a heatmap patients who received tamoxifen to treat breast cancer (Fig. (Fig. 2D, for quantification see Supplementary Fig. S4). To 1; Supplementary Table S2 for patient details). Endometrioid identify any potential transcriptional regulators that interact adenocarcinomas were selected because these are generally with ERa at these sites, conserved chromatin binding events ERa-positive, irrespective of whether the endometrial tumor between endometrial and breast tumors were mined for was spontaneous or tamoxifen associated (7). For all five enriched motifs (Fig. 2E). As expected, ERa binding sites in tumors, ChIP-seq was performed for ERa as well as RNA both endometrial and breast tumors were strongly enriched for polymerase II and H3K27Ac, which marks histones both at ESR1 motifs (log10P ¼ 690.776). Interestingly, FOXA1 motifs active promoters and active enhancers (Fig. 1A). Only for tumor were also found highly enriched at ERa binding sites in both D, H3K27Ac ChIP-seq data were absent. Consistently between tumor types (log10P ¼ 342.067). FOXA1 is classically known all tumors, ERa sites were co-occupied by H3K27Ac and RNA to facilitate ERa/chromatin interactions in breast cancer polymerase II (exemplified in Fig. 1B, shown genome-wide (15, 16), but our findings suggest that FOXA1 may play a in Fig. 1C and quantified in Fig. 1D). For tumors C and E, role for ERa functioning in tamoxifen-associated endometrial RNA polymerase II signal was relatively low at ERa sites cancer as well. (Fig. 1B), but still quantifiably detected and enriched over background (Fig. 1C and D). ChIP-seq peaks were successfully Genomic interactions of FOXA1 and ERa in endometrial validated by ChIP-qPCR for all three factors (Supplementary tumor specimens Figs. S2A–S2C). Experiments were technically reproducible, FOXA1 motifs were enriched at ERa binding sites present in with considerable overlap between replicates for the same both breast and endometrial cancer samples. Immunohis- tumor (ERa: Pearson correlation coefficient ¼ 0.68; H3K27Ac: tochemistry analysis validated ERa and FOXA1 expression in Pearson correlation coefficient ¼ 0.88; see Supplementary Figs. both tumor types (Fig. 3A). This was previously reported by S3A–S3C). Between two sections from the same tumor, RNA others for breast cancer (30, 31) as well as endometrial cancer polymerase II ChIP-seq was more variable (Pearson correlation (32–34). Next, we performed ChIP-seq for FOXA1 in the five coefficient ¼ 0.41), suggesting intratumor heterogeneity of this endometrioid adenocarcinoma specimens that were previously factor. Analogous to what was previously reported in breast used to ChIP ERa, RNA polymerase II, and H3K27ac. We cancer specimens (25, 30), endometrial ERa is rarely found at directly compared these FOXA1 binding patterns with ERa promoters and mainly binds distal intergenic regions and profiles within the same tumor (Fig. 3B). For tumor D, FOXA1 introns (Fig. 1E). Cumulatively, these data illustrate that ERa ChIP-seq signal was not observed (tested in two biologic repli- in tamoxifen-associated endometrial tumor specimens shows cates), nor did we detect its mRNA expression (Supplementary the same pattern as previously identified in breast cancer, Fig. S5). FOXA1 binding sites as identified by ChIP-seq could mainly occupying active enhancer regions, positive for both successfully be validated by ChIP-qPCR (Supplementary Fig. H3K27Ac and RNA polymerase II (Fig. 1F). S2D). Furthermore, FOXA1 ChIP-seq was technically robust (Pearson correlation coefficient ¼ 0.67; Supplementary Fig. Conservation of ERa/chromatin binding between surgical S3D). ERa and FOXA1 were found at overlapping genomic specimens of breast and endometrial cancer patients locations in all tested tamoxifen-associated endometrioid ade- ERa binds active enhancer regions in endometrial tumors, nocarcinomas, which were also shared with the endometrioid analogous to what was found in breast cancer. To assess any adenocarcinoma cancer cell line Ishikawa (Fig. 3B and C, potential overlap in the chromatin binding distributions of quantified in D). For FOXA1, 549 sites were shared between ERa between both tumor types, ChIP-seq data from the five two out of four endometrial tumors. For ERa, 4,623 sites were endometrial cancers (Fig. 1) were compared with publicly shared in at least three out of five tumors (Fig. 3C and D). available ERa ChIP-seq datasets we previously generated from However, raw data analyses illustrated that at ERa binding sites, five breast tumors (Fig. 2A; ref. 25). As exemplified, robust ERa FOXA1 signal was generally observed at the same genomic ChIP-seq signal was detected for both tumor types in all 10 regions. This suggests that false negativity was observed due to tumors (Fig. 2B). Consistent with previous reports in breast the peak-calling threshold. Analogous to these findings, FOXA1 cancer (25, 30), a number of ERa binding sites greatly deviated sites were commonly positive for ERa (Fig. 3C and quantified between tumor samples, as we now also show in endometrial in Fig. 3D). Next, potential interacting transcriptional regulators tumors (Fig. 2C). In total, 9,507 ERa interaction sites were were identified by motif enrichment analysis. As expected, fork- found in at least 2 out of 5 breast cancers, while 12,771 ERa head motifs and ESR motifs were found for regions bound by siteswerefoundsharedinatleast2outof5endometrial ERa as well as FOXA1 (Fig. 3E). Furthermore, additional motifs cancers. Between the two tumor types, 3,074 ERa sites were were identified for transcription factors previously described to

Figure 2. ERa chromatin binding landscape in tamoxifen-associated endometrial cancers and breast tumor samples. A, experimental overview. Tumor samples from five tamoxifen-associated endometrial cancer patients (orange) and five breast cancer patients (pink) were processed for ERa ChIP-seq and profiles were directly compared. B, snapshots of ERa ChIP-seq signal in endometrial tumor tissue (orange) and breast tumor tissue (pink). Genomic locations and read count are shown. C, Venn diagram visualizing shared and unique ERa chromatin binding events in five breast tumor samples (pink) and five endometrial tumor samples (orange). For each tumor type, the ERa peaks shared between at least two tumors were used. The number of overlapping peaks between the two tumor types is shown in red. D, heatmap visualization of the shared ERa binding sites between breast (pink) and endometrial (orange) tumor samples, as definedinC.E,ERa binding sites shared between endometrial and breast tumor samples are enriched for ESR1 and FOXA1 motifs. Motifs sequence logos and P values are shown.

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be involved in endometrial cancer, including androgen receptor were expressed in at least 3 out of 4 tumors and found expres- (35), progesterone receptor (36), RORB (37), as well as SOX sedinIshikawacells.Genesthatcorrespondtogenomicregions proteins (38). Cumulatively, these data implicate that FOXA1 bound by ERa and FOXA1 were identified (<20 kb upstream of binds to ERa sites in tamoxifen-associated endometrial cancer transcriptional start site or within gene body; see Materials and that are potentially co-occupied by other transcription factors. Methods) and used for Ingenuity Pathway Analysis to reveal the network in which these proteins operate. As expected, ESR1 P ¼ fi Endometrial FOXA1/ERa sites mark a regulatory transcription ( 8.71E 08) was identi ed as a top upstream regulator. factor hub at enhancers Accordingly, the top enriched network was centered around Endometrial tumors express ERa as well as FOXA1, which ESR1 (Fig. 4E; Supplementary Table S8). All genes in this a fi share substantial overlap in binding sites. Importantly, ERa and pathway were under control of ER signaling, as identi ed by FOXA1 chromatin binding profiles of Ishikawa cells are shared RNA-seq data integration from Ishikawa cells, treated for 4 with primary endometrial tumor tissue samples, suggesting hours with estradiol or vehicle control (Fig. 4E; Supplementary applicability of this cell line model to analyze the correlation Table S4). a between FOXA1 and ERa binding sites and to identify tran- To investigate the transcription factor context in which ER scriptomic regulation at these sites (Fig. 3). In Ishikawa, and FOXA1 operate, we performed unsupervised hierarchical fi approximately 20% of FOXA1 binding sites overlap with ERa clustering based on the identi ed binding patterns for all binding sites (Fig. 4A), yielding a concise list of 599 shared analyzed transcriptional regulators, which resulted in the iden- fi ERa/FOXA1 binding sites in this cell line. Compared to ERa ti cation of distinct functional genomic clusters (Fig. 4F). unique binding sites and sites shared between ERa and FOXA1, Cluster 1 was enriched for transcriptional regulators found at the genomic regions uniquely bound by FOXA1 were markedly promoter regions (Fig. 4G), such as RNA polymerase II, TAF1, higher enriched at promoter regions (Fig. 4B). Next, we ana- YY1, MAX, EGR1, ZBTB7A, USF1, SRF, and CREB1 (Fig. 4F). a lyzed DNA motifs in genomic regions bound exclusively by Cluster 2, which contained ER and FOXA1 binding regions, ERa, FOXA1, or shared by both transcription factors (Fig. 4C). was enhancer enriched (Fig. 4G) and showed clustering among Sites shared by ERa and FOXA1 showed enrichment for ESR1 p300, FOXM1, NR3C1 (Glucocorticoid Receptor), TEAD4, motifs as well as Forkhead transcription factors. As expected, NFIC, TCF12, and CEBPB. These cell line data are in agreement a ERa unique binding sites were devoid of Forkhead motifs, with primary tumor data where ER was found to bind active while FOXA1 unique sites lacked ESR1 motifs, suggesting no enhancers (Fig. 1). Cumulatively, these data implicate genomic a false negativity for the unique binding site subsets. Apart from locations shared by ER and FOXA1 as central genomic hubs ERa and FOXA1 motifs, other transcription factors were found for enhancer action, where multiple transcriptional regulators selectively enriched between the peak subsets. Substantial bind proximal to genes involved in classical estrogen-regulated overlap was found between these motifs and those found in processes. tumors (Fig. 3E). Out of 106 motifs, 85 transcription factors were expressed in Ishikawa cells, based on publicly available Interplay of FOXA1 and ERa in tamoxifen-associated RNA-seq data, which was generated as part of the ENCODE endometrial cancer consortium (for accession numbers of used datasets see Sup- ERa's functional activity is linked with FOXA1 in tamoxifen- plementary Table S4). To provide experimental validation of associated endometrial cancer. We next determined the potential the identified motifs, publicly available ChIP-seq datasets clinical implications of the interplay of FOXA1 and ERa in this where used from Ishikawa cells (for GEO accession numbers context (Fig. 5), using TMAs from the TAMARISK study (7). The see Supplementary Table S4). In accordance with the motif TAMARISK study involves samples from 230 endometrial tumors analyses, CTCF chromatin binding was found at FOXA1 sites, that were collected from patients who were previously diagnosed but not at ERa sites. Most other transcriptional regulators with breast cancer, half of whom received tamoxifen (Fig. 5A; (TEAD4, EP300, FOXM1, MAX, NFIC, RAD21, SRF, TCF12, Supplementary Table S1). We assessed the interval time between USF1) showed signal for unique regions of ERa and FOXA1, breast cancer diagnosis and endometrial cancer diagnosis as well butwasstrongestatregionsco-occupiedbybothFOXA1and as endometrial cancer-related survival. These clinical variables ERa (Fig. 4D; Supplementary Fig. S6 for quantifications). were tested for association with ERa and FOXA1 levels, as deter- Furthermore, these transcription factors were tested for expres- mined by immunohistochemistry (Fig. 5B for example staining). sion analysis in tamoxifen-associated cancers and Ishikawa Scatter plot visualizations illustrated that FOXA1 and ERa pos- cells (Supplementary Fig. S5). All tested transcription factors itive cells in endometrial tumor specimens do not associate

Figure 3. Chromatin binding patterns of ERa and FOXA1 in tamoxifen-associated endometrial tumor specimens. A, breast and endometrial tissues were stained by immunohistochemistry for ERa and FOXA1. Example of ERa and FOXA1 expression in breast and endometrial tumor tissues. Magnification, 200. B, snapshots of ChIP-seq data for ERa (red; five samples) and FOXA1 (green; four samples) in independent tamoxifen-associated endometrial tumors and the endometrial cancer cell line Ishikawa. Read count and genomic locations are shown. C, heatmap visualization of ERa (red) and FOXA1 (green) binding sites in tumors and the endometrial cancer cell line Ishikawa. For ERa, all 4,623 sites found in at least 3 out of 5 tumors were selected. For FOXA1, 549 sites were identified in at least 2 out of 4 tumors. All peaks identified for ERa (top) and FOXA1 (bottom) were analyzed separately, where raw data for ERa and FOXA1 ChIP-seq are shown. Raw read count of all peaks is vertically aligned on the center of the peak region. D, normalized average read count (CPM) of ChIP-seq data visualized in C. Data were centered on the peak regions and include a 2.5-kb window around the peak. The y-axis shows normalized read count. E, scatter plot showing motif enrichment analysis of ERa and FOXA1 ChIP-seq sites depicted in C and D. The individual dots represent the absolute Z-score of enriched motif in log-scale. Two vertical and horizontal dashed gray lines indicate the absolute Z-score corresponding to P value of 0.05 and 0.01 for ERa and FOXA1 binding sites, respectively.

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(Supplementary Fig. S7), suggesting that ERa and FOXA1 expres- based on a single endometrial cancer cell line, while our study is sion levels may identify different patient groups. Therefore, the first to assess genomic behavior of ERa in several surgical tumors were categorized in groups that express only ERa, only specimens of endometrial cancer patients. We show a significant FOXA1, both ERa and FOXA1, or neither. Most endometrial level of ERa overlap between breast cancer samples and endome- tumors are positive for both ERa and FOXA1, irrespective of trial cancer samples, being enriched for ESR1 and FOXA1 motifs. þ treatment (Fig. 5C). FOXA1 /ERa tumors were enriched among With this, our data illustrate that functional characterization of ERa tamoxifen-treated patients at the expense of the tumors that are genomic action can be accelerated by a joined analysis on cell line positive for both ERa and FOXA1 (Fisher exact test: P ¼ 0.018). studied in conjunction with primary human tissue specimens. ERa expression strongly associated with endometrial cancer Analogous to previous observations in breast cancer, endo- patient survival, in agreement with previous immunohistochem- metrial ERa preferably binds enhancers, which were marked by ical reports (39, 40). In contrast to previous reports (33, 34), H3K27ac and RNA polymerase II, suggesting that these enhan- FOXA1 expression did not associate with survival of endometrial cers are active. Furthermore, ERa binding events in endometrial cancer patients, irrespective whether they did (Fig. 5D) or did not tumor specimens are enriched for motifs of well-established receive tamoxifen (Supplementary Fig. S8). Interestingly, tamox- transcriptional regulators in the field of breast cancer, including ifen-treated patients who developed an endometrial tumor had a FOXA1 (15). FOXA1 was previously reported to be expressed in shorter interval time between breast cancer and endometrial endometrial tissue, and downregulated in poorly differentiated cancer when their endometrial tumor expressed ERa and/or endometrial cancer (32) where low FOXA1 is associated with FOXA1 (ERa: P ¼ 0.02, FOXA1: P ¼ 0.02; ERa/FOXA1: P < poor outcome (33, 34). In line with these previous reports, we 0.001; Fig. 5E). For patients who did not receive tamoxifen show that endometrial tumor cells express FOXA1, even though treatment for breast cancer, no significant differences in interval we do not observe an association of FOXA1 with clinical time between breast cancer and endometrial cancer diagnosis outcome in endometrial cancer in our cohort. Because patients were found, based on ERa and FOXA1 levels in the endometrial in our cohort developed both breast cancer as well as endo- tumor (Supplementary Fig. S9). Our data implicate a coordinated metrial cancer (7), we may have enriched for patients with a action between ERa and FOXA1, associated with a short interval genetic predisposition to developing both tumors types. There- time between breast cancer and endometrial cancer development fore, our study population may be intrinsically different from in the tamoxifen-treated cases, without affecting endometrial sporadic cases that only developed endometrial cancer (33). cancer-related survival. Here, we illustrate that FOXA1 chromatin binding sites are shared with ERa in tamoxifen-associated endometrial cancer. Discussion These sites are not exclusively bound by ERa or FOXA1, but are Tamoxifen is a highly successful drug in the treatment of breast in fact regulated through a large multiprotein transcriptional cancer. However, tamoxifen increases the risk of endometrial network, jointly mediating ERa-driven gene profiles in endo- cancer. This direct link between breast cancer treatment and endometrial cancer. metrial cancer development suggests a crucial role for ERa in both Multiple other proteins found to associate with breast cancer tumor types. Tamoxifen has been reported in endometrial cells to outcome have been implicated in endometrial cancer develop- stimulate the recruitment of coactivators to a subset of genes, ment, including PAX2 (10, 12) and SRC1 (9, 41). Here, we show similar to estrogen (9). Between a breast cancer cell line and an that FOXA1 can be added to this list, exposing a bivalent role in endometrial cancer cell line, limited overlap of ERa sites was response to tamoxifen treatment, dependent on tissue type; while observed (13), giving rise to differential gene expression between high FOXA1 expression is a favorable prognostic factor in breast those cell lines following ligand treatment. More recently, the same cancer (21), its expression levels associate with short interval time lab reported shared ERa sites between these cell lines to be between breast cancer diagnosis and tamoxifen-associated endo- associated with strong EREs, inaccessible chromatin regions and metrial cancer development without affecting outcome in this lack of DNA methylation (29). In contrast, non–ERE-mediated cohort. This closely follows parallel observations made for PAX2, ERa action was found dominant in tissue-type specific interactions, and validates inverse relations of transcriptional regulators which co-occurred with other transcription factors and cell-type between breast and endometrial cancer with respect to tumori- specific chromatin accessibility (29). This previous report was genesis and response to endocrine agents.

Figure 4. Comparative genomics between ERa and FOXA1 in endometrial cancer cells Ishikawa. A, Venn diagram visualizing overlap between chromatin binding events for ERa (red) and FOXA1 (green). B, genomic distribution of sites bound by either ERa alone (red), FOXA1 alone (green), or shared by ERa and FOXA1 (brown). C, radar plot, visualizing DNA motif enrichment for genomic sites bound by either ERa (red) or FOXA1 (green) alone, or by both (brown). TheradialdatapointsrepresenttheabsolutevalueofZ-score. The lower bound of the abs Z-score is 3, which corresponds to a P value of 0.00135. Genes expressed in Ishikawa [genes with fragments per kilobase of exon per million fragments mapped (FPKM) > 1] are in black, while genes not expressed are in blue. Heatmap visualization of ChIP-seq ENCODE data for several transcriptional regulators at sites bound by ERa and/or FOXA1. Raw read count of all peaks was vertically aligned, and data were centered on the center of the peak region. Colors correspond to different treatments; cells were either grown in full medium (purple) or hormone deprived, followed by a 1-hour incubation of either DMSO (green), 10 nM estradiol (E2, brown), ethanol (EtOH, blue), or 100 nM dexamethasone (DEX, black). D, Ingenuity Pathway Analysis depicts the top-network model of genes with a shared ERa/FOXA1 site within the gene body or within 20k upstream of the transcriptional transcription start site. The node color indicates the FPKM fold change in Ishikawa cells upon a 4-hour estradiol (E2) stimulation, being either upregulated (red) or downregulated (green) related to control. E, unsupervised clustering of binding sites of all available transcriptional regulators. FOXA1 unique, ERa unique, and shared ERa/FOXA1 sites are analyzed separately. Red, positive correlation; blue, negative correlations. F, genomic distribution of peaks shared between at least four out of nine proteins in either cluster 1 or cluster 2.

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Figure 5. Immunohistochemistry analysis of FOXA1 and ERa in tamoxifen-associated endometrial cancer. A, overview of the study design. Endometrial tumor tissue was analyzed from patients who were previously treated for breast cancer. Half of these patients received tamoxifen for breast cancer treatment, while the other half did not receive any endocrine treatment. B, immunohistochemistry for ERa and FOXA1 showing no (left), low (middle), or high þ þ þ (right) protein expression. C, bar graph representing percentage and absolute numbers of patients that were categorized as ERa /FOXA1 ,ERa /FOXA1 , þ ERa /FOXA1 ,orERa /FOXA1 and grouped in tamoxifen-treated patients (white) and non–tamoxifen-treated patients (black). For statistics, Fisher exact test was used. D, endometrial cancer–speciﬁc survival of tamoxifen-treated patients, categorized as ERaþ/FOXA1þ,ERaþ/FOXA1,ERa/FOXA1þ, or ERa/FOXA1. E, box plot depicting interval time (years) between breast cancer diagnosis and endometrial cancer diagnosis for patients who received tamoxifen. P values were not corrected for multiple testing. Average interval time in years is shown.

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ERa/FOXA1 Cross-talk in Endometrial Cancer

To conclude, we show that endometrial tumor cells express Writing, review, and/or revision of the manuscript: M. Droog, E. Nevedoms- FOXA1, serving the classical ERa-pioneer factor role as was kaya, Y. Kim, P. Gradowska, M. Hauptmann, H. Hollema, M. Mourits, originally identified in breast cancer (15). H. van Boven, S.C. Linn, L. Wessels, F.E. van Leeuwen, W. Zwart Administrative, technical, or material support (i.e., reporting or organiz- Yet, instead of facilitating the inhibitory potential of tam- ing data, constructing databases): M. Droog, K. Schuurman, G. Dackus, oxifen on ERa activity, FOXA1 may enable receptor activation H. Hollema, P. Nederlof, H. van Boven, W. Zwart through tamoxifen in endometrial tissue in postmenopau- Study supervision: W. Zwart sal women. This function of FOXA1 has far-reaching con- sequences, where it may dictate the stimulatory effects of Acknowledgments tamoxifen treatment on endometrial cancer development. The authors thank Ron Kerkhoven, Shan Baban, and Marja Nieuwlandfrom the NKI genomics facility for sample processing and Arno Velds for bioinformatics support. They acknowledge the NKI-AVL Core Facility Molecular Disclosure of Potential Conflicts of Interest Pathology & Biobanking (CFMPB) for supplying NKI-AVL Biobank material The authors declare no conflict of interest. and lab support. The authors thank Joyce Sanders for help with pathological analyses and Jos Jonkers and Jason Carroll for critically reading the manuscript Authors' Contributions and valuable suggestions. Conception and design: M. Droog, F.E. van Leeuwen, W. Zwart Development of methodology: M. Droog, W. Zwart Grant Support Acquisition of data (provided animals, acquired and managed pati- This work was supported by grants from the Dutch Cancer Society and Pink ents, provided facilities, etc.): M. Droog, K.D. Flach, M. Opdam, Ribbon. K. Schuurman, G. Dackus, H. Hollema, M. Mourits, P. Nederlof, H. van Boven, The costs of publication of this article were defrayed in part by the payment advertisement F.E. van Leeuwen of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biostatistics, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. computational analysis): M. Droog, E. Nevedomskaya, Y. Kim, T. Severson, K.D. Flach, P. Gradowska, M. Hauptmann, M. Mourits, S.C. Linn, L. Wessels, Received June 17, 2014; revised March 24, 2016; accepted April 13, 2016; F.E. van Leeuwen, W. Zwart published OnlineFirst May 6, 2016.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Cancer Correction Research Correction: Comparative Cistromics Reveals Genomic Cross-talk between FOXA1 and ERa in Tamoxifen-Associated Endometrial Carcinomas

In the original version of this article (1), the name of the twelfth author, Marian J. Mourits, is incorrect. The name has now been corrected in the recent online HTML and PDF versions of the article. The authors regret this error.

Reference 1. Droog M, Nevedomskaya E, Kim Y, Severson T, Flach KD, Opdam M, et al. Comparative cistromics reveals genomic cross-talk between FOXA1 and ERa in tamoxifen-associated endometrial carcinomas. Cancer Res 2016;76:3773–84.

Published online May 1, 2018. doi: 10.1158/0008-5472.CAN-18-0561 2018 American Association for Cancer Research.

2446 Cancer Res; 78(9) May 1, 2018 Published OnlineFirst May 6, 2016; DOI: 10.1158/0008-5472.CAN-14-1813

Comparative Cistromics Reveals Genomic Cross-talk between FOXA1 and ER α in Tamoxifen-Associated Endometrial Carcinomas

Marjolein Droog, Ekaterina Nevedomskaya, Yongsoo Kim, et al.

Cancer Res Published OnlineFirst May 6, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-1813

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2017/01/07/0008-5472.CAN-14-1813.DC1

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