Abstract B53: Estrogen receptor beta 2 (ERβ2)- p53- FOXM1 signaling axis in high-grade serous ovarian cancer (HGSOC): Underlying mechanisms and implications for resistance to therapy

Abstract

Abstract Background and Goals: HGSOC is the most aggressive ovarian carcinoma and accounts for two-thirds of deaths from ovarian cancer. The Cancer Genome Atlas (TCGA) analysis showed that p53 is mutated in about 96% of HGSOC and forkhead box M1 (FOXM1) protein signaling network is altered in 87% of cases. These abnormalities significantly contribute to resistance of tumors to chemotherapy. Recent studies have also shown that estrogen receptor beta (ERβ) is expressed at high levels in HGSOC; however, there is no consensus weather ERβ is pro- or anti-tumorigenic. Due to the established roles of FOXM1 in chemotherapy resistance and our observation that ERβ2 regulates FOXM1 expression, we hypothesized that ERβ2 status may be of clinical relevance for the treatment of HGSOC. The objective of the current study was to delineate the mechanisms by which ERβ2-mutant p53-FOXM1 signaling axis impacts resistance to carboplatin therapy. Experimental Procedures: Proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were used to analyze protein-protein interactions. Transcription factor binding to endogenous gene promoters was determined by chromatin immunoprecipitation (Ch-IP) assay. RNAi technology was used to downregulate expression of specific proteins. Endogenous gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Apoptosis was analyzed by Annexin V staining and PARP-cleavage assays. Cell proliferation was measured by colony formation assay. Expression levels of ERβ, p53, and FOXM1 in HGSOC patient tumor tissue microarrays (TMAs) were determined by immunohistochemistry (IHC). Unpublished Results: Co-IP showed that ERβ2 binds to mutant p53. PLA demonstrated that ERβ2 is in a protein complex with mutant p53 in situ and qRT-PCR showed that ERβ2 in concert with mutant p53 upregulates FOXM1 gene transcription. Effect of ERβ2 on FOXM1 transcription was abrogated in cells that are p53-null. ChIP assay showed ERβ2 binding to FOXM1 gene promotor and regulating its gene expression. Concomitantly, expression of downstream targets of FOXM1 that are responsible for cell proliferation and DNA damage repair was upregulated. Furthermore, ERβ and p53 regulate expression of each other in a feedback loop. Knocking down ERβ2 in combination with carboplatin resulted in increased apoptosis and deceased proliferation in HGSOC cells. Importantly, APR-246 (PRIMA-1MET) (a small molecule that binds and converts mutant p53 to wild-type p53 conformation) in combination with ERβ2 knockdown led to decreased FOXM1 protein levels and increased apoptosis. Consistent with our data from cell model studies, IHC data on patient tumor TMAs showed that ERβ2 expression has linear correlation with FOXM1 expression in the primary and metastatic tumors. Analysis of correlation of these data with clinical characteristics such as therapeutic response and patient survival is under way. Conclusions: Our experiments have uncovered a novel (ERβ2)-p53-FOXM1 signaling axis in driving HGSOC and contributing to resistance to therapy. The data suggest that this signaling axis could be a potential therapeutic target. Citation Format: Chetan Oturkar, Austin Miller, Emese Zsiros, Gokul M. Das. Estrogen receptor beta 2 (ERβ2)- p53- FOXM1 signaling axis in high-grade serous ovarian cancer (HGSOC): Underlying mechanisms and implications for resistance to therapy. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B53.