Published OnlineFirst February 11, 2020; DOI: 10.1158/0008-5472.CAN-19-1382 CANCER RESEARCH | GENOME AND EPIGENOME ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells Adriana C. Rodriguez1,2, Jeffery M. Vahrenkamp1,2, Kristofer C. Berrett1,2, Kathleen A. Clark1,2, Katrin P. Guillen1,2, Sandra D. Scherer1,2, Chieh-Hsiang Yang2,3,4, Bryan E. Welm1,2,5, Margit M. Janat-Amsbury 3,4, Barbara J. Graves1,2,6, and Jason Gertz1,2 ABSTRACT ◥ Estrogen signaling through estrogen receptor alpha (ER) plays a factor in controlling the activity of ER and the growth of endometrial major role in endometrial cancer risk and progression, however, cancer cells. the molecular mechanisms underlying ER's regulatory role in endo- Graphical Abstract: http://cancerres.aacrjournals.org/content/ metrial cancer are poorly understood. In breast cancer cells, ER canres/80/6/1234/F1.large.jpg. genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endome- Endometrial cancer cells trial cancer cells remain unknown. We previously identified ETV4 as ER Wild-type a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Cytoplasm Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Nucleus Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 con- ETV4 tributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER ETV4 -/- nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, Cytoplasm where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an Nucleus Hollow organoid formation important role in estrogen signaling in endometrial cancer cells. Reduced in vivo growth Significance: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important Loss of ETV4 diminishes estrogen signaling, which leads to decreased growth of endometrial cancer cells. Introduction the number of fertility cycles, and obesity, which leads to peripheral estrogen production, all indicate a central role for estrogen signaling Endometrial cancer is the fourth most common cancer among in endometrial cancer development (reviewed in ref. 3). Endome- women and the most common gynecologic cancer, leading to over trial cancer mutations in the ligand binding domain of ER (4, 5), 12,000 deaths in the United States each year (1). Type 1 endometrial which likely confer constitutive activity, implicate ER as the main cancer makes up 85% of cases, and the vast majority of these cases mediator of estrogen signaling in endometrial tumors. Endometrial express estrogen receptor a (ER), a steroid hormone receptor (2). cancer is most commonly treated by hysterectomy; however, pro- Risk factors such as unopposed estrogen therapy, tamoxifen treat- gestins, synthetic progesterone, serve as an effective alternative ment for breast cancer, lifetime exposure to estrogens as captured by treatment (6). Progestins bind to and activate progesterone recep- tor, which antagonizes the mitogenic effects of ER (7). These risk factors and treatment strategies for endometrial cancer point to ER 1Department of Oncological Sciences, University of Utah, Salt Lake City, Utah. as a key oncogene in the disease. 2 3 Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah. Division of Although ER plays an important role in endometrial cancer, little is Gynecologic Oncology, Department of Obstetrics and Gynecology, University of understood about ER's molecular actions in endometrial cancer cells. Utah, Salt Lake City, Utah. 4Department of Bioengineering, University of Utah, Salt Lake City, Utah. 5Department of Surgery, University of Utah, Salt Lake City, Many of the molecular details concerning ER have come from Utah. 6Howard Hughes Medical Institute, Chevy Chase, Maryland. extensive work in breast cancer (8–15), where ER has a critical oncogenic role. These findings include regulatory cofactors that ER Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). utilizes to regulate gene expression (16 18). In addition, FOXA1 and GATA3 have been shown to be necessary pioneer factors that maintain Corresponding Author: Jason Gertz, University of Utah, Huntsman Cancer accessible chromatin for ER in breast cancer cells (19–21). ER binds to Institute, 2000 Circle of Hope, Salt Lake City, UT 84112. Phone: 801-213-5662; – fi E-mail: [email protected] the genome in a cell type speci c manner with disparate binding between breast and endometrial cancer cells (14, 22); therefore, Cancer Res 2020;80:1234–45 different transcription factors are likely to control ER binding in doi: 10.1158/0008-5472.CAN-19-1382 endometrial cancer cells. In concordance with this idea, FOXA1 and Ó2020 American Association for Cancer Research. GATA3 exhibit low expression in endometrial tumors (4). As ER has a AACRJournals.org | 1234 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 11, 2020; DOI: 10.1158/0008-5472.CAN-19-1382 ETV4 Is Crucial for Estrogen Signaling in Endometrial Cancer different set of genomic binding sites and FOXA1 only overlaps with were conducted following approved Institutional Animal Care and 8% of ER's binding sites in endometrial cancer cells and tumors, Use Committee protocols. In brief, endometrial cancer patient compared with 57% in breast cancer cells (14, 22), it is likely that specimens were collected in the operating room by the Bioreposi- different transcription factors are responsible for controlling ER's tory and Molecular Pathology Shared Resource and transported in genomic interactions in endometrial cancer cells. ice-cold RPMI-1640 medium (ATCC) to the lab. Each tissue In a previous study, we identified ETV4, an ETS factor, as playing a specimen was mechanically separated into multiple pieces using potential role in endometrial cancer–specific ER binding (14). De novo a sterile disposable scalpel (Andwin Scientific). A minimum of three motif discovery of ER binding sites specific to endometrial cancer cells 6- to 8-week-old, female nude mice (nu/nu; Jackson Laboratories) identified the ETS factor motif. Of the ETS factors, ETV4 is specifically were used for tumor implantation. Animals were anesthetized by expressed in the endometrial cancer cell line Ishikawa, as opposed to intraperitoneal injection of xylazine–ketamine mixture at a dose of the ER-positive breast cancer cell line T-47D. Chromatin immuno- 0.1 mg/kg and a subcutaneous analgesia, buprenophine (Reckitt precipitation sequencing (ChIP-seq) in Ishikawa cells revealed that Benckiser Pharmaceuticals), at a dose of 0.1 mg/kg. A longitudinal ETV4 overlaps with 45% of ER bound sites (14); however, the 1-cm incision was made in the lower midline of the abdominal wall functional importance of this overlap has not been tested. The to penetrate the skin and peritoneum and then the uterus was potential role of ETV4 in controlling ER binding in endometrial exposed. A 2-mm incision was made on one horn of the mouse cancer is an intriguing possibility, as other members of the ETS family uterus. Tumor fragment (4mm3) was implanted into the mouse have been shown to have pioneering capabilities for steroid hormone uterus under the microscope, and the incision was closed using 6-0 receptors. In prostate cancer, overexpression of ETV1, an ETS factor prolene suture (Ethicon). The peritoneum and abdominal wall were closely related to ETV4, has been shown to reprogram androgen sutured using 4-0 PERMA-HAND silk sutures and 4-0 polyglycolic receptor genome binding and associated transcription regulation, acid sutures (Ethicon), respectively, and then disinfected with leading to the formation of prostatic intraepithelial neoplasia (23), alcohol/iodine. The nude mice were not given supplemental estro- and contributing to more aggressive tumors (24). Similar effects have gen. E2 was monitored in the blood and found to be in the range of been shown for ERG, another ETS factor commonly overexpressed in 25 to 50 pmol/L; however, because the patient-derived xenografts prostate cancer (25). The mechanisms underlying these effects on (PDX)wereimplantedintheorthotopicsite,E2levelsmayhave androgen receptor are still unclear; however, a physical interaction has been higher in the tumor environment. Tumor size was measured been observed between ETV1 and androgen receptor (23). Though twice per week. Necropsy was performed once tumor size reached ETS factors in prostate cancer are being widely studied, the functional 2 cm in diameter. Clinical attributes can be found in Supplementary role of ETV4 in ER's genomic binding in endometrial cancer has not TableS1.Modelsareavailableuponrequest. been evaluated. In this study, we use CRISPR/Cas9 to knockout ETV4 in Ishikawa Generation of knockout lines cells in order to determine the importance of ETV4 in controlling ER Ishikawa cells were grown in a 6-well plate in full media and genomic binding. Despite having no effect on ER protein levels, ETV4 cotransfected with the PEA3 (alias for ETV4) CRISPR/Cas9 KO knockout led to greatly reduced ER binding, with the majority of sites plasmid (Santa Cruz, sc-422185) and the PEA3 HDR plasmid (Santa exhibiting a significant reduction in ChIP-seq signal. Consistent with Cruz, sc-422185-HDR) using Lipofectamine 3000. Cells were treated the reduction in ER binding, the transcriptional response to 17b- with puromycin at 1 mg/mL beginning 72 hours after transfection. estradiol (E2) was diminished due to ETV4 knockout. Chromatin RFP-positive single cells were sorted using fluorescence-activated cell accessibility was also reduced, but only at a subset of sites, suggesting sorting into each well of a 96-well plate. Screening for knockout clones a minimal pioneering role for ETV4 in endometrial cancer cells.