P69 IMPACT OF EPIGENETIC REPROGRAMMING ON HEPATIC TUMORIGENIC PROPERTIES

POSTERS P67 PAF-RECEPTOR INHIBITORS HAVE AN ANTI-PROLIFERATIVE EFFECT ON HEPATOCELLULAR CARCINOMA CELLS M. Tarocchi1 , G. Marroncini2 , S. Polvani1 , T. Mello1 , S. Tempesti1 , E. Ceni1 , A. Galli1,2 . 1 Experimental and Clinical Biomedical Sciences, University of Florence, 2 Fiorgen Fondation, Florence, Italy E-mail: mirko.tarocchi@unifi.it Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there is no effective treatment for advance stage HCCs. PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation, growth arrest and massive apoptosis in in murine and human leukemia cells. We investigate the anti-tumor efficacy of WEB treatment in vitro and in vivo models of HCCs. Methods: We evaluated proliferation, apoptosis and migration of different hepatic cancer cell lines (HepG2, Hep3B, HuH7 and Hepa1–6) incubated with WEBs. We tested the in vivo efficacy of the treatment in HBV transgenic mice (TgAlb43Bri), that spontaneously develop hepatic tumors, and in a syngeneic orthotopic murine HCC model, where HCC cells were implanted directly in the animal liver. Results: WEBs were able to reduce the proliferation of cancer cells as assessed by thymidine incorporation, but no notable effects were present on apoptosis as assessed by caspase 3 activity. Furthermore pre-incubating the hepatic cancer cells with WEBs induced cell cycle arrest and impaired migration in a wound healing assay. Treatment with WEB reduced growth and proliferation of the tumors in the transgenic and in the orthotopic murine model. Conclusions: PAF-receptor antagonists, WEB-2086 and WEB-2170, are able to reduce HCC progression in human and murine HCC models blocking cancer cell proliferation and migration. P68 UPREGULATION OF A NOVEL PROTEIN IN HCC ENHANCES CANCER CELL SURVIVAL BY SUPPRESSING SPECIFIC APOPTOTIC EFFECTORS V. Iansante1 , P.M. Choy1 , S.W. Fung2 , J.-G. Chai2 , J. Dyson2 , Y. Liu3 , R. Williams1 , S. Chokshi1 , R.A. Anders3 , C. Bubici2,4 , S. Papa1 . 1 Institute of Hepatology, Foundation for Liver Research, 2 Department of Medicine, Imperial College London, London, United Kingdom; 3 Sol Goldman Pancreatic Cancer Research Center, Division of Gastrointestinal and Liver Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, United States; 4 Division of Biosciences, Brunel University London, London, United Kingdom E-mail: [email protected] Background and Aims: Altered cell survival is a hallmark of cancer, including primary HCC. The aim of our study is to identify novel anti-apoptotic genes promoting liver tumourigenesis. Methods: We used a combination of in vitro and in vivo analyses to identify novel anti-apoptotic genes. We compared the ability of HCC cells stable-expressing either shRNA against HD (a newly-identified anti-apoptotic molecule) or control shRNA to growth in vivo and in vitro. Results: By examining the expression pattern of HD mRNA in a panel of human neoplasms and cancer cells, we demonstrated that HD is highly expressed in both HCC cells and liver biopsies. mRNA profiling showed significantly higher expression of HD transcripts in HCC samples compared to normal livers (**P = 0.0062) and adjacent non-tumor tissues (***P < 0.0001). Elevated HD expression also correlates with HCC progression and poor prognosis. Protein analyses confirmed that HD levels were significantly increased in HCC tissues and hepatoma lines compared to healthy livers. Notably, xenograft experiments demonstrated that suppression of HD strongly impaired tumor formation in vivo. Knockdown

of HD significantly reduced proliferation and markedly increased apoptosis of hepatoma cells. Conclusions: We report that HD mRNA and protein are frequently overexpressed in HCC tissue compared with non-tumour liver tissues, where it correlates with poor patient survival. Enforced expression of HD was sufficient to enhance cell survival, whereas silencing of HD was greatly enhances apoptosis. Notably, HD attenuation in HCC cells also suppressed activation of specific apoptotic markers, thus suggesting that HD is an important novel anti-apoptotic marker for liver tumourigenesis. P69 IMPACT OF EPIGENETIC REPROGRAMMING ON HEPATIC TUMORIGENIC PROPERTIES C. Raggi, V.M. Factor, S. Daekwan, M.C. Gillen, A. Holczbauer, J.U. Marquardt, J.B. Andersen, S.S. Thorgeirsson. Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, United States E-mail: [email protected] Background and Aims: Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of HCC cells. Methods: Established and primary human HCC-derived cell lines were plated in 2D culture at various cell densities and exposed to a transient dose of a DNMT1-inhibitor Zebularine (ZEB). After a 3-day treatment, cells were cultured in 3D nonadherent condition. Differences in self-renewal, gene expression, tumorigenicity and metastatic potential of spheres at generations G1-G5 were examined. Results: Transient ZEB exposure produced differential cell densitydependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes and a stable overexpression of cancer stem cell-related and key epithelial– mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis-related genes. A permanent reduction of the DNMT1 protein using shRNA-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, hepatoblastoma cells expressing low basal levels of DNMT1 also possessed a high self-renewal irrespective of cell density or ZEB exposure. Spheres formed by low density cells treated with ZEB or shDNMT1A displayed a high molecular similarity which was sustained through consecutive generations. Conclusions: These results identify DNA methylation as a key epigenetic regulatory mechanism determining pool of cancer stem cells in liver cancer and possibly other solid tumors. P70 STEM CELL NICHE AND MACROPHAGES IN HUMAN CHOLANGIOCARCINOMA C. Raggi, M. Correnti, P. Invernizzi. Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy E-mail: [email protected] Background and Aims: Tumorigeneicity is modulated by inflammatory microenvironment mainly composed by infiltrating macrophages (MØ). Neoplastic evolution is associated with MØphenotypic switch from M1 (chronic inflammation sites) to M2 subtype (established tumors). Our study aims to highlight the dynamic interactions between stem-like tumor initiating cells (TICs) and MØ in human cholangiocarcinoma (CCA). Methods: Stem-like compartment was identified by sphere forming assay in both transformed (SG231, CCLP-1, HuCCT-1, TFK-1) and

Journal of Hepatology 2014 vol. 60 | S67–S214

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