Abstract 3169: Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes.
Authors: Ranzani Marco , Cesana Daniela , Bartholomä Cynthia C. , Sanvito Francesca , Riba Michela , Pala Mauro , Benedicenti Fabrizio , Gallina Pierangela , Annunziato Stefano , Sergi Sergi Lucia , Merella Stefania , Bulfone Alessandro , Doglioni Claudio , von Kalle Christof , Jun Kim Yoon , Schmidt Manfred , Stupka Elia , Tonon Giovanni , Naldini Luigi , Montini Eugenio 
Published: August 2014

Abstract
Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the 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 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 able to deregulate genes upon integration. Differently from retroviruses and transposons, our replication-defective LV provides a single round of integration shortly after the administration, thus tagging mainly early events in carcinogenesis. A single administration of LV in newborn mice was able to induce hepatocellular carcinoma (HCC) in 3 clinically relevant models of hepatocarcinogenesis: 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 retrieved 172 LV integrations that allowed the identification of Braf, Fign, Sos1 and Rtl1 as candidate cancer loci. The causative role of these 4 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 deregulated by the newly identified cancer genes. HCC induced by integration at the paternally expressed gene Rtl1 (mapping within the imprinted Dlk1-Dio3 region) displayed the peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of the maternally expressed microRNAs encoded within the Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. These findings highlighted a relevant role of Dlk1-Dio3 region in HCCs and in the regulation of metabolism. Additionally, we showed that WNT pathway may represent a target of the new enigmatic oncogene Fign. We then analyzed different human HCCs collections induced by HBV or HCV (N tot of HCC=221). We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in human HCCs. Moreover, we showed that the expression level of the new liver cancer genes or the specific gene expression signature caused by their upregulation can efficiently distinguish HCC patients characterized by poor survival. Overall, we developed a new insertional mutagen by which we 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: Marco Ranzani, Daniela Cesana, Cynthia C. Bartholomä, Francesca Sanvito, Michela Riba, Mauro Pala, Fabrizio Benedicenti, Pierangela Gallina, Stefano Annunziato, Lucia Sergi Sergi, Stefania Merella, Alessandro Bulfone, Claudio Doglioni, Christof von Kalle, Yoon Jun Kim, Manfred Schmidt, Elia Stupka, Giovanni Tonon, Luigi Naldini, Eugenio Montini. Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3169. doi:10.1158/1538-7445.AM2013-3169