Abstract 2653: Direct oncogenic reprogramming of adult mouse hepatocytes into cancer stem cells.
Authors: Holczbauer Agnes , Factor Valentina M. , Andersen Jesper B. , Kleiner David E. , Marquardt Jens U. , Raggi Chiara , Kitade Mitsuteru , Seo Daekwan , Hirofumi Akita , Durkin Marian E. , Thorgeirsson Snorri S. 
Published: August 2014

Abstract
Objective: Primary human liver cancer (PLC), the third most lethal cancer worldwide, is classified into biologically distinct subgroups, which suggests origin from different hepatic lineage cells. The existence of cancer stem cells (CSCs) was reported in PLC, but the cellular origin of liver CSCs has not been elucidated. Our aim was to investigate the contribution of different hepatic lineage cells to the evolution of CSCs and the phenotypic and genetic heterogeneity of PLC. Methods: Three cell types at different levels of differentiation, including primary mouse hepatic progenitor cells (HPCs), lineage committed hepatoblasts (HBs) and terminally differentiated hepatocytes (AHs) were co-transduced with lentiviral vectors carrying oncogenic H-Ras-Luciferase/EGFP and SV40 large T (LT)-mCherry. CSC properties of FACS sorted H-Ras-EGFP+/SV40LT-mCherry+ cells were tested by standard in vitro and in vivo assays. Individual liver tumors derived from intrasplenic injection of transduced HPCs, HBs and AHs were subjected to immunohistochemistry and whole transcriptome profiling. Results: HPCs, HBs and AHs were susceptible to transformation albeit with a different efficiency as shown by the frequency of tumor initiating cells (1/7, 1/26 and 1/42, respectively). All transduced cells acquired similar attributes of liver CSCs in vitro as judged by self-renewal ability, expression of CSC marker CD133, CD24, CD44 and CD90 and high percentage of side population cells. HPC-, HB- and AH-initiated liver tumors commonly showed a multi-lineage differentiation expressing hepatocyte (HNFα), hepatic progenitor cell (EpCAM, cytokeratin 19, A6) and mesenchymal (vimentin) markers and resembled human PLC. Nevertheless, tumors displayed distinct morphophenotypes according to their cell-of origin: AH tumors showed predominantly hepatocellular carcinoma, HB tumors cholangicarcinoma and HPC tumors epithelial-mesenchymal transition (EMT)-like features. Gene expression analyses revealed the activation of EMT- and embryonic cell-related transcriptional programs in all tumors with the highest number of significant genetic changes in AH (2826) versus HB (574) and HPC tumors (906). Hierarchical clustering distinguished tumors of different cellular origin underscoring the contribution of lineage-stage-dependent genetic changes in malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. Stable knockdown of c-Myc in transformed AHs reduced their CSC properties and delayed tumor growth. Conclusions: Our results indicate that liver tumors with dominant CSC features can originate from any cell in the hepatocytic lineage. Identification of common and cell-of-origin specific phenotypic and genetic changes may provide novel therapeutic targets for treatment of PLC.
Citation Format: Agnes Holczbauer, Valentina M. Factor, Jesper B. Andersen, David E. Kleiner, Jens U. Marquardt, Chiara Raggi, Mitsuteru Kitade, Daekwan Seo, Akita Hirofumi, Marian E. Durkin, Snorri S. Thorgeirsson. Direct oncogenic reprogramming of adult mouse hepatocytes into cancer stem cells. [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 2653. doi:10.1158/1538-7445.AM2013-2653