Foxo3a pathway in human intrahepatic cholangiocarcinoma

POSTER PRESENTATIONS mm3). Serum FABP4 levels were significantly lower in mice treated with BMS309403 compared to the controls ( preventive 323.6 ± 117.6 and curative 372 ± 72.9 vs control 553 ± 73.3 ng/mL, p < 0.001). Xenografted tumors obtained for treated mice showed increased necrosis ( preventive 24.3 ± 14.3% & curative 26.8 ± 13.4% vs control 12.7 ± 8.7%, p ≤ 0.01) and decreased FABP4 expression. Conclusions: The study demonstrated the oncogenic effects of FABP4 on hepatoma cell lines and the efficacy of inihibiting this adipokine on tumoral growth in HCC. These results provide significant rationale for developing new targeted strategies in that context of MS. SAT-116 Metformin reduces cell migration and down-regulates epithelial to mesenchymal transition by AMPK / Foxo3a pathway in human intrahepatic cholangiocarcinoma S. Di Matteo1, A. Lustri1, D. Costantini1, L. Nevi1, E. Manzi2, C. Napoletano3, J. Faccioli1, F. Giulitti1, A.M. DeRose4, M.C. Bragazzi1, G. Grazi2, P.B. Berloco5, F. Giuliante4, V. Cardinale1, G. Carpino6, D. Alvaro1. 1Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome; 2Gastroenterology Unit, Regina Elena National Cancer Institute; 3Department of Experimental Medicine, Sapienza University of Rome; 4Hepatobiliary Unit, Catholic University of the Sacred Heart School of Medicine; 5Department of General Surgery and Organ Transplantation, Sapienza University of Rome; 6Department of Health Science, University of Rome foro Italico, Rome, Italy E-mail: [email protected] Background and Aims: Cholangiocarcinom (CCA) is a very aggressive cancer with marked resistance to chemotherapeutics. We have previously demonstrated that CCA is enriched of cancer stem cells expressing epithelial to mesenchymal transition (EMT) traits, these features being associated with aggressiveness and drug resistance. We have recently established primary cell cultures from human intrahepatic CCA subtypes (i.e., mucin and mixed). Treatment with the anti-diabetic drug metformin has been recently associated with reduced cancer incidence. Furthermore, in immortalized cancer cell lines, metformin showed inhibitory effects on EMT by up-regulating Foxo3a signaling in an Akt-dependent manner. We aimed to evaluate the effects of metformin on proliferation, apoptosis, cell migration and the expression of EMT traits in primary cultures of CCA subtypes. Methods: Primary CCA cell cultures were treated with increasing metformin concentrations (from 5 to 1,000 µM, for 1–4 days). Then we evaluated: (i) proliferation by MTS assay; (ii) apoptosis by flow cytometry analysis of Annexin VFITC/Propidium Iodide (PI); and (iii) cell migration by wound-healing assay. The expression of Vimentin, E-Cadherin, SNAIL1, SNAIL2, TWIST1, Cytokeratin19(CK19), FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas ck19, Vimentin, E-Cadherin and Foxo3a were analyzed by Immunofluorescence Assay. Results: Metformin inhibited cell proliferation (MTS assay, population doubling and population doubling time) and induced apoptosis in primary cultures of mucin- and mixed-CCA; the effects were doseand time-dependent ( p < 0.05 vs. controls). The migration of primary human CCA cells, from both mucin and mixed CCA subtypes, was also significantly reduced by treatment with metformin at different concentrations, from 5 to 1,000 µM. The effects of metformin were associated with enhanced gene expression of the epithelial marker ECadherin and decreased expression of Vimentin and EMT specific genes, SNAIL1, SNAIL2 and TWIST1. Metformin also increased the AMPK and Foxo3a mRNA levels. FOXO3a gene expression was negatively correlated with the expression of SNAIL1 and Vimentin genes. Metformin exerted similar effects in mucin- and mixed-CCA human primary cell cultures. Conclusions: In conclusion, we demonstrated that metformin inhibits cell proliferation, enhances apoptosis and impairs the expression of EMT traits by upregulating Foxo3a gene in primary cultures of human CCA. Therefore, metformin could play anticancer effects against human CCAs with relevant therapeutic implications. S636

SAT-117 Molecular basis of Natural Killer cell dysfunction in Hepatocellular Carcinoma V. Regina1, M. Pilli1, A. Olivani1, C. Mori1, C. Carone2, R.D. Valle3, C. Ferrari1, E. Cariani2, G. Missale1. 1U.O. Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria di Parma, Parma; 2 Toxicology and Advanced Diagnostics, Ospedale S. Agostino-Estense, Modena; 3Department of Surgery, University of Parma, Parma, Italy E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) is a complex disease with poor prognosis. Natural Killer (NK) cells play a central role in cell-mediated immune response to cancer. In previous studies number and function of NK-cells have been shown to be positively correlated with HCC outcome. Intra-tumor and circulating NK cells have been shown to be functionally impaired and this may contribute to HCC progression and dismal survival. A better understanding of the molecular basis underlying NK-cell function in HCC, may inform on target molecules and cellular pathways to be restored in immunotherapeutic approaches potentiating NK cell response in HCC. Methods: NK cells (CD56 + CD3−) were derived by fluorescenceactivated cell sorting (FACSAria II) from peripheral blood of 7 patients with Hepatitis C virus (HCV)-related liver cirrhosis, as control, and 11 patients with early stage HCC and HCV-related liver cirrhosis. Gene expression profile was performed by Agilent gene expression microarrays. Differentially expressed genes were defined by GeneSpring and MetaCore for pathway enrichment analysis. Results: Five-hundred and twenty-three genes were differentially expressed. MetaCore pathway analysis allowed the identification of three most relevant pathways: cytoskeleton remodeling, nociceptin expression and immune system, role of the transcription factor AP1 in the regulation of cellular metabolism. Enrichment analysis allowed to identify genes downregulated in NK-cells from HCC patients: actin, vinculin and filamin in the first pathway, nociceptin and prepronociceptin in the second, and genes constituting the transcription factor AP1 in the third. Conclusions: These results may explain a lack of immune surveillance by NK-cells in patients with HCC, in fact formation of a mature, cytolytic synapse between NK cell and target tumor cell may be impaired because of insufficient cytoskeleton assembly and organization. In addition, downregulation of AP1 could lead to a reduced NK-cell activation and differentiation. SAT-118 DNA circulating free in patients with hepatocellular carcinoma V. Dejour1, E.N. Khac1, D. Sefrioui2, L. Beaussire3, P. Michel4, C. Riault1, J. Loreau1, F. Di Fiore2. 1Hepato-Gastroenterology, CHU Amiens, Amiens; 2 Oncology Department; 3INSERM; 4Hepato-Gastroenterology, CHU Rouen, Rouen, France E-mail: [email protected] Background and Aims: Improving the management of patients with hepatocellular carcinoma (HCC) requires better biomarkers. The aim of our study was to evaluate the free circulating DNA (ADNcf) in a cohort of patients with hepatocellular carcinoma. Methods: The clinical data and biological ADNcf of patients with HCC were compared to those of cirrhotic patients without HCC. Extraction of DNA was performed by circulating the Qiamp Nucleic Acid Kit from Qiagen. The ADNcf was then assayed using the kit Quant-iT™ PicoGreen® ds DNA High Sensitivity (Molecular Probes) and “Twinkle LB1970” device (Berthold, Bad Wildbad, Germany). We studied the rate ADNcf depending on tumor characteristics of HCC and those of cirrhosis. Survival analysis was performed with univariate and multivariate Cox model. Results: From 2011 to 2016, 63 patients, mean age 66.95 ± 8.71 years (92% sex M), with 42.9% of alcoholic cirrhosis Child Pugh 6.77 ± 1.91 and HCC BCLC stage A/B/C/D in respectively 30, 31.7, 28.6 and 9.5% of cases were included, and 27 patients with cirrhosis without HCC. The serum ADNcf was not significantly different in HCC on cirrhosis group

Journal of Hepatology 2017 vol. 66 | S543–S750