miR-22 deficiency fosters hepatic tumour development

POSTER PRESENTATIONS Background and Aims: Hepatocellular carcinoma (HCC) occurs as a consequence of malignant transformation of hepatocytes frequently triggered during chronic inflammation and consecutive liver fibrosis. For fibrogenesis chemokines play a crucial role by attracting immune cells. In the current study, the role of CCL5 for fibrogenesis and tumorigenesis was studied in two independent murine models of inflammation triggered hepatocarcinogenesis. Additional, we investigated therapeutic intervention with Evasin-4 – a CCL5/RANTES inhibitor – on hepatic fibrogenesis. Methods: The role of CCL5 in inflammation, fibrosis, tumour initiation and progression was analysed in patients and animal models. In the NEMO model cell populations were studied using NEMOΔhepa/CCL5−/− mice and subsequent bone marrow transplantations (BMT). For therapeutic intervention Evasin-4 was injected either for 24 hours or for 8 weeks. To determine the role of CCL5 during angiogenesis Lx2 and HepG2 as well as a HUVEC based tube formation assay were used in in vitro studies. Results: In patients with chronic liver disease (CLD), CCL5 expression correlates with inflammation stage and fibrosis grade. Interestingly, NEMOΔhepa mice showed CCL5 and CCR5 upregulation as found in CLD patients. Our results identified immune cells in mediating this effect. Genetic inactivation of CCL5 in NEMOΔhepa mice diminished hepatocyte apoptosis, compensatory proliferation and fibrogenesis due to reduced immune cell infiltration. As a consequence late stage NEMOΔhepa/CCL5−/− mice displayed smaller and less malignant tumours, characterized by significantly reduced proliferative capacity and less pronounced angiogenesis in comparison with NEMOΔhepa tumors. Our in vitro experiments demonstrated that CCL5 increases VEGF expression Lx2 and in HepG2 cells leading to increased vessel like formation in a HUVEC based tube formation assay. Mechanistically, we identified hematopoietic cells as main source of CCL5. Short term Evasin-4 treatment significantly reduced the infiltration of CD45+Ly6G+ granulocytes. Subsequently, 8 week treatment of NEMOΔhepa mice resulted in a significant improvement of liver fibrosis. Conclusions: Therapeutic modulation of the CCL5/RANTES pathway with Evasin-4 significantly attenuated hepatic fibrosis progression. Deletion of CCL5 in NEMOΔhepa mice ameliorated hepatic fibrogenesis and HCC progression. Our in vitro observations suggest that CCL5 via VEGF contributes to angiogenesis. Hence, CCL5 is an attractive target to treat chronic liver disease. THU-120 miR-22 deficiency fosters hepatic tumour development A.-S. Ay-Berthomieu1, D. Portius1, D. Abegg2, M. Fournier1, C. Maeder1, A. Adibekian2, M. Foti1. 1Cell Physiology and Metabolism, Faculty of Medecine; 2Organic Chemistry, Faculty of Science, Geneva, Switzerland E-mail: [email protected] Background and Aims: miR-22 was previously reported to be involved in the carcinogenesis of diverse organs. Supporting such a role for miR-22, its expression is deregulated in various human cancers. In the liver, whether miR-22 has an oncogenic or tumour suppressive activity is unknown. The goal of this study is to assess the role of miR-22 in liver carcinogenesis. Methods: Control (CTL) and miR-22 knockout mice (miR22KO) were administered with DEN and fed or not an obesogenic diet. Tumours development was then monitored in lean/obese mice by CT-Scan imaging and in explanted tissues after sacrifice. Spontaneous hepatic tumours development was also examined in mice bearing a tamoxifen-inducible deletion of PTEN or PTEN/miR-22 specifically in hepatocytes (LIPTENKO and LIPTEN/miR22KO mice). The role of miR-22 in cell proliferation and the cell cycle was further assessed in vitro in human HCC cell lines. Finally, a proteomic analysis of hepatic tissues from CTL and miR22KO mice was performed. Results: The onset of tumours appearance was reduced and tumour multiplicity was increased in miR22KO mice administered with DEN as compared to control mice. Diet-induced obesity further aggravated S228

the effect of miR-22 deficiency on tumour onset and multiplicity. We then established a new model of spontaneous hepatic tumour development by generating a mouse strain bearing an inducible deletion of PTEN specifically in hepatocytes (LIPTENKO mice). Induction of PTEN deletion in mouse hepatocytes at two months of age led to the first appearance of tumours at 9 months of age as assessed by CT-scan imaging. However, the simultaneous induction of miR-22 and PTEN deletion in the same conditions (LIPTEN/ miR22KO mice) not only accelerated the onset of tumour appearance (first tumours at 7 months of age), but also significantly increased tumour multiplicity in one-year-old mice. Proteomic analysis of hepatic tissues from CTL and miR22KO mice indicated that miR-22 is associated in vivo with a major translational repression of key effectors of the lipid metabolism and of oncogenic proteins contributing to hepatic carcinogenesis. In vitro, we showed that miR-22 restrains hepatic cancer cell proliferation and block the cell cycle in the G1 phase. Conclusions: miR-22 behaves as a tumour suppressor microRNA in hepatic carcinogenesis. THU-121 Proteomic analysis identifies argininosuccinate synthase 1 as a useful biomarker for hepatocellular adenoma classification and patient management A. Abouhammoud1, E. Henriet2, J.-W. Dupuy3, B. Dartigues4, Z. Ezzoukhry2, N. Senant5, J.-F. Blanc6, B. Lebail7, M. Nikolski8, P. Bioulac-Sage2, C. Balabaud2, A.-A. Raymond2, F. Saltel2. 1U1053; 2 U1053, Inserm; 3Centre de Génomique Fonctionnelle de Bordeaux; 4 Centre de Bioinformatique de Bordeaux, Université de Bordeaux; 5 Plateforme d’Histopathologie expérimentale, INSERM US 005 – TBM CORE; 6U1053 Inserm; 7Service de Pathologie, CHU de Bordeaux; 8 Research Scientist LaBRI, CNRS, Bordeaux, France E-mail: [email protected] Background and Aims: Hepatocellular adenomas (HCA) are rare benign tumors that constitute a heterogeneous entity, divided into 4 groups based on patho-molecular features (1) H-HCA with inactivating mutations of Hepatocyte Nuclear Factor 1A (HNF1α), (2) inflammatory HCA (IHCA) with diverse mutations activating the JAK/STAT pathway, (3) b-HCA with activating β-catenin mutation (4) and finally, the unclassified HCA (UHCA). The identification of a HCA subgroup by default is still a difficulty particularly on a biopsy. Moreover, the risks of bleeding and malignant transformation of UHCA prompt to search new biomarkers for their identification. Aim: The objective of this study was to combine laser microdissection and mass spectrometry analysis to define UHCA proteome in the purpose of identifying specific biomarkers. Methods: After isolation of the tumoral (T) part from non tumoral tissue (NT) on formalin-fixed paraffin-embedded (FFPE) from HCA tissue sections using laser capture methodology, we use mass spectrometry analysis to compare T and NT proteomes of HCA (HHCA, IHCA, b-HCA, UHCA) and focal nodular hyperplasia (FNH). Using this methodology we searched for proteins specifically deregulated in U-HCA. This approach was applied to 9 cases of UHCA and compared with 3 cases of each classified HCA and 3 FNH. Results: First, we validated our approach confirming the known biomarkers expression of each HCA sub-groups (FABP1 for H-HCA, CRP for IHCA and GS for b-HCA and FNH). Indeed, we demonstrated that mass spectrometry analysis allow to discriminate between known HCA subtypes. Moreover proteomic data processing reveal that UHCA form a homogenous group and establish a deregulation of the endogenous synthesis of arginine (R) pathway involving argininosuccinate synthetase (ASS1) and arginosuccinate lyase (ASL). These two enzymes are specifically upregulated in all UHCA tested while there are downregulated in the other HCA subgroups as well as FNH. The systematic overexpression ASS1 in 15 UHCA tumors was then confirmed by immunohistochemistry. Of a total of

Journal of Hepatology 2017 vol. 66 | S95–S332