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

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 gene is methylated in CC cell lines, but the potential diagnostic value and the function of in CC are unknown.

Methods: We aimed to quantitatively assess methylation in resected CC tumor specimens using methylation specific PCR and evaluate the tumor suppressor role of 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 -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 copies were found when compared to matched benign bile duct epithelium from the same patient, with high specificity. expression was absent in cell lines with methylation; this suppression of expression was reversed by treatment with a DNA demethylating agent and histone de-acetylase inhibitor. Transfection of a -expressing construct into a -negative biliary cancer cell line restored 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 in CC and suggest that methylation may be a new biomarker for early detection of CCs. of the peptidome are also involved.