Abstract 4278: Unique cell-based transposon mutagenesis screen for studying EMT process of tumor in the liver

Background and Aims: Epithelial mesenchymal transition (EMT) is a complex differentiation process that epithelial cells lose its own characteristic and acquire mesenchymal property. EMT is reported to contribute vascular invasion, metastasis and poorer prognosis of hepatocellular carcinoma (HCC). However, how and what types of genes are involved in EMT process of HCC is still not fully understood. Here we develop cell-based transposon mutagenesis system to screen genes involved in EMT process in the liver, Methods: Mouse hepatoblasts were isolated from embryos of hepatocyte-specific T2Onc2 transposon transgenic and SB11 transposase knock-in mice (Alb-Cre/T2Onc2/SBase) or their control littermates. They were cultured in vitro and waited for spontaneous immortalization. Multiple immortalized hepatoblast cell lines (IHBCs) were then injected into the flank or the liver of nude mice and monitored for tumor growth. Results: IHBCs from Alb-Cre/T2Onc2/SBase mice showed hepatoblast characteristics with active T2Onc2 transposition and maintained differentiation capacity into mature hepatocytes in vitro. Five of 7 IHBCs developed tumors at flank and 3 of 5 inside the liver, while injection of 8 IHBCs from the control littermates never developed any tumors, indicating that transposon conferred tumorigenic potential on IHBCs. Histological analysis revealed tumors were positive for transposase in addition to Sox9 and EpCAM, indicating that they were originated from hepatoblasts with albumin expression. However, they were spindle-shaped mesenchymal tumors with positive staining for Vimentin, suggesting that transposon induced EMT of hepatoblasts during malignant transformation. qPCR array also showed the strong activation of EMT-related genes in these tumors. We further confirmed these phenotypes by additional injections of IHBCs and collected 52 xenografted tumors from 5 IHBCs. Sequence of transposon insertion sites in these tumors identified 803 candidate cancer genes (CCGs). Trunk driver analysis identified oncogenic activation of Met/Gab1 signaling as the driving force of tumor development. Pathway analysis revealed that CCGs were enriched in known signaling pathways involved in EMT process including Wnt, TGF-beta, MAPK and Notch signaling as well as adherence junction, focal adhesion and regulation of actin cytoskeleton, indicating that transposon targeted these pathways, inducing EMT in these tumors. Furthermore, analysis of RNA-seq data from TCGA human HCC samples showed that CCGs were enriched in genes that show significant correlation between their mRNA levels and those of EMT-related genes, suggesting their involvement in EMT process. Conclusion: Our unique transposon tumor model mimics EMT process of the tumor in the liver. Therefore, transposon-identified CCGs may be a good resource to discover genes involved in EMT process in the liver. Citation Format: takahiro kodama, Justin Newberg, Michiko Kodama, Robert Rangel, Milton J. Finegold, Nancy Jenkins, Neal G. Copeland. Unique cell-based transposon mutagenesis screen for studying EMT process of tumor in the liver. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4278.