Abstract 104: New liver cancer genes identified by lentiviral vector-based insertional mutagenesis in mice are associated to differential survival in hepatocellular carcinoma patients
Authors: Ranzani Marco , Cesana Daniela , Bartholomae Cynthia C. , Sanvito Francesca , Pala Mauro , Benedicenti Fabrizio , Gallina Pierangela , Sergi Lucia Sergi , Merella Stefania , Bulfone Alessandro , Doglioni Claudio , von Kalle Christof , Kim Yoon Jun , Schmidt Manfred , Tonon Giovanni , Naldini Luigi , Montini Eugenio 
Published: August 2012

Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the potential clinical implications of these new findings are still limited, since the identification of cancer driving mutations is hampered by the co-occurrence of several bystander and progression related events. Here we developed a forward genetics approach based on a new lentiviral vector-based insertional mutagen aimed at identifying liver cancer initiating genes that are relevant in human hepatocarcinogenesis. 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 clinically relevant mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (P=0.005 Vs untreated), in 27% of liver-specific Pten deficient mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we could retrieve LV 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. Whole transcriptome gene expression analysis allowed unveiling the molecular pathways on which the new cancer genes have an impact. We showed that tumors with integration within Dlk1-Dio3 imprinted region displayed the overexpression of the paternally expressed gene Rtl1 encoded within the region and a peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. Additionally, upregulation of Fign was associated to the deregulation of Wnt pathway in HCCs. We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in the human HCCs, highlighting a relevant role of these genes in human hepatocarcinogenesis. We also identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 3 different human HCC collections showed that the signatures of all the 4 new cancer genes: 1) are present also in human HCCs; 2) can distinguish different HCC stages; 3) can identify different prognostic groups of patients. Moreover, SOS1 expression levels alone can discriminate HCC patients with good or poor overall survival. This study identified new clinically relevant liver cancer genes that may provide novel prognostic markers and therapeutic targets for the diagnosis and treatment of human HCCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 104. doi:1538-7445.AM2012-104