Hypermethylation of the Prospero-related gene (PROX1) in intra- and extrahepatic cholangiocarcinomas

Aims: The transcription factor Prospero-related gene 1 (PROX1) is an early specific marker for the developing liver and pancreas in the mammalian foregut endoderm. Directed hepatocyte migration into the septum transversum during liver development requires PROX1. Intriguingly, inactivation of PROX1 caused abnormal cellular proliferation via down-regulated expression of the cell-cycle inhibitors CDKN1B and CDKN1C, i.e., p27kip1 and p57kip2, respectively. Aims: We wished to examine whether epigenetic silencing of the PROX1 gene represents a mechanism for the inactivation of this putative tumor suppressor gene in malignant tumors of the bilary system. Methods: Genomic DNA isolated from praffin-embedded cholangiocarcinomatous specimens reviewed by an experienced pathologist was subjected to bisulfite-treatment and methylation-specific polymerase-chain reaction (MSP). Results: Considerable levels of PCR products specifying methylated sequences of the PROX1 promoter were detected in 9 out of 17 clinical cases (53%) analyzed by MSP. Simultaneously, products of unmethylated DNA were obtained from all specimens suggesting hemimethylation of tumor samples or presence of unmethylated immune cells. Additionally, in 2 out of 3 carcinoma cell lines established from the biliary system we identified complete PROX1 methylation. Conclusions: Here, for the first time we present experimental evidence that an abnormal methylation state of CpG islands within the PROX1 gene promoter appears to contribute to the development of cholangiocarcinomas. Reconstitution experiments of PROX1-deficient cell lines should allow for the identification of target genes modulated by this homeodomain transcription factor. Supported by BMBF NBL3 Wilhelm-Roux-Programm (FKZ09/24).

Keywords: Homeobox transcription factor, cholangiocarcinoma, epigenetic silencing, tumor suppressor