Chronic treatment with over-the-counter H1 or H2 histamine receptor blockers decreases cholangiocarcinoma xenograft tumor growth, angiogenesis and EMT

POSTER PRESENTATIONS More specifically, TGFβ1 induced a dose dependent response in healthy scaffolds which was not observed in the cirrhotic scaffolds. Even though TGFβ1 changed hepatocyte specific gene expression i.e. albumin, the cells cultured in both models lack typical EMT markers such as TWIST, vimentin and N-Cadherin. Thus experiments are ongoing by using TWIST/vimentin positive HCC cells to investigate important TGFβ-EMT related responses in 3D scaffolds and the role of ECM in the pathogenesis of EMT-related HCC. Conclusions: This data suggest that acellular liver ECM scaffolds represent a suitable platform for dissecting the biology of liver cancer cells in experimental conditions closer to the natural microenvironment. The various supports and the ECM itself induced different cellular behavior and responses to TGFβ1 treatment. THU-127 microRNA-34a and natural killer cells: an immuno-epigenetic approach to halt hepatocellular carcinoma progression A.K. Abdelhamid1, R.A. Yacoub1, I.O. Fawzy2, A. Gomaa3, K. Hosny4, M.T. Hamza5, G. Esmat6, I. Waked3, A.I. Abdelaziz7. 1Pharmaceutical Biology Department; 2Pharmacology and Toxicology Department, The German University in Cairo, Cairo; 3Hepatology Department, National liver institute Menoufiya University, Menoufiya; 4General Surgery Department, Faculty of Medicine, Cairo University; 5Clinical Pathology Department, Ain Shams University; 6Endemic Medicine and Hepatology Department, Cairo University; 7School of Medicine, NewGiza University, Cairo, Egypt E-mail: [email protected] Background and Aims: MicroRNA-34a is one of the most documented tumor suppressor miRNAs. It was repeatedly found to be downregulated in hepatocellular carcinoma (HCC) and its downregulation was associated with tumor progression. Accordingly, it is currently being studied as a potential therapeutic target. Natural killer cells (NKs), a part of the innate immunity, play a vital role in tumor immune-surveillance; in response, cancers develop mechanisms to evade NK cell mediated lysis. NKG2D is one of the most powerful activating receptors involved in the cytotoxicity and development of NKs; studies have shown that decreasing the expression of NKG2D and its ligands is a strategy commonly adopted by tumors to escape NKs cytotoxicity. Although miR-34a was frequently studied in HCC, its role was never explored in the NKs of HCC patients. Therefore, the aim of this work was to evaluate the expression of miR-34a in NKs of HCC patients and to assess its impact on NKG2D receptor and its ligand ULBP2, as well as NKs cytotoxicity and development. Methods: Liver biopsies were obtained from 22 HCC patients and 19 healthy donors. Primary NKs were isolated from 72 HCC patients and 12 healthy controls using magnetic-assisted cell sorting. The isolated NKs and Huh-7 cells were cultured and transfected with miR-34a oligonucleotides using lipofection. Total RNA was extracted and the expression levels of miR-34a, NKG2D, ULBP2 and Perforin-1 were quantified by qRT-PCR. Percentages of NK subsets were assessed using flow-cytometry. Results: In NKs of HCC patients, overexpression of miR-34a ( p = 0.0317) and NKG2D (p = 0.0374) was observed. Forcing miR-34a expression in NKs of HCC patients significantly increased NKG2D ( p = 0.0474) and cytotoxic perforin-1 mRNA levels ( p = 0.0444). Moreover, the percentage of the more mature CD56dim NK cell subset was decreased ( p = 0.0378) and conversely a significant increase in the percentage of CD56bright NK cell subset was observed ( p = 0.0380). ULBP2 was found to be significantly lower in HCC tissues ( p = 0.0042) and Huh-7 cells ( p = 0.0004) compared to healthy tissues. In Huh-7 cells, mimicking of miR-34a significantly downregulated ULBP2 ( p = 0.0087) expression. Conclusions: This study suggests a possible role for miR-34a in regulating the cytotoxicity and development of the effector NK cells and their target cells in HCC.

THU-128 The anti-inflammatory receptor TREM-2 protects the liver from hepatocellular carcinoma development A. Esparza-Baquer1, I. Labiano1,2, O. Erice1, O. Sharif3,4, F. Oakley5, A. Lapitz1, P. Olaizola1, P.Y. Lee-Law1, E. Hijona1, R. Jiménez-Agüero1, S. Knapp3,4, D.A. Mann5, L. Bujanda1,2, J.M. Banales1,2,6, M.J. Perugorria1,2,6. 1Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV-EHU); 2CIBERehd, San Sebastián, Spain; 3 Research Center for Molecular Medicine of the Austrian Academy of Sciences; 4Department of Medicine I, Laboratory of Infection Biology, Medical University of Vienna, Vienna, Austria; 5Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom; 6IKERBASQUE, Basque Foundation for Science, Bilbao, Spain E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) is the most prevalent primary liver tumor and the third most common cause of cancer related death. To date, this tumor remains highly chemoresistant and shows a poor prognosis. Pro-inflammatory signals and liver regeneration are key contributors to the development of HCC. Toll-like receptor (TLR) derived signaling in non-parenchymal liver cells and accelerates carcinogenesis by modulating inflammatory responses. Therefore, regulation of pro-inflammatory signals arises as a promising therapeutic strategy for HCC. The triggering receptor expressed on myeloid cells-2 (TREM-2) acts as anti-inflammatory receptor, as it inhibits TLR-derived signaling in various tissues, yet its role in the liver and in HCC is still unknown. Methods: TREM-2 mRNA expression was analyzed in liver tissue samples from patients with HCC compared to control individuals. To evaluate the role of TREM-2 in liver regeneration a ∼70% partial hepatectomy (PHx) model was performed in wild type (WT) and Trem-2−/− mice, and animals were sacrificed at 1, 6, 36, 72 hours and 5 days post-surgery. Hepatocyte proliferation and pro-inflammatory cytokines were analyzed following PHx. To study the role of TREM-2 in hepatocarcinogenesis, WT and Trem-2−/− mice were treated with the hepatocarcinogen diethylnitrosamine (DEN) at a dose of 30 mg/ kg and number of tumors and tumor sizes were quantified 30 weeks after the injection. Results: TREM-2 mRNA expression is significantly upregulated in human HCC samples compared to control livers. Similarly, Trem-2 mRNA expression is also elevated in livers of mice after PHx. Hepatocyte proliferation was increased in Trem-2−/− mice compared to WT mice, as shown by PCNA and BrDU incorporation. Likewise, increased pro-inflammatory cytokine expression (TNF-α and IL-6) was observed in the Trem-2−/− livers. Mice lacking Trem-2 develop more tumors in response to the hepatocarcinogenic drug diethylnitrosamine (DEN) as compared to WT mice and this event was accompanied with an increased liver-body weight ratio in the Trem2−/− animals compared to normal controls. Conclusions: TREM-2 halts the initiation of liver regeneration and hepatocyte proliferation following PHx through the inhibition of proinflammatory gene expression. TREM-2 expression is upregulated in human HCC and its absence promotes the development and progression of HCC in mice. In view of these data, TREM-2 arises as a potential therapeutic target for HCC. THU-129 Chronic treatment with over-the-counter H1 or H2 histamine receptor blockers decreases cholangiocarcinoma xenograft tumor growth, angiogenesis and EMT L. Hargrove1, L. Kennedy2, J. Demieville3, H. Francis4. 1Texas A&M University/Baylor Scott & White Hospital; 2Central Texas Veterans Health Care System; 3Central Texas Veterans Healthcare System; 4Central Texas Veterans Healthcare system/Texas A&M HSC, Temple, United States E-mail: [email protected]

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POSTER PRESENTATIONS Background and Aims: Cholangiocarcinoma (CCA) is a fatal liver cancer with limited treatment options. Patients with primary sclerosing cholangitis (PSC) have a higher risk of developing CCA. Mast cells (MCs) infiltrate human CCA via stem cell factor (SCF)/c-kit; and, blocking MC-derived histamine (HA) decreases CCA tumor growth. Chronic treatment with H1 or H2 histamine receptor (HR) blockers decreases PSC progression in Mdr2−/− mice by reducing fibrosis and MC number. The aim of this study was to examine the effects of chronic treatment with H1 or H2 HR blockers on CCA tumor growth. Methods: Immunodeficient nu/nu mice were implanted with MzCha-1 cells into the hind flanks to allow for tumor development. Mice were treated with NaCl (vehicle), mepyramine (H1HR antagonist, 10 mg/kg/BW) or rantidine (H2HR antagonist, 10 mg/kg/BW) by IP injection for 35 days. Tumor growth was measured and extracted tumors were evaluated for the following parameters: proliferation by qPCR and immunoblotting for PCNA; MC presence by toluidine blue staining and qPCR for chymase, tryptase and c-kit; angiogenesis by immunofluorescence for vonWillebrand factor and qPCR for VEGF-A; and EMT by IHC for vimentin and e-cadherin and qPCR for s100A4. HA and tryptase serum levels were detected by EIA. Human tissue arrays were used to evaluate MC presence and the expression of H1HR and H2HR by immunohistochemistry. In vitro, Mz-Cha-1 cell lines were treated with NaCl (vehicle), mepyramine (H1HR antagonist, 25 mM) or rantidine (H2HR antagonist, 25 mM) for up to 96 hours; we measured proliferation by MTS assay, and angiogenesis and EMT by qPCR. Results: Treatment with H1HR or H2HR blockers decreased tumor growth/proliferation when compared to saline. MC infiltration and serum HA and trypatse levels were reduced in H1HR or H2HR treated mice. Angiogenesis and EMT were decreased in tumors from H1HR or H2HR treated mice compared to saline. In vitro CCA cells had decreased proliferation, angiogenesis and EMT gene expression following treatment with H1HR or H2HR antagonists. Conclusions: Inhibition of H1HR or H2HR decreases CCA tumor growth, angiogenesis and EMT. Commonly used over-the-counter HR inhibitors may be potential therapeutics for the treatment of CCA.

Non-invasive assessment of liver disease THU-475 The non-alcoholic fatty liver disease liver fat score is associated with liver disease mortality in the United States population A. Unalp-Arida1, C.E. Ruhl2. 1National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland; 2Social & Scientific Systems, Inc., Silver Spring, Maryland, United States E-mail: [email protected] Background and Aims: Fatty liver disease is common in the United States and worldwide with a global prevalence of 20% to 30%. It can progress to chronic liver disease and cirrhosis which was the 12th leading cause of death in the United States in 2014. The non-alcoholic fatty liver disease (NAFLD) liver fat score (LFS) was derived in a Finnish population and includes alanine aminotransferase (ALT), aspartate aminotransferase (AST), diabetes, metabolic syndrome, and fasting insulin (Kotronen, Gastroenterol, 2009;137:865). The hepatic steatosis index (HSI) was derived in a Korean population and includes ALT, AST, diabetes, sex, and BMI (Lee, Dig Liver Dis, 2010;42:503). We examined whether these two potential markers of liver health were associated with increased overall and causespecific mortality in a U.S. population-based survey with up to 23 years of linked-mortality data. Methods: We studied 8,978 fasted viral hepatitis negative adult participants in the third U.S. National Health and Nutrition

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Examination Survey (NHANES), 1988–1994. Intermediate and high steatosis probabilities were defined based on published cut-offs. Participants were passively followed for mortality, as identified by death certificate underlying or contributing cause diagnoses, by linkage to National Death Index records through 2011. Hazard rate ratios (HR) for mortality were calculated using Cox proportional hazards regression to adjust for common mortality risk factors. Results: The prevalence of intermediate and high steatosis probability using the LFS as a marker was 28.9% and 7.4%, respectively. During follow-up, there were 2,729 deaths from all causes and 89 with liver disease, including primary liver cancer. In age-adjusted analyses, liver disease mortality was increased with an intermediate (HR, 3.2; 95% confidence interval (CI), 1.3–7.4) or high (HR, 9.4; 95% CI, 4.1–21.6) LFS. With multivariate adjustment, the risk was further increased (Figure). There was no association of a higher LFS or HSI with allcause or cardiovascular disease mortality in multivariate-adjusted analyses.

Conclusions: In the U.S. population, a higher steatosis probability using the LFS, but not the HSI, was associated with increased liver disease mortality. Neither score was associated with other mortality outcomes. The performance of liver fat scores may vary among diverse populations. THU-476 The impact of bariatric surgery on nonalcoholic fatty liver disease and cardiovascular risk utilizing non-invasive measures C. Netanel1, D. Goitein2, M. Rubin2,3, M. Shechter3,4, Y. Kleinbaum5, S. Katsherginsky5, H. Hermon2, R. Hemi6, K. Tsaraf1, A. Donuzi1, M. Safran1, I. Tachlytski1, A. Herman3,7, P. Ortiz8, Z. Ben-Ari1,3. 1Liver Disease Center; 2Bariatric and Metabolic Surgery, Sheba Medical Center, Tel Hashomer; 3Sackler School of Medicine, Tel Aviv University, Tel Aviv; 4 Leviev Heart Center; 5Deparmtent of Radiology; 6Institute of Endocrinology; 7Department of Orthopedics, Sheba Medical Center, Tel Hashomer, Israel; 8OWL, Parque Tecnológico de Bizkaia, Derio, Spain E-mail: [email protected] Background and Aims: Bariatric surgery is an effective treatment for morbidly obese patients. Significant improvement in steatosis, inflammation and fibrosis stage is reported in patients with nonalcoholic fatty liver disease (NAFLD). We have prospectively evaluated the effect of weight-loss on liver steatosis grade, nonalcoholicsteatohepatitis (NASH) diagnosis, fibrosis stage and cardiovascular risk before and after sleeve gastrectomy (SG), using non-invasive measures. Methods: 26 patients with NAFLD, diagnosed by liver ultrasound (US), underwent pre- and post-operative evaluation (6–12 months after SG) with anthropometry, biochemical parameters, adiponectin, FibroMAX®, OWLiver® test (a serum based test that can discriminate NAFLD from NASH using metabolomics), US, real-time ShearWave™

Journal of Hepatology 2017 vol. 66 | S95–S332