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Insensitivity to Transforming Growth Factor-ß Results from Promoter Methylation of Cognate Receptors in Human Prostate Cancer Cells (LNCaP)

Departments of Urology (Q.Z., J.N.R., T.L.J., I.P., C.L.), Pathology (M.P., X.Y.), and Preventive Medicine (B.J.), Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611; and Laboratory of Cell Regulation and Carcinogenesis (S.-J.K.), National Cancer Institute, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Chung Lee, Ph.D., Northwestern University Medical School, 303 East Chicago Avenue, Tarry 16-733, Chicago, Illinois 60611. E-mail: c-lee7{at}northwestern.edu or Qiang Zhang, M.D., Ph.D., Department of Urology, Northwestern University, 303E, Chicago Avenue, Tarry 16-726, Chicago, Illinois 60611. E-mail: q-zhang2{at}northwestern.edu.

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

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