279 EPIGENETIC REGULATION IN INHIBITION OF PROSTATE-DERIVED ETS FACTOR, A TUMOR METASTASIS SUPPRESSOR, IN ADVANCED PROSTATE CANCER A TUMOR METASTASIS SUPPRESSOR, IN ADVANCED PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: There is, at present, no effective treatment for intervention in metastatic prostate cancer. In our recent studies we demonstrated that Prostate-derived Ets factor (PDEF) expression is decreased, and even lost in high grade prostate cancer. Using in vitro assays we show that reintroduction of PDEF results in phenotypic reversal from aggressive to a less morbid phenotype in prostate cancer cells. Since a common mechanism of tumor suppressor inactivation is by promoter hyper-methylation, the objective of this study was to determine if and how PDEF is regulated epigenetically through promoter methylation. METHODS: LNCaP cells (Androgen dependent), LNCaP C4-2B (Androgen un-responsive) and PC3 (Androgen independent) prostate cancer cell lines were maintained in their respective growth media supplemented with 10% Fetal Bovine Serum and antibiotics. PDEF was over-expressed using bicistronic vectors and delivered by retroviral transfection. Where indicated cells were pretreated with 5-aza cytidine (5-azaC) for various time points prior to measurement of PDEF expression by RTPCR method. Cellular RNA was isolated, reversetranscribed into cDNA, and PCR was performed using PDEF-specific primers. Migration (scratch assays and Boyden chambers without Matrigel) and invasion (Boyden chambers with Matrigel) were performed on cells treated with or without 5-azza-2’-deoxycytidine. RESULTS: We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Treatment of prostate cancer cells (PC3 cells) that do not show any PDEF expression with DNA methyl transferase inhibitor, 5-azaC, led to expression of PDEF in a time dependent fashion, suggesting epigenetic mechanisms in suppression of PDEF in advanced prostate cancer. Our studies suggest that treatment with 5-azaC results in decreased cell migration and invasion, concordant with an increase in PDEF expression. CONCLUSIONS: These studies demonstrate for the first time that inhibition of PDEF expression in aggressive prostate cancer cells is modulated by epigenetic mechanisms. Based on these exciting results, we propose that epigenetic regulations are critical for progression of prostate cancer to aggressive phenotype and that demethylating agents like 5-azaC may serve as effective agents to prevent prostate cancer progression.