Abstract LB-85: TGF-ß regulates its own expression and down-regulates its own receptors in prostate cancer (CaP) cells through a positive feedback pathway mediated by DNA methyltransferase

Abstract

Abstract Introduction: Over expression and loss insensitivity to TGF-ß are two features which are coincident with CaP progression. In present study, we investigated whether a positive feedback pathway exists in which TGF-ß activates DNA methyltransferase (DNMT), which induces promoter hypermethylation. This hypermethylation may down-regulate TGF-ß receptors and in turn stimulate increased expression of TGF-ß. Materials and Methods: Human CaP cells (including the less aggressive lines, PC-3 and PC-3M-Pr04; and more aggressive lines, PC-3M and PC-3M-LN4) were rendered insensitive to TGF-ß using a retroviral vector containing a dominant negative TGF-ß type II receptor gene (TßRIIDN). ELISA were performed to quantify the secreted TGF-ß 1 (pg/105 cells/48 hours) in the pooled conditioned medium from CaP cells expressing T RIIDN and mock transfected. Semi-quantification western blot analyses were performed to evaluate the expression of DNA methyltransferase (DNMT), TßRI and TßRII. A [3H]-Thymidine Incorporation Assay was performed to determine cell proliferation rates in relation to TGF-ß. Results: Compared to PC-3 and PC-3M-Pr04, PC-3M and PC-3M-LN4 secreted almost 2 folds higher baseline levels of TGF-ß1 and DNMTs (1, 3α and 3ß). In contrast, TßRI and TßRII expression was significantly reduced in these same cell lines. In all cell lines, TßRIIDN expression was associated with significantly higher (∼1-2×) TGF-ß secretion, a greater than 3-fold decrease in the expression of DNMT1, DNMT3a, and DNMT 3b, and a correspondingly increased expression of both TßRI and TßRII (naïve intracellular domain). Although there was no significant difference in the original proliferation rate among PC3 variants, PC-3 and PC-3M-Pr04 cells were more sensitive to growth inhibition (>30%) by TGF-ß1 compared to PC-3M-LN4 and PC-3M cells (<7.5%). The growth inhibitory effects of TGF-ß1 disappeared when these cells were rendered insensitive to TGF-ß by infection with TßRIIDN. Conclusion: Higher expression of TGF-ß and lower expression of its own receptors are associated with more aggressive CaP phenotypes. Overexpression of tumor derived TGF-ß can activate DNMTs which will then mediate promoter methylation and down regulation of its receptors, subsequently, blockade of TGF-ß signaling may activate a feedback system which can stimulate the secretion of more TGF-ß. This novel mechanism has yet been reported, and may provide a new insight on TGF-ß regulation in prostate cancer. This study was supported in part by grants from the American Cancer Society, Illinois (#08-22), Department of Defense (W81XWH-09-1-0311), Portes Center/Institute of Medicine of Chicago (QZ), American Cancer Society Institutional Research Grant (ACS-IRG 93-037-12), American Urological Association Foundation (AUA Foundation, QZ), National Cancer Institute (P50CA090386), Illinois Department of Public Health (Contract #:83284034), a grant from the Genzyme Corporation, and a gift from Mr. Fred L. Turner. 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 LB-85.