Article

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

Department of Urology, Northwestern University Feinberg School of Medicine, 303E, Chicago Avenue, Tarry 16-726, Chicago, Illinois 60611, USA.
Molecular Endocrinology (impact factor: 4.54). 10/2005; 19(9):2390-9. DOI:10.1210/me.2005-0096 pp.2390-9
Source: PubMed

ABSTRACT Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta 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 (TbetaRI), and type II (TbetaRII) TGF-beta 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-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII 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 TbetaRI promoter and of Sp1 in the TbetaRII 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 TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.

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Keywords

activator protein
 
AP)-2 box
 
benign prostate cell line
 
Chromatin immunoprecipitation analysis
 
clinical prostate cancer specimens
 
DNA binding activity
 
frequent event
 
Gleason's scores
 
LNCaP cells
 
methylation-specific PCR
 
phosphorylated Smad-2
 
promoter methylation
 
prostate cancer cell line
 
prostate cancer cells
 
Prostate cancers
 
sequence analysis
 
Sp1 boxes
 
TbetaRI promoter
 
TbetaRII promoter
 
type II