Intra-hepatic cholangiocarcinoma (IHCC) is thought to arise from the intra-hepatic biliary system based on common histological and molecular properties (1
). These tumors are characterized by an aggressive course and early metastasis. The incidence of IHCC is on the rise and while effective palliative chemotherapy has recently been defined, treatment options for the majority of patients remain limited (2
). Many aspects of its biology and genetics remain incompletely defined including key questions relating to 1) the cell(s)-of-origin, 2) the precursor lesions in the liver, their molecular profiles, and their relationship to fully established IHCC and 3) the functional impact of oncogenes and tumor suppressor genes on malignant progression.
While the genetic basis for IHCC has not been fully elucidated, mutations in a number of established oncogenes and tumor suppressors are well described. Recurrent mutations have been observed in the KRAS oncogene, which is activated in 20% to 50% of tumors (4
). Mutations in BRAF were described in two European cohorts but have not been reported in the US studies (1
). p53 inactivation is the most common tumor suppressor lesion, observed in 37% of IHCC (7
). Additionally, subsets of IHCC show mutations or deletions of SMAD4 and p16INK4A
Pathologic studies of the biliary system in diseased livers and of biliary lesions adjacent to IHCC have led to a proposed multi-stage progression model for the development of invasive cancers from the normal hepatic epithelium (9
). In particular, lesions known as biliary intraepithelial neoplasia (BilIN) and intraductal papillary biliary neoplasms (IPBN) are thought to be precursors of IHCC and have been graded according to the degrees of architectural distortion and cellular atypia (10
). Additional lesions of the biliary tract include malformations of the ductal systems referred to as biliary hamartomas or Von Meyenburg complexes, although the relationship of these lesions to IHCC is less clear (12
). Importantly, despite these detailed pathologic descriptions, the genetic features of the different biliary ductal lesions and the capacity of these lesions to give rise to IHCC remain undefined.
Experimental model systems have been central to providing basic and preclinical insights into many cancer types. While a number of advances have been made in this regard in IHCC, there is currently an incomplete array of systems for the study of this malignancy. For example, only a small number of IHCC cell lines are reported in the literature, with most published experimental studies employing no more than two or three lines. Alternatively, studies often employ a combination of IHCC, EHCC, and gall bladder cell lines, although these different types of biliary cancer carry distinct mutational profiles. A number of carcinogen-induced models of primary liver tumors in mammalian systems have been described (14
) and the transduction of viral onco-proteins has also enabled transformation of carcinogen treated hepatic epithelium both in vitro and in vivo (18
). Genetically engineered mouse (GEM) models have also been developed to model tumors with similarities to IHCC. p53 mutant mice develop cholangiocarcinomas upon repeated carcinogen exposure, although there is long latency in this model (20
). Liver targeted delivery of mouse polyoma virus middle T antigen (PyMT) using a transgenic avian retroviral system induces focal regions of IHCC as well as more prominent hepatocellular carcinoma (HCC) lesions in Trp53 and Ink4a/Arf knockout mice (21
). Mixed HCC/IHCC histology is also seen in mice with liver specific inactivation of the NF2, Sav1, and Mst1/Mst2 tumor suppressor genes, although HCC is the predominant component in each case (reviewed in (22
)). Combined homozygous deletion of conditional Smad4
alleles in the liver via crosses to the Albumin-Cre strain causes tumors histologically similar to IHCC (23
). While providing important systems to study malignant transformation of liver cells, these models have not been reported to exhibit progressive precursor lesions of the biliary tract nor do they accurately incorporate the most common genetic lesions seen in the human disease.
GEM models designed to mimic both genetic and pathologic aspects of cancer have proven critical to drug development efforts, biomarker identification, and the study of early disease (24
). In order to create a model of IHCC based on oncogenic mutations commonly observed in the human disease we generated compound mutant mice with Albumin-Cre mediated somatic activation of KrasG12D
and deletion of p53 in the hepatic parenchyma. We report that cooperation between these two relevant genetic alterations in the hepatic epithelium leads to a model of IHCC that recapitulates the histologic and molecular features of multistage progression of human IHCC. We employ this model to study the role of pre-invasive lesions as precursors to IHCC, and use a panel of IHCC-derived cell lines to show that autophagy may be an important targetable pathway in this malignancy as in some other Kras-driven carcinomas. Thus, our work establishes a relevant and faithful preclinical model system with which to study this challenging disease.