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Background  Although cholangiocarcinoma (CC) is an uncommon and highly lethal malignancy, early detection enables the application of potentially curative therapies and improves survival. Consequently, tools to improve the early diagnosis of CC are urgently needed. During a screen for genes epigenetically suppressed by methylation in CC that might serve as methylation markers for CC, we found that the  BMP3  gene is methylated in CC cell lines, but the potential diagnostic value and the function of  BMP3  in CC are unknown.    Methods  We aimed to quantitatively assess  BMP3  methylation in resected CC tumor specimens using methylation specific PCR and evaluate the tumor suppressor role of  BMP3  in biliary cancer cell lines in comparison to an immortalized normal cholangiocyte cell line. Expression of BMP3 was quantified by mRNA levels before and after treatment with 5-Aza-2’-deoxycytidine and trichostatin A. After transfection with a  BMP3 -containing plasmid, cell viability was measured using the bromodeoxyuridine incorporation assay and apoptosis quantified by caspase assay.    Results  In primary CC tumor tissue specimens significantly more methylated  BMP3  copies were found when compared to matched benign bile duct epithelium from the same patient, with high specificity.  BMP3  expression was absent in cell lines with  BMP3  methylation; this suppression of  BMP3  expression was reversed by treatment with a DNA demethylating agent and histone de-acetylase inhibitor. Transfection of a  BMP3 -expressing construct into a  BMP3 -negative biliary cancer cell line restored  BMP3  mRNA expression and reduced cell proliferation and cell viability while increasing the rate of apoptosis.    Conclusion  These findings strongly support a tumor suppressor role for  BMP3  in CC and suggest that  BMP3  methylation may be a new biomarker for early detection of CCs. of the peptidome are also involved.

Methylated Bone Morphogenetic Protein 3 (BMP3) Gene: Evaluation of Tumor Suppressor Function and Biomarker Potential in Biliary Cancer