Abstract 3185: Expression of DNA methyltransferase (DNMT) induced by transforming growth factor-ß (TGF-ß) promotes progress of prostate cancer

Abstract

Abstract Purpose: Loss of sensitivity to TGF-ß are features associated with advanced prostate cancer. Previously, we reported that TGF-ß induced DNMT are associated with aggressive prostate cancer (CaP) cell lines. However, the mechanisms underlying this association, and the association between DNMT expression and clinical outcome following radical prostatectomy has yet to be defined. Here we report that TGF-ß-induces Erk activation mediates DNMT expression in aggressive prostate cancer which is associated with worse outcome in clinic. Methods: Human CaP cell lines PC-3 serials including the less aggressive lines, PC-3 and PC-3M-Pr04; and more aggressive lines, PC-3M and PC-3M-LN) were cultured in fresh media for 24 hours, then exposed for 24 hours to either: 1) external recombinant TGF-ß 1 (10ng/ml), 2) anti-TGF-ß neutralizing monoclonal Ab (1D11; 5μg/ml), or 3) MEK inhibitor U0126 (5μM). Western blot and Real-time PCR analyses were performed to compare Erk, DNMTs and TGF-β receptors (TβRs) expression under the different treatments over time. Immunofluorescence staining was performed to examine the localization of phosphorylated Erk (p-Erk) and DNMTs. A total of 287 radical prostatectomy specimens were obtained from NU Prostate SPORE tissue bank to determine the association between DNMT expression in radical prostatectomy specimens and clinical outcome. Levels of TGF-β, TβRs, and DNMT were analyzed using a tissue microarray analysis. Disease recurrence was defined as PSA >0.2 ng/ml and based on patient follow-up information from the Prostate SPORE clinical-linked database. Median follow-up was 60 months. Results: TGF-ß1 treatment resulted in an increase in p-Erk AND DNMTs expression (4-fold) in all PC-3 cell lines in a time dependent manner. When PC-3 cells were rendered insensitive to TGF-ß by transfection with TβRIIDN, levels of both p-Erk and DNMTs were significantly decreased in 50.5% and 61%. Further, co-localization of p-Erk and DNMTs were found in all PC-3 cells with or without treatment of TGF-ß. Treatment with a 1D11 or UO126, led to 42.2% and 60.5% decrease in DNMTs expression respectively. TGF-β1 and DNMTs correlated with increasing Gleason score. DNMT1 was inversely correlated to TβRI and TβRII. Kaplan Meier curves show that expression levels of TGF-β1, age, Gleason Score and DNMT1were associated with PSA recurrence. In COX proportional hazards model, DNMT1 <3 or 3 was found to be independently predictive of PSA recurrence. Conclusions: TGF-ß activates Erk and DNMTs in CaP. Erk activation is a major mediator for TGF-ß induced DNMTs which may result in promoter DNA hypermethylation of its own receptors and lead to down-regulation of Tβßs in CaP. This mechanism may eventually contributes to the insensitivity in growth inhibition by this cytokine, and may independently predict disease recurrence following radical prostatectomy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3185.