Abstract 4038: Human prostate cancer invasion could be suppressed by 5-Aza-2′-deoxycytidine which can inhibit the TGF-β induced DNA methyltransferase (DNMT)
Authors: Zhang Qiang , Helfand Brian , Lin Xiao , Jang Thomas , Guo Yinglu , Khandekar Janardan , Kuzel Timothy Michael , Lee Chung 
Published: May 2015

Introduction and Objective: We previously reported that loss of sensitivity to TGF-ß growth inhibition is partially due to promoter methylation and subsequent down regulation of TGF-ß receptors (TßRs) expression; a characteristic of aggressive PCa phenotypes. In this study, we address whether TGF-ß induced DNMT expression contributes to its receptors promoter methylation and loss of sensitivity to this cytokine, and a more agressive phenotype. We also explored the effects of 5-Aza-2′-deoxycytidine (5-Aza), a DNMT inhibitor, on CaP cell invasion.

Methods: PC3 human PCa line and its variants with different invasive abilities were used. Expression of TßRs, DNMT and vimentin under the treatment of TGF-ß (10ng/mL), or 5-aza was evaluated by Western Blot and Real-Time PCR analysis. Methylation specific PCR (MSP) was performed to evaluate the status of TßRs promoters. Matrigel cell invasion assays were conducted using a 24-well transwell chamber (8-um pore size). Four different treatment groups were assigned to each cell line: Group 1- no treatment (control); Group 2- treatment with TGF-ß (10ng/mL) for 24 hours; Group 3- treatment with 5-Aza (2 µg/ml) for 24 hours; and Group 4- treatment with TGF-ß plus 5-Aza for 24 hours. The invaded cells were stained and counted under high magnification objective (100x). [3H] Thymidine incorporation assay were used to evaluate cell growth under the treatment of 5-Aza.

Results: Both TßRI and TßRII expresion was significantly increased (2-2.5 folds) after treatment with 5-Aza in PC-3 cells. In contrast, treatment of TGF-ß signficantly increased the expression of DNMT by 3.2 fold and suppressed the expression of TßRI and TßRII by 46% and 29% respectively. This suppression and promoter methylation was recovered following treatment with 5-Aza. 5-Aza treatment also resulted in a significant inhibition of cell proliferation, regardless of whether cells were exposed to exogenous TGF-ß. An average of 82 cells/field were found to invade in the control group (Group 1). In Group 2, there was a significant increase in the number of invaded cells (139/field) with TGF-β treatment. Invasion of PC-3 cells was significantly inhibited by treatment of 5-aza, (only 10 /field). After treatment with 5-Aza, the expression of vimentin decreased by 50% fold, as well as DNMTs was inhibited significantly.

Conclusion: Tumor derived TGF-ß-induced DNMTs mediates promoter hypermethylation and downregulation of its receptors of its own receptors. Knockdown of DNMT by 5-aza result in the demethylation of the TGF-ß receptors gene promoters, restoration of TGF-ß inhibition of cell growth, and inhibition of invasion of CaP cells by inhibiting the expression of DNMT and vimentin induced by TGF-ß. This results indicated 5-Aza may be a candidate for novel antitumor metastasis strategies in men with aggressive CaP.

Citation Format: Qiang Zhang, Brian Helfand, Xiao Lin, Thomas Jang, Yinglu Guo, Janardan Khandekar, Timothy Michael Kuzel, Chung Lee. Human prostate cancer invasion could be suppressed by 5-Aza-2′-deoxycytidine which can inhibit the TGF-β induced DNA methyltransferase (DNMT). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4038. doi:10.1158/1538-7445.AM2014-4038