Abstract 5191: Functional role of EZH2 in neuroendocrine prostate cancer

Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive malignant variant of prostate cancer (PCa). Since there is no effective treatment available for NEPC to date, devising a therapeutic intervention requires identifying the mechanisms of how prostate cancer cells gain neuroendocrine phenotype. Evidence suggest that neuroendocrine differentiation is associated with elevated expression of Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2) that functions to regulate histone H3 methylation and causes transcriptional repression of target genes. We hypothesize that the elevated expression of EZH2 in NEPC may render the tumors cells an undifferentiated state which enables the lineage switch and trans-differentiation into neuroendocrine malignancies. Method and Results: First, we extracted microarray data to analyze the expression of polycomb genes during PCa progression, using Gene Expression Omnibus (GEO) database from NCBI. The expressions of Polycomb genes (EZH2, EED, SUZ2, CBX1) were up-regulated in metastatic castrate-resistant prostate cancer and the increased EZH2 expression was in concert with elevated expressions of NEPC marker (CHGA, FOXA2, SOX2) and decreased expression of FOXA1, a prostate differentiation marker. Additionally, we conducted IHC staining in sections derived from benign prostate hyperplasia, low- and high- grade prostate adenocarcinoma, patient-derived NEPC xenograft tumors (LuCaP), and mouse models of NEPC (12T10 LADY and TRAMP). EZH2 and FOXA2 were overexpressed in all NEPC tumors while FOXA1 was abundant in prostate adenocarcinoma but decreased in NEPC. Also, we cultured prostate adenocarcinoma, LNCaP cells in androgen-depleted media and analyzed the gene expression of major polycomb genes and NEPC markers by qPCR. The expressions of Polycomb genes (BMI1, RING1a, CBX1) were induced by androgen depletion, concurrent with the induction of NEPC gene, CD56. Furthermore, we stably knocked down EZH2 in DU145 and PC3 cell lines (both have NEPC features) using shRNA and measured both the RNA and protein level of EZH2, PCa and NEPC markers using qPCR and western blot. We found that the levels of AR, FOXA1 and E-cadherin are upregulated in both DU145 and PC3 cells when EZH2 was knocked down. Conclusion: Our data support that EZH2 is functionally involved in the development of NEPC. Future direction: We will overexpress EZH2 in LNCaP cells to study if elevated EZH2 expression promotes NEPC. We will also perform RNA-seq and ChIP-seq to study how the altered expression of EZH2 affects the global transcriptome and identify the downstream target genes of EZH2 in PCa. Additionally, we will conduct in vivo animal experiments to determine EZH2’s role in NEPC using NEPC PDX models and transgenic mouse models. Findings from this research would provide detailed insights into developing therapeutics against EZH2 mediated NEPC. (Funding source: R03 and FWCC). Citation Format: Md Imtiaz Khalil, Shu Yang, Anthony Blankenship, Zachary Connelly, Xiuping Yu. Functional role of EZH2 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5191.