Cadherin-6 is a putative tumor suppressor and target of epigenetically dysregulated miR-429 in cholangiocarcinoma

ABSTRACT

Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.

KEYWORDS:

• Biliary tract cancer
• DNA methylation
• epigenomics
• microRNA
• patient survival

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The excellent technical assistance of Veronika Geissler, John Moyers (Institute of Pathology, University of Heidelberg), Monika Helf and Oliver Mücke as well as helpful discussions with Daniel Lipka, Christopher Oakes, Yassen Assenov and Daniela Mancarella (Division of Epigenomics and Cancer Risk Factor, German Cancer Research Center) are gratefully acknowledged. We would also like to thank the Genomics and Proteomics Core Facility of the German Cancer Research Center for providing the CDH6 ORFeome clone.

The work was supported by the Biobank of the National Center of Tumor Diseases (NCT) Heidelberg and, in part, by the Helmholtz Association.

Funding

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to P. Schirmacher (SFB/TRR77) and by the German Cancer Consortium to C. Plass and P. Schirmacher.