Identification Of New Human Liver Cancer Genes By A Novel Lentiviral Vector-Based Insertional Mutagenesis Approach In Three Mouse Models Of Hepatocarcinogenesis

Retrovirus or transposon-based in vivo insertional mutagenesis triggers cancer by activating nearby oncogenes upon integration and led to the discovery of many candidate oncogenes important in human cancer. However, these insertional mutagens constantly undergo integration cycles throughout the life of the animal, thus resulting in several bystander and progression-related integrations that hamper the identification of cancer-initiating events. To overcome these limitations, we generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers capable to induce hepatocellular carcinoma (HCC) upon a single administration in 3 different mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (0 in controls, P=0.005), in 27% of liver-specific Pten deficient mice (6% in controls, P=0.04) and in 75% of wild type mice coupled to CCl4 administration (0 in controls, P=0.002). From 30 LV-induced HCCs we could retrieve 172 unique integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. The causative role of these genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. The LV integration in Braf and Sos1 generated constitutively active proteins previously found in human tumors from tissues other than liver. Genome-wide gene expression analyses identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 2 different human HCC collections showed that these signatures: 1) are present also in human HCCs; 2) discriminate among normal liver, non-tumoral diseased liver and HCC; 3) can distinguish different HCC stages. All the newly identified cancer genes were significantly upregulated and/or mutated in the human HCCs, underscoring a relevant role of these genes in human hepatocarcinogenesis. Integration of LV at Dlk1-Dio3 locus resulted in the upregulation of the paternally expressed gene Rtl1 and induced HCCs characterized by a peculiar upregulation of oxidative phosphorylation genes. Oppositely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 locus in HCCs which display a canonical dowregulation of oxidative phosphorylation genes. The latter expression pattern was confirmed in a cohort of human HCCs. Our data indicate that the Dlk1-Dio3 locus plays a relevant role in hepatocarcinogenesis, energy metabolism and stem cell maintenance in mice and humans.This study uncovered a role in human HCCs of some previously known and other completely new oncogenes that altogether may provide novel candidate therapeutic targets and diagnostic markers for human HCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2011-4982