- Email: [email protected]
CXCR4 RESULTING IN MYOFIBROBLAST ACTIVATION, GR1+ CELL INFILTRATION, AND DESMOPLASIA IN HEPATOCELLULAR CARCINOMA
POSTERS was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. Methods: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN, respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. Results: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). Conclusions: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients. P92 NEWLY ESTABLISHED MURINE HEPATIC ANGIOSARCOMA CELL LINES: IN VITRO AND IN VIVO MODEL TO STUDY ANGIOSARCOMA BIOLOGY AND TARGETED THERAPIES S. Rothweiler1 , M. Dill1 , L. Terracciano2 , Z. Makowska1 , L. Quagliata2 , M. Heim1,3 , D. Semela1,4 . 1 Department of Biomedicine, University Basel, 2 Institute of Pathology, University Hospital Basel, 3 Division of Gastroenterology and Hepatology, University Hospital Basel, 4 Division of Gastroenterology and Hepatology, Kantonsspital St. Gallen, Basel, Switzerland E-mail: [email protected] Background and Aims: Liver angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin. Studies of the molecular biology of AS are limited since animal models are missing. We have established three cell lines from Notch1 KO animals developing spontaneously liver AS. The aim of this study is to study the molecular pathogenesis and to evaluate the effect of targeted therapies for the treatment of AS. Methods: Transcriptome analysis in AS cell lines vs. normal and Notch1 KO liver sinusoidal endothelial cells including gene set enrichement analysis (GSEA) were performed. Effects of sorafenib (1–20mM) were analysed: cell migration, proliferation, and viability. AS cells were injected s.c. with matrigel in NOD/Scid mice. Results: Transcriptome analysis showed massive changes in gene expression and GSEA revealed that six of the top20 upregulated chemical and genetic perturbation gene sets were related to myc targets (FDR < 0.25). Timelapse imaging showed that sorafenib treatment dramatically reduced migration of AS cells as well as filopodia dynamics (p = 0.0201 after 6 h). Sorafenib inhibited cell proliferation in a time and dose-dependant manner, but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of CyclinD2. Injection of AS cells into NOD/Scid mice resulted in formation of undifferentiated tumors confirming the tumorigenic potential of these cells. Conclusions: Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, which support further evaluation of sorafenib as novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.
P93 HYPOXIA UNDER SORAFENIB TREATMENT UPREGULATES SDF1a/CXCR4 RESULTING IN MYOFIBROBLAST ACTIVATION, GR1+ CELL INFILTRATION, AND DESMOPLASIA IN HEPATOCELLULAR CARCINOMA T. Reiberger1,2 , Y. Chen1 , P. Huang1 , G.Y. Lauwers1 , A.X. Zhu1 , R.K. Jain1 , D.G. Duda1 . 1 Edwin L. Steele Lab, Radiation Oncology, MGH/Harvard Medical School, Boston, MA, United States; 2 Internal Medicine III, Div. of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Current EASL guidelines recommend sorafenib for treatment of advanced hepatocellular carcinoma (HCC). The effects of sorafenib on HCC desmoplasia – a potential mechanism of drug resistance have not been assessed. Methods: Orthotopic murine HCA1 model and the GEMM MST1−/− MST2F/− model. Liver fibrosis was induced by CCl4 . Liver fibrosis and tumor desmoplasia were assessed by masson trichrome and collagen I staining. HSC activation was evaluated by aSMA and GFAP staining. Gr1+ cell infiltration was determined by flow cytometry. Results: Sorafenib exerted differential effects on tumor desmoplasia versus liver fibrosis in orthotopic HCC models. Sorafenib intensified hypoxia, which increased stromal-derived factor 1a (SDF1a) expression in cancer and stromal cells, and subsequently Gr-1+ myeloid cell infiltration. The SDF1a/CXCR4 pathway promoted hepatic stellate cell (HSC) differentiation and activation via MAP kinase pathway. This was consistent with the colocalization of SDF1a expression in fibrotic septa of cirrhotic human livers as well as in desmoplastic regions of human HCC samples. After treatment with sorafenib, SDF1a increased the survival of HSCs and their aSMA and collagen I expression, thus increasing HCC desmoplasia. CXCR4 inhibition using AMD3100 in combination with sorafenib treatment prevented the increase in tumor desmoplasia – despite persistently elevated hypoxia. In addition, Gr-1+ myeloid cells infiltration mediated HSC differentiation/activation in a paracrine manner. CXCR4 inhibition or antibody blockade of Gr-1 also reduced Gr-1+ myeloid cell infiltration and inhibited HCC growth by reducing HCC desmoplasia. Conclusions: Blocking SDF1a/CXCR4 or Gr-1+ myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase efficacy of sorafenib treatment. P94 c-Rel AND p27Kip ARE DOWNSTREAM TARGETS OF ERK5 IN HEPATOCELLULAR CARCINOMA (HCC) IN VIVO AND IN VITRO G. Di Maira1 , E. Rovida2 , N. Navari1 , S. Cannito3 , P. Della Sbarba2 , M. Parola3 , F. Marra1 . 1 Medicina Sperimentale e Clinica, 2 Patologia Oncologia Sperimentale, Firenze, Firenze, 3 Medicina Oncologia Sperimentale, Torino, Torino, Italy E-mail: giovanni.dimaira@unifi.it Background and Aims: The molecular mechanisms underlying the development and progression of HCC remain poorly understood. The MAP kinase ERK5 has been implicated in tumor development. The goal of this study was to evaluate the relevance of this pathway ERK5 in HCC biology and to establish its downstream targets. Methods: In Huh-7 and HepG2, ERK5 was silenced by siRNA transfection or with lentiviral vectors encoding specific shRNA. The specific ERK5 inhibitor XMD8–92 was also used. In vivo development of HCC was evaluated using a xenograft model with Huh-7 in nude mice. Results: In vitro, ERK5 silencing or inhibition caused growth arrest of HCC cells, affecting the G1/S transition. This phenotype was associated with an increased expression of p27Kip, a negative regulator of cell cycle progression. Upon ERK5 knockdown, expression of c-Rel, a member of the NF-kappaB family required
Journal of Hepatology 2014 vol. 60 | S67–S214
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P93 HYPOXIA UNDER SORAFENIB TREATMENT UPREGULATES SDF1a/CXCR4 RESULTING IN MYOFIBROBLAST ACTIVATION, GR1+ CELL INFILTRATION, AND DESMOPLASIA IN HEPATOCELLULAR CARCINOMA T. Reiberger1,2 , Y. Chen1 , P. Huang1 , G.Y. Lauwers1 , A.X. Zhu1 , R.K. Jain1 , D.G. Duda1 . 1 Edwin L. Steele Lab, Radiation Oncology, MGH/Harvard Medical School, Boston, MA, United States; 2 Internal Medicine III, Div. of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Current EASL guidelines recommend sorafenib for treatment of advanced hepatocellular carcinoma (HCC). The effects of sorafenib on HCC desmoplasia – a potential mechanism of drug resistance have not been assessed. Methods: Orthotopic murine HCA1 model and the GEMM MST1−/− MST2F/− model. Liver fibrosis was induced by CCl4 . Liver fibrosis and tumor desmoplasia were assessed by masson trichrome and collagen I staining. HSC activation was evaluated by aSMA and GFAP staining. Gr1+ cell infiltration was determined by flow cytometry. Results: Sorafenib exerted differential effects on tumor desmoplasia versus liver fibrosis in orthotopic HCC models. Sorafenib intensified hypoxia, which increased stromal-derived factor 1a (SDF1a) expression in cancer and stromal cells, and subsequently Gr-1+ myeloid cell infiltration. The SDF1a/CXCR4 pathway promoted hepatic stellate cell (HSC) differentiation and activation via MAP kinase pathway. This was consistent with the colocalization of SDF1a expression in fibrotic septa of cirrhotic human livers as well as in desmoplastic regions of human HCC samples. After treatment with sorafenib, SDF1a increased the survival of HSCs and their aSMA and collagen I expression, thus increasing HCC desmoplasia. CXCR4 inhibition using AMD3100 in combination with sorafenib treatment prevented the increase in tumor desmoplasia – despite persistently elevated hypoxia. In addition, Gr-1+ myeloid cells infiltration mediated HSC differentiation/activation in a paracrine manner. CXCR4 inhibition or antibody blockade of Gr-1 also reduced Gr-1+ myeloid cell infiltration and inhibited HCC growth by reducing HCC desmoplasia. Conclusions: Blocking SDF1a/CXCR4 or Gr-1+ myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase efficacy of sorafenib treatment. P94 c-Rel AND p27Kip ARE DOWNSTREAM TARGETS OF ERK5 IN HEPATOCELLULAR CARCINOMA (HCC) IN VIVO AND IN VITRO G. Di Maira1 , E. Rovida2 , N. Navari1 , S. Cannito3 , P. Della Sbarba2 , M. Parola3 , F. Marra1 . 1 Medicina Sperimentale e Clinica, 2 Patologia Oncologia Sperimentale, Firenze, Firenze, 3 Medicina Oncologia Sperimentale, Torino, Torino, Italy E-mail: giovanni.dimaira@unifi.it Background and Aims: The molecular mechanisms underlying the development and progression of HCC remain poorly understood. The MAP kinase ERK5 has been implicated in tumor development. The goal of this study was to evaluate the relevance of this pathway ERK5 in HCC biology and to establish its downstream targets. Methods: In Huh-7 and HepG2, ERK5 was silenced by siRNA transfection or with lentiviral vectors encoding specific shRNA. The specific ERK5 inhibitor XMD8–92 was also used. In vivo development of HCC was evaluated using a xenograft model with Huh-7 in nude mice. Results: In vitro, ERK5 silencing or inhibition caused growth arrest of HCC cells, affecting the G1/S transition. This phenotype was associated with an increased expression of p27Kip, a negative regulator of cell cycle progression. Upon ERK5 knockdown, expression of c-Rel, a member of the NF-kappaB family required
Journal of Hepatology 2014 vol. 60 | S67–S214
S97