Abstract 1429: TGF-β mediates epigenetic regulation of DNA damage in the prostate cancer associated stroma

Abstract

Abstract DNA damage of stromal fibroblastic cells is known to promote tumorigenesis in number of tissues including the breast and pancreas. 69% of prostate cancer patients lose expression of the Tgfbr2 in the stromal compartment. We previously described, transforming growth factor-beta (TGF-ß) responsiveness of the prostate stromal fibroblastic cells can promote tumorigenesis in transgenic mice. It is known TGF-ß promotes DNA stability, however the mechanism is not known. We found that human prostate cancer stroma has significant number of cells with DNA double stranded breaks, as determined by phosphorylated Ser139-histone H2 (γH2AX) immunolocalization. Similarly, prostate stromal cells from mice with a conditional Tgfbr2-knockout were positive for γH2AX staining. Further promoter methylation array analysis of prostate stromal cells from Tgfbr2-knockout and Tgfbr2-flox (control) indicated 17 DNA damage repair genes to be methylated in Tgfbr2-knockout cells versus the control. Thus, we hypothesized that the loss of stromal TGF-ß responsiveness facilitates stromal DNA damage accumulation through epigenetic regulation. We sought to test the role of stromal TGF-ß signaling in epigenetic regulation. Fourteen of 17 genes were RT-PCR verified to be silenced in Tgfbr2-KO cells and re-expressed following 5-azaDC (de-methylating agent) treatment. Homologous genes were found to be epigenetically silenced in human prostate CAF (carcinoma associated fibroblasts) cultured from primary tumors compared to NAF (normal prostate fibroblasts) from 10 prostate cancer patients. A downstream mediator of TGF-ß signaling includes Smad3 activation. Thus, a similar methylation array analysis of Smad3-knockout prostate stromal cells was performed. We found none of the DNA damage repair genes to be silenced in Smad3-KO cells. Next, we measured DNA methyltransferase-1 (DNMT1) mRNA, protein and activity in Tgfbr2-KO and Tgfbr2-flox cultured prostate stromal cells. Interestingly, protein expression and activity of DNMT1 was 3-fold greater in Tgfbr2-KO stroma over Tgfbr2-flox cells, in the absence of DNMT1 mRNA expression differences. DNMT3b differed little between the two cell types. The use of proteasome inhibitor, MG-132, suggested DNMT1 to be down regulated by TGF-ß in a post-translational manner. Finally, tissue recombinant experiments of Tgfbr2-KO prostate stromal cells with LNCaP, human prostate cancer epithelia, developed large tumors in orthotopic grafts. Similar grafts with wild type and Smad3-KO stromal cells with LNCaP epithelia developed small tumors. Strikingly Tgfbr2-KO stromal cells treated with 5-azaDC and subsequently grafted with LNCaP cells developed smaller tumors. These results for the first time support the role of TGF-ß signaling on stromal DNA stability in tumorigenesis and support a mechanism for TGF-ß responsiveness results in epigenetic silencing of DNA damage repair genes. 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 1429.