Abstract 4181: Comparison of tumorigenic proficiency at different stages of hepatocyte differentiation
Authors: Holczbauer Agnes , Marquardt Jens U. , Durkin Marian , Seo Daekwan , Andersen Jesper B. , Choi Dongho , Raggi Chiara , Factor Valentina M. , Thorgeirsson Snorri S. 
Published: February 2011

BACKGROUND: Current evidence suggests that any cell that can proliferate repeatedly is susceptible to neoplastic transformation. In the liver, these include all cells within the lineage from hepatoblast and adult liver stem cell to hepatocyte and cholangiocyte. Lentiviral vectors with their ability to transduce non-dividing cells are effective tools to introduce oncogenes at different stages of lineage commitment. AIM: To address whether (1) the transforming potential of defined oncogenes is dependent on the stage of hepatocytic differentiation and (2) the genotype and phenotype of developed tumors reflect the stage of differentiation. METHODS: To generate genetically defined liver carcinomas, constitutive lentiviral vectors were developed carrying oncogenic H-Ras and luciferase/EGFP double reporter as well as SV40 large T (LT) antigen and mCherry fluorescent reporter. The E-cadherin-positive hepatoblasts (ED16.5) and newborn (1day) hepatocytes as well as adult (3month) hepatocytes were transduced ex vivo by lentiviral vectors and FACS-sorted before intrasplenic transplantation into NOD/SCID mice. Liver tumors larger than 3mm derived from fetal (n=20), newborn (n=18) and adult hepatocytes (n=10) transfected with H-Ras and SV40 LT were macrodissected and subjected to immunohistochemistry and microarray analyses. RESULTS: Infection with H-Ras alone was not sufficient to transform hepatocytes at any state of differentiation. SV40 LT was capable to transform only fetal hepatocytes but not hepatocytes as judged by robust anchorage-independent growth in vitro and rapid tumor formation in vivo. The combination of activated H-Ras and SV40 LT significantly accelerated the tumor potential of all infected hepatocytes. In all cases, tumors resembled the histopathological subtypes of human hepatocellular carcinoma (HCC) albeit with variable degrees of malignancy. HCCs derived from hepatoblasts were more aggressive with a predominance of pseudoglandular component and more frequent extrahepatic growth. The pseudoglandular structures were strongly positive for cytokeratin 19 (CK19). Unsupervised hierarchical clustering of the expression profiles grouped the tumors according to CK19 associated gene expression signature with highest levels found in fetal- to lowest in adult hepatocyte-derived tumors. HCCs established from hepatoblasts were genetically more heterogeneous and clustered with tumors derived from both newborn and adult hepatocytes. CONCLUSIONS: There is a reciprocal relationship between the transforming potential of defined oncogenes and extent of commitment to hepatocyte lineage. Transformation of hepatocytes at the different stages of differentiation may induce a cancer stem cell-like phenotype that displays the markers characteristic of hepatic stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4181.