Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu

Abstract

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.