- Email: [email protected]
P0400 : Microrna signature of early-stage hepatocellular carcinoma arising in HCV-related cirrhosis
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
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POSTERS collected one sample of tumor and two samples of tumor-free cirrhotic tissue (near and at a distance from the tumor) and used them to construct 30 small RNA libraries for next-generation sequencing. The reads that emerged were clustered and aligned against the MiRBase with CLC Genomic Workbench software. Results: A total of 2,329 mature microRNAs were found. Annotated microRNAs with a mean read number per sample of >20 underwent parametric and randomized statistical analyses. Those associated with P values of ≤0.01 in both analyses were considered differentially expressed between tissue types. Of the 617 microRNAs subjected to statistical analysis, 35 exhibited expression levels in HCC that were significantly different from those found in one or both tumor-free cirrhotic tissues; two of the 35 were also differentially expressed in the two types of cirrhotic tissue. Eighteen of the 35 had never been reported as dysregulated in HCC. Conclusions: We identified an HCC-signature set of 35 microRNAs, which distinguishes HCCs from background cirrhotic tissues. It should provide a good starting point for subsequent research aimed at identifying biomarkers correlating with HCC outcome. P0401 THE SERINE PROTEASE FSAP (FACTOR VII ACTIVATING PROTEASE) MAINTAINS THE DIFFERENTIATED STATE OF MOUSE HEPATOCYTES A. Martinez-Palacian1 , S. Kanse1 . 1 Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, 0372 Oslo, Norway E-mail: [email protected] Background and Aims: FSAP, encoded by the Habp2 gene, is a serine protease secreted by hepatocytes as an inactive zymogen that circulates in the blood. We have recently demonstrated a novel anti-fibrotic role for FSAP in the liver. The aim of this study was to analyze the effect of FSAP on hepatocyte differentiation and proliferation. Methods: Primary hepatocytes isolated from BALB/c wild type (WT) or FSAP-deficient mice and the mouse hepatocyte cell line AML-12 were used as in vitro models. The use of knockout hepatocytes was further supported by the use of siRNA-mediated knock-down of the gene for FSAP and the addition of exogenous FSAP protein. Transcript levels were analyzed by qRT-PCR, protein levels by Western blotting and DNA synthesis by BrdU incorporation. Results: Primary hepatocytes and AML-12 cells lacking expression of endogenous FSAP show a reduction in HNF4a (Hepatocyte Nuclear Factor 4a) and an increase in AFP (alphafetoprotein) transcript levels compared to WT cells. Besides, Transforming Growth Factor beta-1 (TGFb) almost completely abolishes HNF4a mRNA levels in FSAP-depleted cells, while the reduction in WT cells is around 50%. Basal reduction on HNF4a mRNA is not due to an Epithelial-Mesenchymal Transition (EMT): silencing of the gene for FSAP does not down-regulate E-cadherin protein levels nor up-regulates mRNA levels of the EMT-inducing transcription factors (TFs) SNAI1, ZEB1 and ZEB2. Moreover, FSAP depletion does not impair the TGFb-induced up-regulation of these TFs. Preliminary results show that exogenous FSAP induce a 30% increase in basal BrdU incorporation in hepatocytes. Exogenous FSAP can overcome the anti-proliferative effect of TGFb in FSAPdeficient hepatocytes, but not in WT cells, suggesting that lack of endogenous FSAP decreases the sensitivity to TGFb inhibitory effects. Conclusions: We have for the first time described a novel role for FSAP in hepatocyte differentiation. It contributes to mantain HNF4a expression and AFP repression but does not affect EMT. This, together with its potential role on proliferation, provides a mechanistic insight into the effect of FSAP on hepatocytes that is relevant for hepatocellular carcinoma. S464
P0402 COMBINATION THERAPY FOR HEPATOCELLULAR CARCINOMA: A SYSTEMS BIOLOGY PERSPECTIVE ON THE SYNERGISTIC ANTITUMOR ACTIVITY OF SORAFENIB WITH PI3K/AKT PATHWAY INHIBITORS T. Ersahin1 , N. Tuncbag2 , A. Acar2 , R. Cetin-Atalay2 . 1 Department of Molecular Biology and Genetics, Bilkent University, 2 Graduate School of Informatics, ODTU, Ankara, Turkey E-mail: [email protected] Background and Aims: Sorafenib, a multi-kinase inhibitor with anti-angiogenic functions, is the only FDA-approved moleculartargeted agent for the treatment of patients with advanced Hepatocellular carcinoma (HCC). Yet, Sorafenib shows limited overall survival benefit associated with resistance and tumor recurrence. Current mono-target- or single pathway-centric drug designs are not sufficient for effective therapy of advanced HCC. Since Sorafenib targets angiogenic VEGFR and PDGFR kinases and RAF/MEK/ERK signaling, the primary mechanism of resistance to Sorafenib and tumor recurrence in HCC patients emerges to be the compensatory PI3K/AKT signaling. In this study, we analyzed the synergistic effects of Sorafenib and PI3K/AKT inhibitors on HCC cell growth, determined possible mechanisms underlying synergistic mechanism of action by transcriptome analysis, and further showed that combination therapy leads to tumor regression in HCC xenografts in vivo. Methods: Cytotoxic activities of the PI3K/AKT pathway small molecule inhibitors were shown by SRB and RT-CES assays in HCC cell lines having normal or hyperactive AKT kinase. Apoptotic cell death and suppression of cell cycle progression were shown by flow cytometry, immunofluorescence and western blots experiments. Transcriptome profiling of inhibitor treated cells were performed with RNA-sequencing. Therapeutic efficacy of combination therapy was shown in vivo in athymic mice bearing HCC xenografts. Results: We showed the cytotoxic activities of isoform specific PI3K inhibitors and AKT inhibitors (Akti-1,2 and Akti-2) alone and in combination with Sorafenib in vitro. We revealed the molecular mechanisms of action of single agent and combination therapies using transcriptome profiling. We determined the predominant role of PI3K isoform p110a in PTEN-deficient HCC cells. We showed that combining Sorafenib with PI3K/AKT inhibitors enhances anti-tumor efficacy of Sorafenib and results in tumor regression in vivo. Conclusions: Our results provide in vitro and in vivo experimental evidence of the therapeutic potential of combination therapies with Sorafenib and PI3K/Akt inhibitors for the treatment of advanced HCC. P0403 STEATOTIC LIVER AS A SOURCE OF HEPATIC PROGENITOR CELLS WITH THERAPEUTIC POTENTIAL W.-Y. Lu1 , A. Tsuchiya2 , L. Boulter3 , R. Guest1 , D. Wojtacha1 , T. Bird1 , T.Y. Man1 , D. Hay1 , J. Iredale4 , S. Forbes1 . 1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom; 2 Division of Gastroenterology and Hepatology, Niigata University, Niigata, Japan; 3 Institute of Genetics and Molecular Medicine, 4 Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom E-mail: [email protected] Background and Aims: Ductular reactions (DRs) are observed in chronic liver injuries. In mice, ductular reactions have been studied extensively in models such as Choline-Deficient Ethionine (CDE) supplemented diet and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. On contrary, DRs in models of fatty liver disease is less studied. We aim to investigate the DR in fatty liver disease and the plausibility to isolate Hepatic Progenitor Cells (HPCs) from fatty liver to use as an alternative to whole organ transplant. Methods: C57BL6 mice were supplemented with High Fat Diet, Choline Deficient (CD) diet, Methionine-Choline Deficient (MCD)
Journal of Hepatology 2015 vol. 62 | S263–S864
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
S463
POSTERS collected one sample of tumor and two samples of tumor-free cirrhotic tissue (near and at a distance from the tumor) and used them to construct 30 small RNA libraries for next-generation sequencing. The reads that emerged were clustered and aligned against the MiRBase with CLC Genomic Workbench software. Results: A total of 2,329 mature microRNAs were found. Annotated microRNAs with a mean read number per sample of >20 underwent parametric and randomized statistical analyses. Those associated with P values of ≤0.01 in both analyses were considered differentially expressed between tissue types. Of the 617 microRNAs subjected to statistical analysis, 35 exhibited expression levels in HCC that were significantly different from those found in one or both tumor-free cirrhotic tissues; two of the 35 were also differentially expressed in the two types of cirrhotic tissue. Eighteen of the 35 had never been reported as dysregulated in HCC. Conclusions: We identified an HCC-signature set of 35 microRNAs, which distinguishes HCCs from background cirrhotic tissues. It should provide a good starting point for subsequent research aimed at identifying biomarkers correlating with HCC outcome. P0401 THE SERINE PROTEASE FSAP (FACTOR VII ACTIVATING PROTEASE) MAINTAINS THE DIFFERENTIATED STATE OF MOUSE HEPATOCYTES A. Martinez-Palacian1 , S. Kanse1 . 1 Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, 0372 Oslo, Norway E-mail: [email protected] Background and Aims: FSAP, encoded by the Habp2 gene, is a serine protease secreted by hepatocytes as an inactive zymogen that circulates in the blood. We have recently demonstrated a novel anti-fibrotic role for FSAP in the liver. The aim of this study was to analyze the effect of FSAP on hepatocyte differentiation and proliferation. Methods: Primary hepatocytes isolated from BALB/c wild type (WT) or FSAP-deficient mice and the mouse hepatocyte cell line AML-12 were used as in vitro models. The use of knockout hepatocytes was further supported by the use of siRNA-mediated knock-down of the gene for FSAP and the addition of exogenous FSAP protein. Transcript levels were analyzed by qRT-PCR, protein levels by Western blotting and DNA synthesis by BrdU incorporation. Results: Primary hepatocytes and AML-12 cells lacking expression of endogenous FSAP show a reduction in HNF4a (Hepatocyte Nuclear Factor 4a) and an increase in AFP (alphafetoprotein) transcript levels compared to WT cells. Besides, Transforming Growth Factor beta-1 (TGFb) almost completely abolishes HNF4a mRNA levels in FSAP-depleted cells, while the reduction in WT cells is around 50%. Basal reduction on HNF4a mRNA is not due to an Epithelial-Mesenchymal Transition (EMT): silencing of the gene for FSAP does not down-regulate E-cadherin protein levels nor up-regulates mRNA levels of the EMT-inducing transcription factors (TFs) SNAI1, ZEB1 and ZEB2. Moreover, FSAP depletion does not impair the TGFb-induced up-regulation of these TFs. Preliminary results show that exogenous FSAP induce a 30% increase in basal BrdU incorporation in hepatocytes. Exogenous FSAP can overcome the anti-proliferative effect of TGFb in FSAPdeficient hepatocytes, but not in WT cells, suggesting that lack of endogenous FSAP decreases the sensitivity to TGFb inhibitory effects. Conclusions: We have for the first time described a novel role for FSAP in hepatocyte differentiation. It contributes to mantain HNF4a expression and AFP repression but does not affect EMT. This, together with its potential role on proliferation, provides a mechanistic insight into the effect of FSAP on hepatocytes that is relevant for hepatocellular carcinoma. S464
P0402 COMBINATION THERAPY FOR HEPATOCELLULAR CARCINOMA: A SYSTEMS BIOLOGY PERSPECTIVE ON THE SYNERGISTIC ANTITUMOR ACTIVITY OF SORAFENIB WITH PI3K/AKT PATHWAY INHIBITORS T. Ersahin1 , N. Tuncbag2 , A. Acar2 , R. Cetin-Atalay2 . 1 Department of Molecular Biology and Genetics, Bilkent University, 2 Graduate School of Informatics, ODTU, Ankara, Turkey E-mail: [email protected] Background and Aims: Sorafenib, a multi-kinase inhibitor with anti-angiogenic functions, is the only FDA-approved moleculartargeted agent for the treatment of patients with advanced Hepatocellular carcinoma (HCC). Yet, Sorafenib shows limited overall survival benefit associated with resistance and tumor recurrence. Current mono-target- or single pathway-centric drug designs are not sufficient for effective therapy of advanced HCC. Since Sorafenib targets angiogenic VEGFR and PDGFR kinases and RAF/MEK/ERK signaling, the primary mechanism of resistance to Sorafenib and tumor recurrence in HCC patients emerges to be the compensatory PI3K/AKT signaling. In this study, we analyzed the synergistic effects of Sorafenib and PI3K/AKT inhibitors on HCC cell growth, determined possible mechanisms underlying synergistic mechanism of action by transcriptome analysis, and further showed that combination therapy leads to tumor regression in HCC xenografts in vivo. Methods: Cytotoxic activities of the PI3K/AKT pathway small molecule inhibitors were shown by SRB and RT-CES assays in HCC cell lines having normal or hyperactive AKT kinase. Apoptotic cell death and suppression of cell cycle progression were shown by flow cytometry, immunofluorescence and western blots experiments. Transcriptome profiling of inhibitor treated cells were performed with RNA-sequencing. Therapeutic efficacy of combination therapy was shown in vivo in athymic mice bearing HCC xenografts. Results: We showed the cytotoxic activities of isoform specific PI3K inhibitors and AKT inhibitors (Akti-1,2 and Akti-2) alone and in combination with Sorafenib in vitro. We revealed the molecular mechanisms of action of single agent and combination therapies using transcriptome profiling. We determined the predominant role of PI3K isoform p110a in PTEN-deficient HCC cells. We showed that combining Sorafenib with PI3K/AKT inhibitors enhances anti-tumor efficacy of Sorafenib and results in tumor regression in vivo. Conclusions: Our results provide in vitro and in vivo experimental evidence of the therapeutic potential of combination therapies with Sorafenib and PI3K/Akt inhibitors for the treatment of advanced HCC. P0403 STEATOTIC LIVER AS A SOURCE OF HEPATIC PROGENITOR CELLS WITH THERAPEUTIC POTENTIAL W.-Y. Lu1 , A. Tsuchiya2 , L. Boulter3 , R. Guest1 , D. Wojtacha1 , T. Bird1 , T.Y. Man1 , D. Hay1 , J. Iredale4 , S. Forbes1 . 1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom; 2 Division of Gastroenterology and Hepatology, Niigata University, Niigata, Japan; 3 Institute of Genetics and Molecular Medicine, 4 Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom E-mail: [email protected] Background and Aims: Ductular reactions (DRs) are observed in chronic liver injuries. In mice, ductular reactions have been studied extensively in models such as Choline-Deficient Ethionine (CDE) supplemented diet and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. On contrary, DRs in models of fatty liver disease is less studied. We aim to investigate the DR in fatty liver disease and the plausibility to isolate Hepatic Progenitor Cells (HPCs) from fatty liver to use as an alternative to whole organ transplant. Methods: C57BL6 mice were supplemented with High Fat Diet, Choline Deficient (CD) diet, Methionine-Choline Deficient (MCD)
Journal of Hepatology 2015 vol. 62 | S263–S864