Hepatocellular Carcinoma Differentially Modulates AMPK Activity and Induces Autophagy in Hepatic Stellate Cells in a Paracrine Manner

POSTER PRESENTATIONS Conclusions: The EGFR pathway is involved in maintaining an epithelial, non-migratory, phenotype in HCC cells and, in this way, it counteracts the TGF-β effects on EMT and cell migration. [Grants from the MINECO (cofounded by FEDER funds), Spain (BFU2012-35538 and ISCIII-RTICS RD12-0036-0029) and AGAUR-Generalitat de Catalunya (2014SGR0334)] FRI-058 MIR-21 AND MIR-150 DOWNREGULATION IS ASSOCIATED WITH OXALIPLATIN-RELATED SINUSOIDAL OBSTRUCTION SYNDROME AND IMPAIRED SURVIVAL J. Zhao1, S. Rensen1, C. Vreuls2, M. van den Broek1, A. Driessen2, M. van Herwijnen3, M. Jetten3, D. Jennen3, C. Dejong1,4, S.O. Damink1,5. 1 Department of Surgery, Maastricht University Medical Centre and NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University; 2Department of Pathology, Maastricht University Medical Centre; 3Department of Toxicogenomics, Maastricht University; 4 Grow School for Oncology and Developmental Biology, Maastricht University, Maastricht, Netherlands; 5Institute for Liver and Digestive Health, University College London, London, United Kingdom E-mail: [email protected] Background and Aims: Oxaliplatin-related sinusoidal obstruction syndrome (SOS) is associated with increased morbidity and impaired survival. Hence, identification of the molecular mechanisms underpinning the development of SOS is pivotal. Methods: This case-matched cohort study of patients who underwent partial hepatectomy for colorectal liver metastases between 2008 and 2009 included 28 patients with preoperative oxaliplatin treatment and SOS grade 0 or 2/3; ten patients with moderate/severe SOS were matched with ten without SOS by number of oxaliplatin cycles, cumulative oxaliplatin dose, sex, age, body mass index, and liver transaminases. Non-tumor liver microRNA expression was analyzed using Agilent arrays for the 20 patients that could be matched and validated by quantitative PCR for all 28 patients included. MicroRNA expression was subsequently related to overall survival and recurrence free survival. Results: Moderate/severe SOS was not associated with upregulation of any microRNA. However, miR-21 and miR-150 were significantly downregulated in the moderate/severe SOS group (array: 1.34-fold, p = 0.028; 1.46-fold, p = 0.035, respectively; qPCR: 1.32-fold, p = 0.001; 1.34-fold, p = 0.014, respectively). Low hepatic expression of both miR-21 and miR-150 was associated with impaired overall survival (n = 28, p = 0.032 and p = 0.010, respectively). Likewise, both overall recurrence free survival and liver recurrence free survival were reduced in subjects with low hepatic miR-21 ( p = 0.010 and p = 0.046, respectively) and miR-150 expression ( p = 0.025 and p = 0.031, respectively). Conclusions: The strong association between reduced hepatic miR21/miR-150 levels and SOS, tumor recurrence, as well as survival indicates that miR-21 and miR-150 regulated genes may represent the molecular links explaining the impact of SOS on survival. FRI-059 NUCLEAR LOCALIZATION OF IMPDH2, THE PRIMARY TARGET OF MYCOPHENOLIC ACID, CONSTRAINS HEPATOCELLULAR CARCINOMA K. Chen1,2, K. Sideras1, B. Ma1, W. Cao1, L.J. van der Laan3, D. Sprengers1, R. Smits1, H.J Metselaar1, J. Kwekkeboom1, M.P Peppelenbosch1, Q. Pan1. 1Department of Gastroenterology and Hepatology, Erasmus MCUniversity Medical Center, Rotterdam, Netherlands; 2College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China; 3Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, Netherlands E-mail: [email protected] Background and Aims: Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in purine-nucleotide synthesis. Mycophenolate mofetil (MMF)/Mycophenolic acid (MPA), a widely

used immunosuppressant in organ transplantation, is a reversible and uncompetitive IMPDH inhibitor, preferentially targeting the IMPDH2 isoform. This study aimed to investigate the function of IMPDH2 in hepatocellular carcinoma (HCC) and its role in mediating the effects of MPA. Methods: Immunohistochemical (IHC) staining of IMPDH2 was performed in tissue microarrays of patient HCC tissue. Three HCC cell lines and immunodeficient mice were used for functional experiments. Retrospective analysis was performed to investigate the association of MMF therapy with HCC recurrence in liver transplantation patients. Results: Cytoplasmic IMPDH2 protein level was significantly lower in HCC tissues compared with adjacent liver tissues (n = 131, p < 0.01). High IMPDH2 level in tumor tissue was significantly associated with better cumulative survival (n = 131, p < 0.01). Conversely, downregulation of IMPDH2 by RNAi in HuH7 cells significantly increased the ability of single cell colony formation in vitro (n = 6, p < 0.01), and the efficiency of tumor initiation (100% vs 67%) in mice. Furthermore, HuH7 cells with IMPDH2 down-regulation formed significantly larger tumors compared to the control group (1.40 g ± 0.23 vs 0.29 g ± 0.16, mean ± SEM, n = 6, p < 0.01). Interestingly, nuclear IMPDH2 was observed in a subset of patient HCC tissues, which was significantly associated with better cumulative survival (n = 16, p < 0.05). In HCC cell lines, MPA potently inhibited cell proliferation. In HCC related liver transplantation patients (n = 42), the use of MMF was significantly associated with lower recurrence rates ( p < 0.05) and higher cumulative survival rates ( p < 0.05). Mechanistically, the inhibitory effects of MPA on HCC cells were independent of cellular nucleotide synthesis. However, MPA was able to drive nuclear translocation of IMPDH2 forming rod/ring structures in the nucleus. Conclusions: High expression of IMPDH2, in particular its nuclear localization, is associated with less colony formation in cell lines, smaller and less aggressive tumors in mice and a better clinical outcome in HCC patients. The inhibitory effects of MMF/MPA on HCC is independent of cellular nucleotide synthesis, but likely attributed to its ability of driving nuclear translocation of IMPDH2. FRI-060 HEPATOCELLULAR CARCINOMA DIFFERENTIALLY MODULATES AMPK ACTIVITY AND INDUCES AUTOPHAGY IN HEPATIC STELLATE CELLS IN A PARACRINE MANNER K. Schölzel1, L. Longato1, M. Pinzani1, K. Rombouts1. 1Institute for Liver and Digestive Health, University College London UCL, London, United Kingdom E-mail: [email protected] Background and Aims: AMP-activated protein kinase (AMPK) is a critical regulator of cell metabolism and autophagy in hepatocellular carcinoma (HCC) and hepatic stellate cells (HSC) and has recently been implicated in HCC development and progression. Autophagy exerts both pro-fibrogenic effects on HSC as well as tumourpromoting effects on HCC, supporting tumour survival and metabolic adaptation. In HCC, tumour-stromal interactions are considered to be a potential target for novel anti-cancer therapies. Therefore, the paracrine effect of HCC on HSC behaviour with special focus on AMPK signalling and autophagy was assessed in this study. Methods: Conditioned medium of HepG2 and PLC/PRF/5 cells was obtained after 48 hours incubation in serum-free medium. Activated, serum-starved, primary human HSC were exposed to HCC conditioned medium with or without the AMPK activators AICAR, Metformin or Phenformin, or the AMPK inhibitor Compound C (CC) for 24 hours. Proliferation (BrdU incorporation) and metabolic activity (MTS assay) of HSC were assessed. Expression levels of kinases up- and downstream of AMPK and markers of autophagy were investigated by western blot and qRT-PCR. Results: All AMPK activators induced activation/phosphorylation of AMPK-Thr172 and significantly inhibited HSC proliferation and

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POSTER PRESENTATIONS metabolic activity. HSC proliferation was significantly stimulated by HepG2 conditioned medium, accompanied by inhibition of AMPK activity ( p-AMPK-Ser485/491). This effect was reversed by the AMPK activator AICAR. In contrast, PLC/PRF/5 conditioned medium did not affect HSC proliferation and induced activation/phosphorylation of AMPK-Thr172. Moreover, PLC/PRF/5 conditioned medium induced autophagy as assessed by LC3B protein expression as well as induction of pro-inflammatory cytokine mRNA expression in HSC. Interestingly, the AMPK inhibitor CC induced phosphorylation of AMPK-Ser485/491, but inhibited HSC proliferation to the same extend as the AMPK activator AICAR. Furthermore, CC but not AICAR induced autophagy in HSC. In order to identify factors in HCC conditioned media which influence AMPK activation and induction of autophagy, mass spectrometry analysis is performed. Conclusions: These data indicate that diverse HCC cell types can differentially modulate AMPK activity in HSC in a paracrine manner, resulting in both HSC activation and induction of autophagy. Therefore, modulating AMPK activity in HCC and HSC may lead to the development of novel approaches for both anti-cancer and antifibrogenic therapy. FRI-061 STANNIOCALCIN 1 OVEREXPRESSION PROMOTES METASTASIS IN HEPATOCELLULAR CARCINOMA K. Chan1, C. Leung1, C. Wong1,2, I. Ng1,2, R. Lo1,2. 1Pathology; 2State Key Laboratory for Liver Research, THE UNIVERSITY OF HONG KONG, Hong Kong, Hong Kong, China E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) is an aggressive cancer with characteristically rapid tumor growth, frequent recurrence and metastasis, and resistance to conventional chemotherapy. Recently, Stanniocalcin 1 (STC1) was underlined as a proto-oncogene and a potential prognostic biomarker in human cancers. In addition, STC1 was found to be induced by hypoxia. Hypoxia is a well-characterized microenvironment of HCC. Hypoxiarelated genetic alterations profoundly contribute to tumor progression and chemoresistance. Thus identification of hypoxiaresponsive genes is an integral part of understanding the tumor biology of HCC. In this study, we postulate that STC1 plays an important role in HCC development and aim at investigating the functional significance of STC1 in HCC. Methods: Expression level and localization of STC1 were assessed in clinical HCC samples and HCC cell lines using quantitative real-time PCR, Western blotting and immunofluorescence microscopy. To unveil the roles of STC1 in HCC, we established stable STC1 knockdown clones in SMMC-7721, Huh-7 and MHCC-97L HCC cell lines using lentiviral-based approach and performed transwell migration and matrigel invasion assays. The role of STC1 in metastasis was further examined with an orthotopic mouse model. Results: STC1 mRNA expression level was significantly up-regulated in human HCC samples (T), when compared to the corresponding non-tumorous (NT) liver tissues ( p = 0.002, n = 75). STC1 overexpression at transcript level (T/NT ≥ 2-fold) was observed in 41% of cases (31/75), and was associated with direct liver invasion ( p = 0.034) on statistical correlation with clinicopathological parameters. STC1 was expressed in multiple HCC cell lines MHCC97L, MHCC-97H, SMMC-7721, BEL-7402 and Huh-7, while the immortalized liver cell line MIHA showed no detectable expression of STC1. Moreover, STC1 expression was induced by hypoxia (1% O2) in HCC cells, possibly in a HIF1a-dependent manner. Silencing of STC1 in both SMMC-7721 and Huh-7 cells suppressed HCC cell invasion and migration ability under both normoxic and hypoxic conditions in vitro. Knockdown of STC1 in MHCC-97L cells inhibited tumor growth and reduced lung metastasis in vivo. Conclusions: STC1 expression is up-regulated by hypoxia in HCC and promotes tumor metastasis.

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FRI-062 INFLAMMATION AND FIBROSIS IN THE LIVERS OF TNFR1/MDR2KO MICE L.K. Berkhout1, T. Krech2, G. Tiegs1, R. Barikbin1. 1Institute of Experimental Immunology and Hepatology; 2Department of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany E-mail: [email protected] Background and Aims: One third of the cancer related deaths is attributed to the hepatocellular carcinoma (HCC), which in almost 90% of all cases arises in a setting of chronic inflammation and advanced fibrosis. The multidrug resistance protein 2 knockout (Mdr2ko) mice are widely used as a model for chronic hepatitis and inflammation-associated HCC. Tumor necrosis factor receptor-1 (TNFR1) mediated signaling is known to induce hepatic epithelial cell death, inflammation, and fibrosis. Aim of this study was to analyze whether an additional knockout of TNFR1 in an Mdr2ko mouse model would suffice to significantly reduce hepatic inflammation and subsequent fibrosis which could consequently delay HCC development. Methods: Tissue injury was assessed by plasma analysis of ALT and ALP levels. TNFα Hepatic immune cell composition was determined by FACS analysis. Inflammation and fibrosis were evaluated by histological analysis of H&E stained liver slices by a pathologist. ECM deposition was further analyzed through quantification of the hepatic hydroxyproline content. Real time RT–PCR was applied to analyze hepatic expression levels of different targets involved in inflammation (IL-1β, TNFα), matrix remodeling (Collagen, MMPs, TIMPs, α-sma, PDGFs), tumor development (AFP, A20, OPN), and tissue regeneration (CcnA2, CcnE2, CDK1, CDK2). Results: TNFR1/Mdr2ko mice showed elevated plasma levels of ALT and ALP which are indicative of more severe tissue damage. TNFR1/ Mdr2ko mice showed significantly increased hepatic hydroxyproline contents and expression levels of collagen type III. FACS analysis revealed, that the tissue damage in the TNFR1/Mdr2ko mice is highly correlated to the hepatic infiltration of granulocytes, this correlation could not be shown for the Mdr2ko mice. Real time RT-PCR showed elevated expression levels of genes involved in fibrosis, inflammasome activity, necroptosis and possibly malignant transformation. The absence of TNFR1 mediated signaling seemed to have no effect on the regenerative capacity of the liver in the Mdr2ko mouse model. Conclusions: The absence of TNFR1 mediated signaling does not lead to an improvement of the pathological phenotype of the Mdr2ko mouse model. It instead leads to exacerbated tissue damage possibly through alternative forms of cell death, which results in an increased fibrotic response in these mice. Long-term studies will have to show whether this will affect the tumor incidence in the Mdr2ko mouse model. FRI-063 RESTORING MIR122 IN HUMAN STEM-LIKE HEPATOCARCINOMA CELLS, PROMPTS TUMOR DORMANCY THROUGH SMADINDEPENDENT TGF-Β PATHWAY L. Boix1, J.M. Lopez-Oliva2, A.C. Rhodes3, J. Bruix4. 1BCLC group. Liver Unit Hospital Clinic Barcelona, CIBEREHD. IDIBAPS; 2BCLC group Liver Unit Hospital Clinic Barcelona; 3BCLC group. Liver Unit Hospital Clinic Barcelona, FCRB IDIBAPS; 4BCLC group Liver Unit Hospital Clinic Barcelona, CIBEREHD. IDIBAPS, Barcelona, Spain E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in the world. Early diagnosis and new therapeutic strategies have significantly improved patient prognosis but disease recurrence and treatment resistance are still a problem. Cancer stem cell hypothesis establishes that tumours are initiated and maintained by a minority of cells with stem cell properties that are responsible for disease recurrence and treatment failure. miR122 is the prevalent miRNA in adult healthy liver and it is

Journal of Hepatology 2016 vol. 64 | S425–S630