301 RAGE IS REQUIRED FOR OVAL CELL ACTIVATION AND INFLAMMATION-ASSOCIATED MOUSE LIVER CARCINOGENESIS

POSTERS in this study we tested the hypothesis that due to its growth promoting function, SIRT1 expression may be a positive factor in the growth of HCC and therefore inhibiting its activity may be a means to limit the growth of HCC. Methods: HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for mRNA expression of SIRT1–7. To inhibit SIRT1, cells were either transduced with lentiviruses expressing shRNA sequences targeting SIRT1 or treated with small molecule inhibitors, sirtinol or cambinol. Genetic deletion was confirmed by RT-PCR and Western-blot. Effect of SIRT1 inhibition was monitored by changes in morphology, proliferation and cell cycle in vitro. Half a million wild type and SIRT1 knock-down HCC cells were transplanted under the liver capsule of Rag2gc−/− immunodeficient mice and tumor growth was monitored by bioluminescence. Results: SIRT1 mRNA and protein was significantly higher expressed in HCC cell lines compared to normal liver cells, whereas other family members, SIRT2–7, were expressed at equal or lower levels. Inhibition of SIRT1 in HepG2 and Hep3B cells either by shRNA or with sirtinol or cambinol altered cell morphology, impaired proliferation and in HepG2 resulted in a decrease of dedifferentiation markers AFP and glypican. SIRT1 inhibition in Hep3B cells resulted a senescent morphology and expression of SA b-gal. Intrahepatic tumors developed in 84% of mice injected with control cells, while only 33% of the animals developed tumors with SIRT1 knock-down cells. In addition, cambinol reduced xenograft growth in mice compared to vehicle treated controls. Conclusion: Targeted deletion of SIRT1 expression impairs the growth of HCC cells in vivo and vitro. Activation of SIRT1 may have a beneficial role in non-malignant liver tissue, however in malignant HCCs, inhibiting SIRT1 activity may be a novel therapeutic option. 301 RAGE IS REQUIRED FOR OVAL CELL ACTIVATION AND INFLAMMATION-ASSOCIATED MOUSE LIVER CARCINOGENESIS T. Pusterla1 , J. Nemeth1 , I. Stein2 , L. Wiechert1 , D. Knigin2 , S. Marhenke3 , T. Longerich4 , A. Vogel3 , A. Bierhaus4 , E. Pikarsky2 , J. Hess1 , P. Angel1 . 1 Signal Transduction and Growth Control, DKFZ, Heidelberg, Germany; 2 Hebrew University Hadassah, Jerusalem, Israel; 3 Hannover Medical School, Hannover, 4 University Hospital Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: The Receptor for Advanced Glycation-End products (Rage) is a multiligand receptor and member of the immunoglobulin super-family of surface receptors, mainly involved in tissue damage and acute and chronic inflammatory disorders. Rage and its ligands (S100 proteins and HMGB1) have been shown to be overexpressed in tumors, enhancing tumor progression and metastasis by still unknown mechanisms. We investigated the role played by RAGE in the Mdr2−/− mouse model of inflammation-driven liver carcinogenesis. Methods: Liver lesion size and number, tumor grading and multiplicity were compared in 15 month-old Mrd2−/− and Rage−/− Mrd2−/− (dKO) mice. Liver inflammation, damage, fibrosis, oval cell activation and presence of RAGE ligands was characterized in premalignant Mrd2−/− and dKO livers (3 and 6 months of age). The role played by RAGE in oval cell activation and its engagement with the RAGE ligand HMGB1 was further characterized in vitro in an immortalized oval cell line. Results: dKO mice were enriched in pre-malignant dysplastic nodules and developed hepatocellular carcinomas (HCC) that were smaller in size and number compared to Mrd2−/− mice. Interestingly, pre-malignant dKO mice did not display a reduced recruitment of inflammatory cells to the liver. Nevertheless, dKO mice showed reduced liver damage and fibrosis, in association with decreased oval cell activation. In fact, oval cells expressed high levels of RAGE and displayed reduced cell proliferation upon knock-down S124

of RAGE expression in vitro, whereas treatment with the RAGE ligand HMGB1 promoted an ERK1/2-cyclin D1-dependent oval cell proliferation. Conclusions: Our results provide experimental evidence of the crucial role played by RAGE signaling in the pathogenesis of HCC, most likely through the regulation of the liver progenitor cell pool. 302 A POLYMORPHISM IN THE OSTEOPONTIN PROMOTER REGION ASSOCIATED WITH PROGNOSIS OF HUMAN HEPATOCELLULAR CARCINOMA Q. Dong, X. Zhang, Y. Zhao, H. Jia, Q. Ye, L. Qin. Liver Cancer Institute & Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China E-mail: [email protected] Background and Aims: In hepatocellular carcinoma (HCC), increased osteopontin (OPN) levels are associated with metastasis, poor prognosis, and early tumor recurrence. Our previous study reported that osteopontin (OPN) plays a crucial role in hepatocellular carcinoma (HCC) metastasis. In the present study, we therefore determined the effects of OPN promoter polymorphisms on OPN expression and HCC prognosis. Methods: OPN promoter polymorphisms were assessed in HCC tissues of 827 patients (Cohort 1, n = 510; Cohort 2, n = 317) who underwent radical liver resection at the Liver Cancer Institute and Zhongshan Hospital in Shanghai, China. We determined the association of these polymorphisms and related haplotypes with HCC prognosis and OPN expression in HCC tissue. Results: Single nucleotide polymorphisms (SNPs) were identified at four loci (−1748, −616, −443, and −155). Three major haplotypes [Ht1: −1748G/−616T/−443T/−155G; Ht2: −1748A/−616G/−443T/−155− (− indicates base deletion); Ht3: −1748A/−616G/−443C/−155−] accounted for more than 90%. Only one nucleotide at −443 differed between Ht2 and Ht3. We found that only the −443T/C genotype and its related haplotypes(Ht2 and Ht3) was significantly associated with OPN transcription and HCC prognosis. In an independent cohort of 317 HCC patients, we determined that the −443C/C genotype, which significantly decreased transcription and OPN expression, were associated with a decreased incidence of recurrence and a prolonged overall survival compared with other genotypes (77.0%, 64.9% and 64.9% versus 47.0%, 38.0% and 30.2% in 3-, 5-and 9-year cumulative recurrence rates respectively, P = 0.005; 77.0%, 68.6% and 68.6% versus 59.9%, 51.2% and 37.0% in 3-, 5-and 9-year survival rates, respectively, P = 0.027). Moreover, both the −443C/C genotype and Ht3 were independent predictors for time to recurrence and overall survival (P = 0.005 and P = 0.024; P = 0.013 and P = 0.036, respectively). Conclusions: The OPN promoter polymorphisms at locus −443 and related haplotypes influencing OPN protein levels were identified as prognostic factors for HCC. 303 PLASMINOGEN K1–5 STRONGLY DECREASES INTRAHEPATIC SPREAD AND TUMOUR GROWTH IN AN EXPERIMENTAL MOUSE FIBROSIS MODEL E. Raskopf, T. Sauerbruch, V. Schmitz. Department of Innere Medicine I, University of Bonn, Bonn, Germany E-mail: [email protected] Background and Aims: Fibrosis and cirrhosis is the main cause of liver cancer. This is partly due to increased expression and secretion of proinflammatory and extracellular matrix proteins. This altered liver structure contributes to a protumoural and prometastatic environment. Previous studies showed that the angiostatin derivate PlgK1–3 decreases tumour cell homing in an experimental CRCmetastasis model. For another plasminogen derivate, PlgK1–5, it was shown that tumour growth was nearly completely blocked

Journal of Hepatology 2012 vol. 56 | S71–S224