Diabetes augments obesity in accelerating liver tumour development: role of oxidative stress-induced JNK signalling and DNA damage response

ORAL PRESENTATIONS Conclusions: Hepatic LGR5 stem cells are only induced following liver injury and importantly contribute to DEN-induced liver carcinogenesis but not to tissue repair. Thus targeting LGR5-positive cells appears promising as an anti-cancer strategy in the liver. PS-142 Immune gene expression profile in hepatocellular carcinoma and surrounding tissue predicts time to tumor recurrence C. Carone1, A. Olivani2, R.D. Valle3, T. Trenti1, G. Missale2, E. Cariani1. 1 Toxicology and Advanced Diagnostics, Ospedale S. Agostino-Estense, Modena; 2U.O. Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria di Parma; 3Department of Surgery, University of Parma, Parma, Italy E-mail: [email protected] Background and Aims: The anti-tumor immune response may play a major role on the clinical outcome of hepatocellular carcinoma (HCC). We characterized the liver immune microenvironment by direct hybridization of RNA extracted from HCC and non-tumorous tissues. Methods: RNA was extracted from frozen liver tissue samples of HCC (T, n. 30) and non-tumorous tissues (NT, n. 33) obtained from 38 patients. Matched samples were available for 25 patients. The immune gene expression profile was analyzed by the nCounter GX Human Immunology v2 system (NanoString Technologies) that detects the expression levels of 579 immune response-related genes simultaneously. Results: The immune gene expression profile of T and NT tissues was significantly different ( p < 0.05). The possible prognostic relevance of liver immune microenvironment was therefore evaluated in T and NT samples separately. Unsupervised clustering detected two main clusters of immune gene expression both in T and in NT liver samples. In both cases, expression clusters identified groups of patients with significantly different median time to HCC recurrence. Based on T tissue, two groups with median TTR of 18.5 and 127 months, respectively, were detected ( p = 0.005). Expression of genes related to inflammation, T and B cell activation were associated with longer TTR. The analysis of NT tissues discriminated subsets of patients with median TTR of 19 and 68 months (p = 0.032). By contrast to T tissue, a predominant inflammatory immune environment was associated with shorter TTR. The liver immune gene profile was not statistically related to overall survival in this series. Conclusions: We evaluated immune gene expression by direct hybridization of RNA extracted from liver tissue. Immune gene expression profiles predictive of TTR could be identified both in HCC and in adjacent cirrhotic tissues. Longer TTR was associated with overexpression in T tissue and downregulation in NT tissue of inflammation-related genes. PS-143 Chk2 DNA damage response protein mislocalization further enhances chromosomal instability and human hepatocellular carcinoma progression V. Carloni1, M. Lulli2, S. Madiai1, T. Mello3, A. Hall4, T.V. Luong4, M. Pinzani5, K. Rombouts5, A.G. Galli3. 1Department of Experimental and Clinical Medicine; 2Department of Experimental and Clinical Biomedical Sciences, Pathology Unit; 3Department of Experimental and Clinical Biochemical Sciences, Gastroenterology Unit, University of Florence, Florence, Italy; 4Department of Cellular Pathology, Royal Free Hospital; 5Division of Medicine, Institute for Liver &Digestive Health, University College London (UCL), London, United Kingdom E-mail: [email protected] Background and Aims: High levels of genomic instability correlate with progression in hepatocellular carcinoma (HCC) of which the most common form is chromosomal instability (CIN), resulting in heterogeneity, with drug resistance and immunity escape as a consequence. CIN perse is an important factor of DNA damage sustaining numerical/structural chromosome abnormalities S78

but the underlying mechanisms are not well characterized. In this study, the role of Chk2, a DNA damage response kinase was investigated. Methods: An animal model of diethylnitrosamine-induced HCC was employed known to induce DNA damage and elevated mitotic errors. DNA damage response kinase Chk2 localization was determined in two cohorts of human HCC specimens. To assess the functional role of Chk2, gain on-and loss-of-function, mutation analysis with Chk2 variants, karyotyping, immunofluorescence/ live imaging were performed. Three different cell lines were used: HCT116, a near-diploid cell line, Huh7 a stable hyperdiploid karyotype, and human hepatocytes (HuS) immortalized with TERT gene. Results: Tumours of DEN-treated animals showed nuclear upregulation of Chk2 and P-H2A.X known to be activated in the presence of DNA damage. In vitro, defective chromosome segregations caused DNA damage per se and induced Chk2 overexpression. Chk2 overexpression/phosphorylation-activation and mislocalization occurred exclusively within mitotic components of HuS30gen. This coincided with an increased mitotic index which was reversed by knockdown of Chk2. The forkhead-associated (FHA) domain of Chk2 is uniquely essential for proper localization to mitotic structures. Aurora B kinase and P-Histone H3 colocalized with Chk2 when mislocalized, thus sustaining a constant mitotic activity. Retinoblastoma phosphorylation contributed to defective mitoses, but not p53. Chk2 expression was investigated in two cohorts of HCC tissues. A strong cytoplasmic and perinuclear presence in Grade I and Grade II was observed. In contrast, grade III HCC tissues were marked by a strong and exclusive nuclear Chk2. RNA-Seq-based transcriptomes of 188 HCC tissues (TCGA) were analysed; 102 with TP53 mutations and 86 with CNNB1 mutations. Chk2 was identified to be significantly associated with HCC TP53 mutations characterized by CIN. Conclusions: The study reveals a new mechanistic insight in the co-involvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients. PS-144 Diabetes augments obesity in accelerating liver tumour development: role of oxidative stress-induced JNK signalling and DNA damage response E. Arfianti1,2, V. Barn1, S. Pok1, C.Z. Larter1, N.C. Teoh1, G.C. Farrell1. 1 Liver Research Group, The Australian National University, Canberra, Australia; 2Department of Medical Biology, Riau University, Pekanbaru, Indonesia E-mail: [email protected] Background and Aims: Obesity and diabetes are independent risk factors for hepatocellular carcinoma (HCC) yet the contribution of each metabolic condition has not been clarified. Earlier, we demonstrated that obese diabetic foz/foz mice exhibit accelerated diethylnitrosamine (DEN)-induced HCC. To establish whether obesity itself or diabetes is more relevant to enhanced HCC development, we compared chemically induced HCC between equally obese diabetic Alms1 mutant ( foz/foz) NOD.B10 and nondiabetic foz/foz BALB/c mice. Methods: Male foz/foz and Wt NOD.B10 and BALB/c mice were injected with DEN (10 mg/kg) at 12–15 days, controls with saline. Hepatic protein expression was assayed by immunoblotting and immunohistochemistry. Results: Both strains of foz/foz mice developed equivalent obesity, but metabolic complications of obesity, including hepatomegaly, insulin resistance with hyperinsulinemia, and hyperglycemia, occurred only in foz/foz NOD.B10 mice. At 6 mths, the incidence of liver tumours was significantly higher in foz/foz NOD.B10 than foz/foz BALB/c mice (100% vs 40%). Liver nodules were also more

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ORAL PRESENTATIONS numerous and larger in foz/foz NOD.B10 compared with BALB/c counterparts. Increased serum ALT and hepatic Bax expression occurred in diabetic obese NOD.B10 but not in non-diabetic obese BALB/c mice. Despite the obvious difference in levels of serum insulin in response to obesity, insulin-mediated mammalian target of rapamycin (mTORC1) activation was similar between groups. Instead, activation of c-Jun N-terminal kinase (JNK) signalling was clearly enhanced in foz/foz NOD.B10 mice but unaltered in foz/foz BALB/c mice. This was associated with the up-regulation of c-Myc. There was a parallel increase in activation of nuclear factor E2related factor 2 (NRF2) in foz/foz NOD.B10 vs foz/foz BALB/c mice, inferring enhanced oxidative stress in obese mice with diabetes. Consistent with increased liver injury, DNA damage sensors, ataxia-telangiectasia-mutated (ATM) and p53, were up-regulated in livers from foz/foz NOD.B10 (vs foz/foz BALB/c), but the cell cycle checkpoint protein, CHK2, was less activated. Conclusions: Diabetes accelerates liver tumour development in obese mice. The pathogenic mechanisms may include increased liver injury related to metabolic obesity and inadequate cell-cycle checkpoint control in response to DNA damage. In addition, oxidative stress-induced JNK activation may be important in the acceleration of hepatocarcinogenesis. PS-145 Hepatitis C virus mediated chronic inflammation and tumorigenesis in the Humanised Immune system and Liver mouse model F.Z. Zheng1, C.W. Sze2, C.T. Keng1, M. Al-Haddawi1, M. Liu1, S.Y. Tan1, H.L. Kwek1, Z. Her1, X.Y. Chan1, B. Barnwal1, E. Loh3, T.C. Tan4,5, K.T.E. Chang3,5, Q. Chen1,2,6, Y.J. Tan1,2. 1Institute of Molecular and Cell Biology; 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore; 3Department of Pathology and Laboratory Medicine; 4Department of Obstetrics & Gynaecology, KK Women’s and Children’s Hospital; 5Duke-NUS Graduate Medical School; 6National Cancer Centre, Singapore, Singapore E-mail: [email protected] Background and Aims: Currently, 130–150 million individuals are estimated to be infected by the hepatitis C virus (HCV) globally. While the development of direct acting antivirals against HCV infection have proven to be highly efficacious and it is hopeful that we may achieve sustained virologic response in most HCV infected patients, the precise mechanisms of hepatitis C pathogenesis are still not well understood. One of the main obstacles for studying HCV mediated pathogenesis is lack of small animal models that can accommodate HCV infections and recapitulate the disease process. To address this, we have developed a humanised mouse model termed the Humanised Immune system and Liver (HIL) mice with matching human immune systems and hepatocytes. We have previously described successful infections of the HIL mice with HCV, resulting in HCV-specific immune responses and clinical symptoms such as liver inflammation and fibrosis by 9 weeks post infection. Here, we investigated the long term effects of HCV pathogenesis in HIL mice up to 28 weeks post infection. Methods: HIL mice were infected with the J6/JFH1-P47 strain of HCV and monitored for abnormal liver pathologies over a period of 20–28 weeks. Results: HCV RNA could be detected in infected mice at 9 weeks post infection but not at 20–28 weeks post infection. HCV infected mice developed increased incidences of liver fibrosis, granulomatous inflammation compared to control mice. Chronic liver inflammation in infected mice was mediated by the human immune system and was accompanied by expansions of monocytes/macrophages and T cells. A small percentage (6%) of HCV infected mice developed tumours in the form of hepatocellular adenomas or hepatocellular carcinomas by 28 weeks post infection. Further analyses revealed that

while the liver tumours were comprised of human and mouse hepatocytes, a majority (93%) of actively dividing cells within the tumours were human hepatocytes. Conclusions: HIL mice can recapitulate some of the clinical symptoms seen in chronically infected HCV patients. Persistence of HCV-associated liver disease such as chronic liver inflammation and tumorigenesis appear to require initial infections of HCV and immune responses but not long term HCV viraemia.

Alcohol and drug-induced liver injury PS-146 Efficacy of grannulocyte colony stimulating factor in the management of steroid non-responsive severe alcoholic hepatitis – A double blind randomised control trial S.M. Shasthry1, M. Sharma1, S. Sarin1. 1Hepatology, ILBS, New Delhi, India E-mail: [email protected] Background and Aims: Severe alcoholic hepatitis (SAH) (Maddrey’s score >32) carries high mortality with limited response to corticosteroid therapy. Treatment options for steroid non-responsive (Lille score >0.45) SAH are limited. Impaired hepatic regeneration and failure of commitment of regenerative cells has been considered partly responsible for this. We evaluated the efficacy of Grannulolyte colony stimulating factor (G-CSF) in this group of patients. Methods: Consecutive patients with SAH patients were treated with 40 mg prednisolone per day and were classified as steroid responder and non-responder (Lille score >0.45) at day 7. Responders were continued with the Prednisolone and non-responders were randomized into GCSF vs. placebo.

Results: Thirty-three of 132 (25%) SAH patients were steroid nonresponders and 28 [mean age 40.2 ± 10.3 yr.] consented to receive either GCSF or placebo. SAH patients were sick [mean bilirubin 27.7 ± 23.4 mg/dL, INR of 2.4 ± 1.9, S.creatinine was 0.59 ± 0.66 mg/dL. Baseline hepatic venous pressure gradient was 19.1 ± 5.5 mmHg with CTP 10.8 ± 1.2, MELD 26.2 ± 4.3, GAH 8.5 ± 0.9, Discriminant factor 81.1 ± 26 and Lille score was 0.68 ± 0.2] and were comparable in 2 groups. In the G-CSF group, 7 (50%) patients survived compared to 3 (21.4%) in the placebo group, with the mean follow up of

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