Abstract 4800: Epigenetic changes in normal and cancer prostate cells grown as prostatospheres

Background: Normal and cancer prostate cells grown in suspension cultures or low attachment media grow as spheroids and are called Prostatospheres. These cultures when maintained as self-renewing spheres express many of the stem cell markers and are useful models to understand stem cell differentiation and pathways that could be responsible for cancer cell growth and/or resistance to therapy. We are interested in studying the epigenetic signatures that may be different from monolayer cultures to cells grown as sphere cultures such that we can identify prostate specific genes in prostate stem cell differentiation and growth. Methods: We have established successful culture systems to grow and maintain Prostatospheres (PSs) from normal (RWPE1/2) and prostate cancer (DU-145, PC3, LnCaP) cell lines. After several generations in sphere cultures, the cells were collected and subjected to analysis for promoter DNA methylation. We used a 24-gene, 96 well Methyl Profiler TM DNA methylation PCR platform array from SABiosciences which provides a fast and reliable quantitative DNA methylation analysis without bisulfite conversion. The percent of methylated, unmethylated and intermediate methylated genes were determined in both monolayer and sphere cultures. The array comprises of prostate cancer related genes that belong to the family of tumor suppressors, apoptosis, cell cycle, and genes involved with growth, differentiation and development. Finally, we confirmed the methylation status of candidate genes from the array analysis using Methylated Specific Primers (MSP) corresponding to the candidate gene. Next we examined the effect of demethylating agent AZA (5-aza-2’-deoxycytidine) and Histone deacytlase inhibitor TSA (Trichostatin A) to ascertain the reversal of methylation of candidate genes and their function in monolayer and sphere cultures. Results: Methylation specific array data show a significant shift toward increase in methylation of several genes in PSs compared to monolayer cultures. Of the 24 genes examined, more are hypermethylated in DU145 PSs (82%) than they are in DU145 monolayers (52%). Of the 10 hypermethylated genes, 7 genes (BRCA1, CDH1, CDKN1C, CDKN2A, PRDM2, RASSF1, and TP73) are tumor suppressor genes, 1 gene (PYCARD) is an apoptotic gene, 1 gene (CCND2) is a cell cycle regulation gene and 1 receptor/transcription factor (ERS1). MSP-PCR confirmed the reversal of methylation upon AZA and TSA treatment. RT-PCR data showed that several genes tested in PSs showed significant increase in their mRNA expression upon AZA or TSA treatment as single agents. Conclusion: Our preliminary data confirm that several genes are hypermethylated in PSs compared to monolayer cultures and more predominantly are those that belong to the family of tumor suppressor and apoptotic genes. Suggesting that silencing of these genes may be an important adaptation for prostate cancer stem cells during differentiation and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2011-4800