P0399 : Notch1 is a master regulator of the senescence secretome through repression of CEBPβ

POSTERS P0398 CHANGE OF FIBROSIS PATTERN AFTER AUTOLOGOUS BONE MARROW CELL INFUSION IN PATIENTS WITH ADVANCED LIVER CIRRHOSIS J.K. Kim1 , Y.N. Park2 , J.I. Lee1 , D.Y. Kim1 , S.H. Ahn1 , K.-H. Han1 , K.S. Lee1 . 1 Dept. of Internal Medicine, 2 Dept. of Pathology, Yonsei University College of Medicine, Seoul, Korea, South E-mail: [email protected] Background and Aims: Although we previously published positive clinical results of human clinical trial of autologous bone marrow cell infusion (ABMI) therapy for advanced liver cirrhosis (LC) (Kim JK et al. Cell Transpl 2010), change of fibrosis stage could not be shown. Because the patients had thick advanced fibrotic band, small change could not be evaluated by current staging system. Recently, enhanced detection and quantification of collagen fibers are possible using the nonlinear optical microscopy. The aim of this study is to show the change of fibrosis after ABMI in patients with advanced LC. Methods: Patients with Child–Pugh B or C LC and no viable hepatocellular carcinoma (HCC) underwent ABMI. Autologous BMCs were harvested and infused into peripheral vein after RBC depletion and mononuclear cell concentration. Patients were followed up every month during first 6 months of study period after the ABMI. Liver biopsy was performed before ABMI, and 1, 3, and 6 months after ABMI, if the patient agreed. Repeated biopsy samples were imaged by Genesis™ system (Histoindex, Singapore), a second harmonic generation and two-photon excitation fluorescence technology-based commercial devise. Collagen features were divided into portal, septal or fibrillar collagen as previous report (Xu et al. J Hepatol 2014). Results: Twenty patients were screened and 19 patients (M:F=9:10) were enrolled. Mean age was 52 year-old. Repeated biopsy samples were available in 8 patients for this study. Seven patients had B-viral LC and one had alcoholic LC. Only at 3 months after ABMI, aggregated collagen, string area, number of crosslinks, portal collagen percentage, portal aggregated collagen percentage, portal string area, portal and aggregated string area, portal and aggregated number of thin strings, portal number of crosslinks, septal number of thin strings, septal string area, septal and aggregated string area, fibrillar number of strings, fibrillar string area, fibrillar and aggregated string area were significantly decreased compared to baseline (p < 0.05, respectively). Conclusions: ABMI improved not only hepatic function but also collagen deposition. However, significant collagen decrease was noted only at 3 months after ABMI. P0399 Notch1 IS A MASTER REGULATOR OF THE SENESCENCE SECRETOME THROUGH REPRESSION OF CEBPb M. Hoare1 , Y. Ito1 , T.-W. Kang2 , S. Menon1 , R. Salama1 , L. Zender2 , M. Narita1 . 1 Cambridge Institute, Cancer Research UK, Cambridge, United Kingdom; 2 Department of Internal Medicine I, University Hospital, Tuebingen, Tuebingen, Germany E-mail: [email protected] Background and Aims: Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of SASP, the senescence-associated secretory phenotype. Major components of the SASP include TGFb1 and pro-inflammatory cytokines, such as IL1, IL6, and IL8, that have pleiotropic context-dependent effects. Methods: We utilised the well validated ER:RasG12V IMR90 HDF in vitro model which undergo Ras-induced senescence (RIS) with 4OHT. Genetic manipulation was achieved through retroviral gene transfer; transcriptional profiling by mRNA-seq; validation through qPCR and immunoblotting.

Results: Previously, we have shown that Notch1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of Notch1, downstream signaling is dynamically regulated: the cleaved, active intracellular domain of Notch1 (N1ICD) and Notchtarget genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression pattern of N1ICD and TGF-b1 expression were nearly identical, and inversely correlated with the cytokines, IL1 and IL8. Inhibition of Notch1 signaling, through expression of a dominant-negative form of the Notch1 binding partner MAML1, led to a reduction in TGF-b1, but increased IL1 and IL8 expression during RIS. In addition, ectopic restoration of N1ICD in established RIS cells drove reciprocal secretome changes with reduced IL1 and IL8 and increased TGF-b1, suggesting Notch1 signaling plays a critical role in secretome switching. It has been shown that the SASP in RIS is regulated by two major transcription factors, NFkB and CEBPb. Strikingly, over-expression of N1ICD strongly down-regulated CEBPb, but not NFkB, in fully established RIS cells. Further, expression of ectopic CEBPb in N1ICD-expressing cells partially restored levels of IL6/8. These data indicate that Notch1 represses pro-inflammatory cytokines by down-regulating CEBPb. Finally, Notch1 up-regulation in RIS was confirmed in several mouse models: the murine Kras-driven pancreatic intraepithelial neoplasia and Nras-driven hepatocyte senescence models. Conclusions: We propose that the transition to OIS is correlated with a switch from Notch1-driven TGFb1-rich secretome to a CEBPb-driven IL1/8 rich secretome, and that dynamic Notch1 signaling modulates senescence and its long-term fate strictly through a non-cell-autonomous fashion.

P0400 MICRORNA SIGNATURE OF EARLY-STAGE HEPATOCELLULAR CARCINOMA ARISING IN HCV-RELATED CIRRHOSIS G. Ghittoni1 , L. Veronese1 , F. Torello Viera1 , V. Curti2 , M. Ghitti3 , L.L. Rosa1 , V. Ravetta1 , L. Siciliani1 , S. Rossi1 . 1 VI Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation, 2 Department of Earth and Environmental Sciences, Laboratory of Immunology and Genetic Analysis, University of Pavia, 3 Department of Earth and Environmental Science, Laboratory of Statistics and Bioinformatics, University of Pavia, Pavia, Italy E-mail: [email protected] Background and Aims: Systems currently used to stage hepatocellular carcinomas (HCCs) perform poorly in prognostic settings. The availability of tumor biomarkers that reliably correlate with disease outcome would facilitate treatment and follow-up strategy planning. To identify such markers, we analyzed the microRNA profiles of HCCs and the cirrhotic tissue in which they develop. Methods: Ten patients with HCV-related cirrhosis underwent resective surgery for solitary, early-stage HCCs. In each case, we

Journal of Hepatology 2015 vol. 62 | S263–S864

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