Author Manuscript Published OnlineFirst on January 10, 2017; DOI: 10.1158/1078-0432.CCR-16-1501 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Active estrogen receptor-alpha signaling in ovarian cancer models and clinical specimens Courtney L. Andersen1,2,3,12, Matthew J. Sikora1,3, Michelle M. Boisen3,4, Tianzhou Ma5, Alec Christie3, George Tseng5, Yongseok Park5, Soumya Luthra6, Uma Chandran6, Paul Haluska7, Gina M. Mantia- Smaldone8, Kunle Odunsi9, Karen McLean10, Adrian V. Lee1,3, Esther Elishaev11, Robert P. Edwards4, Steffi Oesterreich1,2,3 Affiliations: 1Department of Pharmacology & Chemical Biology, University of Pittsburgh; 2Molecular Pharmacology Training Program, University of Pittsburgh; 3Women’s Cancer Research Center, University of Pittsburgh Cancer Institute; 4Department of Obstetrics, Gynecology, & Reproductive Sciences, Magee- Womens Hospital of UPMC; 5Department of Biostatistics, University of Pittsburgh; 6Department of Biomedical Informatics, University of Pittsburgh; 7Oncology, Merck Research Laboratories; 8Department of Surgical Oncology, Fox Chase Cancer Center; 9Department of Gynecologic Oncology, Roswell Park Cancer Institute; 10Division of Gynecologic Oncology, University of Michigan; 11Department of Pathology, Magee- Womens Hospital of UPMC; 12Present address: IMED Oncology, AstraZeneca Corresponding Author: Steffi Oesterreich, PhD.: B411 Womens Cancer Research Center, University of Pittsburgh Cancer Institute, Magee-Womens Research Institute, 204 Craft Ave, Pittsburgh, PA 15213. Tel: (412)641-8555, Fax: (412)641-2458, [email protected] Keywords: ovarian cancer, high-grade serous cancer, estrogen receptor-alpha, endocrine therapy, tumor explants, ultra-low attachment, fulvestrant, predictive biomarkers Microarray data (GEO): GSE81612 COI: Dr. Andersen is now an employee of AstraZeneca Pharmaceuticals. Abbreviations: HGSOC, high-grade serous ovarian cancer; ERα, estrogen receptor-alpha; PDX, patient- derived xenograft; 4OHT, 4-hydroxytamoxifen; AI, aromatase inhibitor Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 10, 2017; DOI: 10.1158/1078-0432.CCR-16-1501 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 Translational Relevance High-grade serous ovarian cancer (HGSOC) is a malignancy with extremely poor prognosis and limited therapeutic options. Targeting estrogen receptor-alpha has shown promise in laboratory models and in clinical trials but identification of the appropriate patient subset has remained elusive. We characterized endocrine response in cell line and patient-derived HGSOC models to identify features associated with estrogen-responsive HGSOC. In these studies, we observed that a subset of HGSOC models require estrogen for growth and survival. Further, we identified genes (e.g. the ER-alpha target IGBP3) which were associated with clinical endocrine response. We also determined that fulvestrant may be more effective than tamoxifen at blocking cell proliferation in HGSOC. Our data may enable the identification of patients with ovarian cancer who would benefit from endocrine therapy. Abstract Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha in ~80% of HGSOC and some small but promising clinical trials of endocrine therapy, estrogen receptor-alpha has been understudied as a target in this disease. We sought to identify hormone-responsive, estrogen receptor-alpha-dependent HGSOC. Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions (2- D, 3-D, forced suspension) and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of estrogen receptor-alpha target genes. Expression of this panel and estrogen receptor-alpha H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response. Results: Proliferation is estrogen receptor-alpha-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3-D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as estrogen receptor-alpha targets. The selective estrogen receptor-alpha down-regulator (SERD) Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 10, 2017; DOI: 10.1158/1078-0432.CCR-16-1501 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 fulvestrant is more effective than tamoxifen in blocking estrogen receptor-alpha action. Estrogen receptor- alpha H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3. Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g. IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 10, 2017; DOI: 10.1158/1078-0432.CCR-16-1501 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 1 Introduction 2 High-grade serous ovarian cancer (HGSOC) is an aggressive and often lethal disease with limited 3 options for therapy. HGSOC typically responds to surgical debulking and platinum-based chemotherapy as 4 first-line treatment but the majority of patients relapse and ultimately succumb to the disease (1). Identifying 5 targeted, individualized treatment strategies for ovarian cancer will be essential for improving patient 6 survival. 7 One promising but understudied therapeutic target for HGSOC is estrogen receptor-alpha (ERα). 8 ERα is expressed in ~80% of HGSOC (2–4), and estrogen exposure (e.g. oral contraceptive use, hormone 9 replacement therapy) affects the risk of ovarian cancer (4–6). Preclinical studies have shown that estrogen 10 can promote proliferation and migration of HGSOC cell lines and mouse models and in part these effects 11 are blocked by antiestrogens (7–12). 12 Several clinical trials have evaluated endocrine therapy in ovarian cancer. Trials were small (n=14- 13 105 patients), patients were heavily pre-treated with chemotherapy, and ERα status was infrequently used 14 as an inclusion criterion (4). Nevertheless, in each trial a subset of patients benefited from tamoxifen (~20% 15 of patients (13–20)), aromatase inhibitors (~17% (21–24)), or fulvestrant (~40% (25)). Though consistent 16 inclusion of ERα-status may improve response rates, superior biomarkers for ERα function and endocrine 17 responsiveness are needed. 18 We sought to identify HGSOC likely to be ERα-dependent and endocrine responsive. Therefore, we 19 comprehensively characterized estrogen and antiestrogen response with regards to growth, survival, and 20 gene expression in HGSOC cell lines and patient-derived xenograft (PDX) explants. Based on those data, 21 we built an assay for endocrine response and profiled tumors from patients with ovarian cancer who 22 received endocrine therapy to identify genes associated with clinical response. Here we show that ERα H- 23 score with expression of other biomarkers (e.g. IGFBP3) can identify patients with HGSOC who benefit from 24 endocrine therapy. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 10, 2017; DOI: 10.1158/1078-0432.CCR-16-1501 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 5 25 Materials and Methods 26 Antibodies and reagents 27 Chemicals: estradiol (E2) (Sigma-Aldrich, St. Louis, MO); 4-hydroxytamoxifen (4OHT) (Sigma- 28 Aldrich); ICI 182,780 (fulvestrant) (Tocris Bioscience, Bristol, UK); staurosporine (STS) (Tocris Bioscience); 29 and Z-VAD (Tocris Bioscience). E2, 4OHT, and fulvestrant were solubilized in 200-proof ethanol prior to 30 use. STS and Z-VAD were solubilized in sterile DMSO. 31 Antibodies: ERα 6F11 clone (Leica Biosystems, Buffalo Grove, IL), ER (SP1 Clone, Biocare 32 Medical, Concord, CA), BrdU (Bu20a clone, Cell Signaling Technology, Danvers, MA), Ki67 (M1B clone, 33 Dako, Carpinteria, CA), Tubulin (Sigma-Aldrich), beta-actin (Sigma-Aldrich). 34 35 Cell lines and culture conditions 36 PEO1, PEO4, OVSAHO, and MCF-7 cells were maintained in DMEM (Invitrogen) + 10% fetal bovine 37 serum (FBS) (Gibco). OVCA432 cells were maintained in RPMI-1640 + 10% FBS. Cell line identity was 38 verified by short tandem repeat (STR) profiling. PEO1 and PEO4 cells were derived from the same patient 39 (26), PEO1 after her first recurrence and PEO4 after the tumor became platinum-resistant. 40 Hormone deprivation was performed as described previously (27) using IMEM + 10%