TARGETING ESTROGEN RECEPTOR AS A STRATEGY FOR PERSONALIZED MEDICINE IN OVARIAN CANCER by Courtney Lynn Andersen A.A., Liberal Arts & Sciences, Middlesex Community College, 2008 B.S., Biological Sciences, University of Massachusetts Lowell, 2011 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of PhD in Molecular Pharmacology University of Pittsburgh artin 2016 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Courtney L. Andersen It was defended on February 16, 2016 and approved by Don DeFranco, PhD, Professor, Pharmacology & Chemical Biology Adrian Lee, PhD, Professor, Pharmacology & Chemical Biology, Anda Vlad, MD, PhD, Professor, Obstetrics, Gynecology, & Reproductive Sciences Robert Edwards, MD, Chair, Obstetrics and Gynecology, Magee-Womens Hospital of UPMC Dissertation Advisor: Steffi Oesterreich, PhD, Professor, Pharmacology & Chemical Biology ii Copyright © by Courtney L. Andersen 2016 iii TARGETING ESTROGEN RECEPTOR AS A STRATEGY FOR PERSONALIZED MEDICINE IN OVARIAN CANCER Courtney L. Andersen, PhD University of Pittsburgh, 2016 Ovarian cancer comprises a diverse set of diseases that are difficult to detect and treat successfully. Improving outcomes for ovarian cancer patients is contingent upon identifying targeted, individualized therapeutic strategies. One promising but under-utilized target is estrogen receptor-alpha (ER). ER is expressed in ~70% of epithelial ovarian cancers and epidemiologic studies implicate a role for estrogen in ovarian tumorigenesis. Further, clinical data suggest that a subset of ovarian cancer patients benefit from endocrine therapy. We hypothesized that ER drives development and progression of a subset of ovarian tumors and that outputs of ER function would identify patients who respond to endocrine therapy. We assessed endocrine response and mechanisms of ER signaling in models and clinical samples of high- grade serous ovarian cancer (HGSOC). These studies suggested that expression of ER target genes (ERTGs) reflect active ER in HGSOC and correspond with endocrine responsiveness. In light of this, we profiled ERTG expression to evaluate changes in ER signaling during the progression from benign endometriosis to endometriosis-associated cancer (EAOC). This analysis suggested that canonical ER signaling becomes largely inactivated during this transformation and that de-repressed genes (e.g. FGF18, ESR2) may contribute to the evolution of EAOC. Finally, we compared ER and ERTG expression between serous and mucinous low malignant potential (LMP) tumors. Serous LMP tumors have high expression of ER and several ERTGs (e.g. GREB1). Taken together, our findings describe biomarkers that could identify iv ovarian cancer patients across multiple disease subtypes who would benefit clinically from endocrine therapy. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 OVARIAN CANCER .......................................................................................... 1 1.1.1 Heterogeneity of epithelial ovarian cancer ................................................. 2 1.1.2 Ovarian cancer treatment paradigms ......................................................... 3 1.1.3 Targeted therapeutics in ovarian cancer .................................................... 4 1.1.4 Opportunities for personalized medicine in ovarian cancer ..................... 6 1.2 ESTROGEN IN OVARIAN CANCER PATHOBIOLOGY ........................... 7 1.2.1 Targeting estrogen receptor in cancer ........................................................ 8 1.2.2 Estrogen exposure and ovarian cancer risk ............................................... 9 1.2.3 Clinical trials of endocrine therapy in ovarian cancer ............................ 11 1.2.4 Identifying endocrine-responsive ovarian cancer .................................... 13 1.3 ESTROGEN REPEPTOR SIGNALING IN OVARIAN CANCER ............. 14 1.3.1 Overview of estrogen receptor signaling................................................... 14 1.3.2 Interplay between ER and PR in ovarian cancer .................................... 16 1.3.3 Preclinical studies of ER signaling in ovarian cancer.............................. 17 1.3.4 Novel models of ovarian cancer ................................................................. 19 vi 1.4 IMPLICATIONS FOR PERSONALIZED THERAPY FOR OVARIAN CANCER ............................................................................................................................. 21 2.0 BIOMARKERS OF ER FUNCTION REFLECT ENDOCRINE RESPONSE IN HIGH-GRADE SEROUS OVARIAN CANCER ..................................................................... 22 2.1 INTRODUCTION ............................................................................................. 22 2.2 MATERIALS AND METHODS ...................................................................... 23 2.2.1 Cell culture .................................................................................................. 23 2.2.2 Hormone deprivation.................................................................................. 24 2.2.3 In vitro proliferation and viability assays ................................................. 24 2.2.4 Cell line gene expression analyses ............................................................. 25 2.2.5 Xenograft studies ........................................................................................ 26 2.2.6 Immunoblots ................................................................................................ 26 2.2.7 Ex vivo cultures for rapid assessment of endocrine response ................. 27 2.2.8 Immunohistochemistry ............................................................................... 28 2.2.9 Development of the E2sig and EndoRx panel .......................................... 29 2.2.10 Analysis of clinical specimens .................................................................... 30 2.2.11 Statistical methods ...................................................................................... 31 2.3 RESULTS ........................................................................................................... 31 2.3.1 Endocrine response in HGSOC cell lines.................................................. 31 2.3.2 Endocrine response in HGSOC patient-derived xenografts ................... 36 2.3.3 Identifying genes associated with clinical endocrine response ............... 41 2.4 DISCUSSION ..................................................................................................... 45 3.0 UNDERSTANDING ER SIGNALING IN OVARIAN CANCER ........................ 48 vii 3.1 INTRODUCTION ............................................................................................. 48 3.2 MATERIALS AND METHODS ...................................................................... 49 3.2.1 Cell culture .................................................................................................. 49 3.2.2 Transient knockdown ................................................................................. 50 3.2.3 Gene expression analyses in HGSOC cell lines ........................................ 50 3.2.4 Immunoblots ................................................................................................ 50 3.3 RESULTS ........................................................................................................... 51 3.3.1 Evaluating DEPTOR as a potential mediator of endocrine response .... 51 3.3.2 Using changes in ER signaling between 2-D and ULA to identify ERTGs critical to growth ........................................................................................................ 52 3.3.3 Identifying pathways that crosstalk with ER ........................................... 56 3.3.4 Evaluating crosstalk between ER and IL-6/STAT signaling .................. 57 3.4 DISCUSSION ..................................................................................................... 59 4.0 EVOLUTION OF ESTROGEN SIGNALING IN THE PROGRESSION FROM ENDOMETRIOSIS TO ENDOMETRIOSIS-ASSOCIATED OVARIAN CANCER ......... 63 4.1 INTRODUCTION ............................................................................................. 63 4.2 MATERIALS AND METHODS ...................................................................... 64 4.2.1 Clinical specimens ....................................................................................... 64 4.2.2 RNA extraction and NanoString analysis ................................................. 65 4.2.3 Immunohistochemistry ............................................................................... 66 4.2.4 Statistical analysis ....................................................................................... 66 4.2.5 Gene set enrichment analysis (GSEA) ...................................................... 67 4.3 RESULTS ........................................................................................................... 67 viii 4.3.1 Identification of differentially expressed genes between normal, endometriosis, and cancer tissues ............................................................................. 67 4.3.2 Changes in hormone receptor expression and canonical ER signaling in EAOC…