Molecular and Cellular Pathobiology Comparative Cistromics Reveals Genomic Cross-talk between FOXA1 and ERa in Tamoxifen-Associated Endometrial Carcinomas Marjolein Droog 1 , Ekaterina Nevedomskaya 1,2 , Yongsoo Kim 1,2 , Tesa Severson 1 , Koen D. Flach 1 , Mark Opdam 1 , Karianne Schuurman 1 , Patrycja Gradowska 3 , Michael Hauptmann 3 , Gwen Dackus 1 , Harry Hollema 4 , Marian J. Mourits 5 , Petra Nederlof 6 , Hester van Boven 6 , Sabine C. Linn 1,7 , Lodewyk Wessels 2,8 , Flora E. van Leeuwen 3 , and Wilbert Zwart 1 Abstract Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERa) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERa transcriptional action in breast and endome- trial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assess- ment of ERa-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERa was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERa binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERa binding sites proximal to genes involved in classical ERa target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERa and FOXA1 together with the enhancer- enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERa expression was associated with a longer interval between breast cancer and the emergence of endome- trial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERa in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERa are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773–84. Ó2016 AACR. Introduction Of all breast tumors, roughly 75% depend on ERa for cell proliferation and tumor progression. Consequently, most breast cancer treatment modalities are aimed to inhibit ERa activity. Tamoxifen is the most widely applied hormonal therapy, which acts through competitive inhibition of ERa's natural ligand estro- gen (1). Although tamoxifen inhibits ERa in breast cells, it stimu- lates ERa in certain other tissues, including the endometrium, specifically in a low estrogen environment, i.e., postmenopausal women (2–6). Due to tissue-selective action of tamoxifen, postmenopausal breast cancer patients on this drug have an increased risk of endometrial cancer development by 2- to 7-fold, depending on the duration of use (4, 5, 7, 8). This increased ERa activity was linked with an altered expression level of coactivator SRC1 (9) and PAX2 (10) in endometrial cancer cells. However, manip- ulating expression levels of either SRC1 or PAX2 in breast cancer cell line models did not support these findings (11, 12), implying that these proteins are not the sole drivers of agonistic features of tamoxifen observed in endometrial tissue. In addition, ERa transcriptional regulation in endometrial cancer has exclusively been studied using cell lines (13) while ERa genomic behavior in primary human endometrial tumors remains unexplored. For ERa to bind the chromatin in breast cancer cells, it requires FOXA1 (14–16). FOXA1 is a pioneer factor, regulating chromatin accessibility and thereby enables ERa-dependent gene activation and proliferation of breast cancer cells (14). FOXA1 was previously identified as one of the luminal breast cancer–defining transcription factors (17, 18), and its expres- sion correlates with a favorable outcome in breast cancer (19–21). In ERa-negative breast cancer cells, exogenous intro- duction of ERa and FOXA1 along with GATA3 was sufficient to reprogram these cells toward hormone responsiveness (22). 1 Department of Molecular Pathology, Netherlands Cancer Institute, Amsterdam, the Netherlands. 2 Department of Molecular Carcinogen- esis, Netherlands Cancer Institute, Amsterdam, the Netherlands. 3 Department of Epidemiology, Netherlands Cancer Institute, Amster- dam, the Netherlands. 4 Department of Pathology, University Medical Center Groningen, Groningen, the Netherlands. 5 Department of Gyne- cological Oncology, University Medical Center Groningen, Groningen, the Netherlands. 6 Department of Pathology, Netherlands Cancer Insti- tute, Amsterdam, the Netherlands. 7 Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands. 8 Faculty of EEMCS, Delft University of Technology, Delft, the Netherlands. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). E. Nevedomskaya and Y. Kim contributed equally to this article. Corresponding Author: Wilbert Zwart, the Netherlands Cancer Institute, Department of Molecular Pathology, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Phone: 31-20-512-2101; E-mail: w.zwart@nki.nl doi: 10.1158/0008-5472.CAN-14-1813 Ó2016 American Association for Cancer Research. Cancer Research www.aacrjournals.org 3773 Downloaded from http://aacrjournals.org/cancerres/article-pdf/76/13/3773/2734002/3773.pdf by guest on 29 May 2023