Over-expression of polycomb group protein EZH2 relates to decreased expression of p16INK4a in cholangiocarcinogenesis in hepatolithiasis

Polycomb group protein EZH2 and Bmi1are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi-step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor gene p16(INK4a). We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN-1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN-3 (carcinoma in situ), 12 BilIN-2 (high-grade dysplasia), 32 BilIN-1 (low-grade dysplasia) and 37 non-neoplastic biliary epithelia from these livers. Expression of p16(INK4a), EZH2 and Bmi1 were surveyed in these foci. P16(INK4a) promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT-1 and TFK-1) and small interfering RNA (si RNA), the effects of the knockdown of EZH2 on p16(INK4a) methylation of CC cells were examined. Expression of p16(INK4a) which was frequent in BilIN1, was decreased in BilIN-2/3 and invasive carcinoma, while EZH2 expression showed step-wise increase from BilIN-1, -2 and -3 to invasive carcinoma (p < 0.01). P16(INK4a) promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16(INK4a) methylation and decreased binding of EZH2 to the p16(INK4a) gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p(16INK4a) gene. Our data suggest that over-expression of EZH2 may induce hypermethylation of p16(INK4a) promoter followed by decreased expression of p16(INK4a) in the multi-step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.