Overexpression of C–Neu and C–Met During Rat Liver Cholangiocarcinogenesis: A Link Between Biliary Intestinal Metaplasia and Mucin–Producing Cholangiocarcinoma

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Abstract
Based on limited but compelling immunohistochemical data demonstrating individual overexpression of the tyrosine kinase growth factor receptors, c–erbB–2 and c–met, in significant percentages of human cholangiocarcinoma (ChC), we investigated if combined overexpression of both c–neu, the rat homologue of c–erbB–2, and c–met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), might represent a characteristic, early event associated with furan–induced cholangiocarcinogenesis in rat liver. Specifically, through the use of immunohistochemistry, in situ hybridization (ISH), and Western and Northern blotting, we found that both c–neu and c–met are prominently overexpressed in intestinal metaplastic lesions in early putative precancerous cholangiofibrotic tissue formed in the livers of rats after 6 weeks of furan treatment when compared with normal and hyperplastic intrahepatic biliary epithelia. We further demonstrated that c–neu and c–met are concordantly overexpressed in neoplastic glandular epithelia in later–developed primary “intestinal–type” of ChC formed in the livers of furan–treated rats, as well as in subsequently derived transplantable mucin–producing tumors. Overexpression of c–neu and c–met correlated with increased proliferating cell nuclear antigen (PCNA)–labeling indices, which were determined to be three to four times higher in intestinal metaplastic glands in precancerous cholangiofibrotic tissue and in neoplastic glands in the primary “intestinal type” of ChC than in hyperplastic bile ductular structures within either cholangiofibrotic or bile duct-ligated (BDL) livers. The c–neu and c–met receptor proteins overexpressed in different in vivo passages of a transplantable ChC each contained immunoreactive phosphotyrosines, indicating an activated state. However, we did not detect evidence of either gene amplification of c–neu or c–met or of a common transmembrane–activating mutation in c–neu expressed in transplantable ChC. Our findings indicate that altered expression of c–neu and c–met occurs relatively early in the process of furan–induced cholangiocarcinogenesis in rat liver and may play a potentially important role in its pathogenesis. They further indicate a common alteration in tyrosine kinase growth factor receptor expression linking early putative precancerous intestinal metaplastic lesions in liver to later–developed mucin–producing biliary cancer.