Induction of cholangiocellular carcinoma by liver specific disruption of Smad4 and Pten in mice.

5108

Cholangiocarcinoma, or cholangiocellular carcinoma (CC) is a malignant epithelial neoplasm with bile duct epithelial differentiation. CC accounts for about 15% of total liver cancer cases in the world with significant variations from country to country and is associated with poor prognosis and most patients die soon after diagnosis. Indeed, CC is the most common primary liver cancer-related cause of death in the United Kingdom. It has been shown that CC harbors alterations of a number of tumor suppressor genes and oncogenes, including p53, p16, p27, p57, SMAD4, cyclin D1, ERK, Ras, AKT, and c-Myc. In part because of the paucity of proper animal models, critical roles of these alterations in tumor initiation, progression and metastasis have not been studied. To study mechanisms underlying CC formation, we generate a mouse model that develops CC with high penetrance using a Cre-loxP mediated liver specific disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from bile ducts of mutant mice at two months of age and continue to grow leading to tumor formation in all animals at 4-7 months of age. We show that CC formation follows a multistep progression of histopathological changes that are associated with significant molecular alterations, including high levels of phosphorylated AKT, GSK-3beta, mTOR, and ERK, and increased levels of expression of c-Myc and cyclin D1. Conversely, reconstitution of SMAD4 and PTEN in cultured tumor cells suppresses most of these alterations. We further demonstrate that SMAD4 and PTEN regulate each other through a novel feedback mechanism to maintain an expression balance and synergistically repress CC formation. These findings elucidate the relationship between SMAD4 and PTEN and extend our understanding of CC formation. Because the mice carrying liver specifis SMAD4 and PTEN double knockout developed CC at defined time points with high penetrance, this animal model should be useful in identifying markers associated with the earliest changes during CC initiation, drug screening, chemoprevention, and therapeutic treatment of the disease.