Global alterations of DNA methylation in cholangiocarcinoma targets the Wnt signaling pathway

Goeppert B, Konermann C, Schmidt CR, Bogatyrova O, Geiselhart L, Ernst C, Gu L, Becker N, Zucknick M, Mehrabi A, Hafezi M, Klauschen F, Stenzinger A, Warth A, Breuhahn K, Renner M, Weichert W, Schirmacher P, Plass C, Weichenhan D

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled ten intrahepatic (ICC) and eight extrahepatic (ECC) CCs in comparison to non-tumorous biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments using 5-aza-2'deoxycytidine (DAC) treatment of two CC cell lines. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were non-randomly distributed and enriched for genes involved in cancer-related pathways including Wnt, TGF-β, and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2 and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially downregulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the fundament for future studies on epigenetic changes in cholangiocarcinogenesis.

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September, 2013


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