Insensitivity to transforming growth factor-β results from promoter methylation of cognate receptors in human prostate cancer cells (LNCaP)

Abstract

Prostate cancers often develop insensitivity to TGF-β to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-β receptors (TβRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-β. Sensitivity to TGF-β was restored in cells treated with 5-Aza-2′-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TβRI), and type II (TβRII) TGF-β receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-β. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TβRI and 33 sites in TβRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TβRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TβRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TβRI promoter and of Sp1 in the TβRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TβRI or TβRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TβRI (six sites) and TβRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TβRI and/or TβRII, especially those with high Gleason's scores. In those specimens showing a loss of TβR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-β in some prostate cancer cells is due to promoter methylation in TβRs. Copyright © 2005 by The Endocrine Society.