- Email: [email protected]
110 MOLECULAR AND CLINICAL CHARACTERIZATION OF RICTOR (MTORC2) AS A CANDIDATE ONCOGENE IN HEPATOCELLULAR CARCINOMA
Parallel Session 13: LIVER REGENERATION AND CARCINOGENESIS an inflammatory process, initiated by the production of TNF alpha and interleukin-6 (IL-6). Since cannabinoid receptors 2 (CB2) play a major role in the regulation of inflammation, we investigated herein whether they regulate liver regeneration. Liver regeneration was studied in a model of acute hepatitis induced by a single injection of carbon tetrachloride (CCl4 ) or following two-third hepatectomy. Experiments were performed in CB2 knockout (CB2−/−) and wild type (WT) mice, or in WT mice pretreated with the CB2 agonist JWH-133. Hepatic CB2 receptor expression was markedly induced following partial hepatectomy and CCl4 -induced liver injury. Liver injury was increased in CB2−/− mice compared to WT mice following acute CCl4 administration, as assessed by serum ALT and TUNEL labelling. In addition, PCNA expression was significantly delayed in CB2−/− animals as compared to WT mice (72 vs 48 hrs). Conversely, treatment of WT mice with a single dose of CB2 specific agonist, JWH-133, reduced liver injury and accelerated liver regeneration in response to CCl4 exposure. Similar delay in PCNA expression was observed in CB2−/− animals submitted to partial hepatectomy. Impaired regeneration in CB2−/− mice was associated with a reduction in the expression of IL-6; consistent with these data, IL-6 administration to CB2−/− mice partially restored PCNA expression. Finally, MMP-2 activity was enhanced in CB2−/− mice injected with CCl4 , in keeping with the reported effects of MMP-2 in liver injury. These data demonstrate that the CB2 receptors reduce liver injury and promote liver regeneration following acute insult, by a mechanism involving IL-6 production and inhibition of MMP-2 activity. These results suggest that CB2 agonists elicit useful hepatoprotective properties, in addition to their antifibrogenic effects.
109 DEPLETION OF CASPASE-8 IN MICE MODULATES TNF-INDUCED COMPLEX FORMATION AND CELL CYCLE SIGNALING IN HEPATOCYTES FOLLOWING PARTIAL HEPATECTOMY J. Freimuth1 , D. Riethmacher2 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, Germany; 2 Division of Human Genetics, University of Southampton School of Medicine, Southampton, UK E-mail: [email protected] Background and Aims: Binding of the cytokine TNF-a to TNF-Receptor 1 mediates pleiotropic effects via two different signaling complexes. Complex I triggers activation of NF-úB and activation of c-Jun N-terminal kinases (JNK) leading to inflammation and/or cell proliferation, whereas complex II induces apoptosis through caspase-8. TNF signaling is crucial for the priming phase of liver regeneration following partial hepatectomy (PH) which directly contributes to gene activation of cyclin D via NF-úB, c-jun/AP1 and E2F1. The aim of this study was to investigate the consequences of caspase-8 depletion for TNF signaling following PH. Methods: Hepatocyte-specific conditional knockout mice for caspase-8 (Casp8Dhep ) and wildtype (WT) animals were subjected to PH and investigated for cell cycle progression and TNF complex formation. Results: Following PH, Casp8Dhep mice showed accelerated liver regeneration and hepatocyte proliferation due to earlier activation of cyclin D, which is regulated by a signaling cascade involving transcription factors c-Jun/AP1 and E2F. Consistently, in Casp8Dhep mice expression of E2F1 was amplified at all timepoints between 24−60 hours (h) post PH and phosphorylation and nuclear translocation of c-jun was strongly accelerated compared to controls. In WT controls, maximal but transient JNK recruitment to complex I occurred 2 h after PH, whereas in Casp8Dhep mice strong interaction of JNK with complex I was already detectable 0.5 h after surgery for up to 6 h post PH. Interestingly, in Casp8Dhep mice we also detected sustained and prolonged NF-úB activation following PH. Accordingly, cFLIP, a NF-úB target gene and caspase-8 antagonist, was up to 6fold higher expressed in Casp8Dhep mice compared to WT littermates. The adaptor kinase RIP1 is a component of complex I and was shown to be crucial for activating NF-úB and JNK. Control mice showed a transient expression and up-regulation of RIP1 2 h post PH. In
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contrast, depletion of caspase-8 resulted in constitutive expression of RIP1 with further, prolonged up-regulation immediately after surgery (0.5 h post PH). Conclusion: Caspase-8 performs a novel, non-apoptotic function in affecting TNF-receptor complex formation and subsequent regulation of NF-úB and c-Jun. Therefore, accelerated liver regeneration in Casp8Dhep mice is a unique consequence of dysregulated NF-úB and JNK activation. 110 MOLECULAR AND CLINICAL CHARACTERIZATION OF RICTOR (MTORC2) AS A CANDIDATE ONCOGENE IN HEPATOCELLULAR CARCINOMA A. Villanueva1 , H. Cornella1 , V. Tovar1 , C. Alsinet1 , D. Chiang2 , J. Peix1 , B. Minguez3 , T. Sara3 , R. Savic3 , Y. Hoshida2 , A. Lachenmayer3 , S. Thung4 , S. Roayaie5 , M. Schwartz5 , B. Jordi1 , V. Mazzaferro6 , S. Friedman3 , J. Llovet1,3 . 1 BCLC-Group, HCC Translational Research Lab, IDIBAPS, CIBEREHD, Hospital Clinic, Barcelona, Spain; 2 Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, 3 Division of Liver Diseases, 4 Pathology, 5 Surgical Oncology, Mount Sinai School of Medicine, New York, New York, USA; 6 Gastrointestinal Surgery and Liver Transplantation Unit, National Cancer Institute, Milan, Italy E-mail: [email protected] Introduction: Recent data implicate mTOR signaling in human hepatocellular carcinoma (HCC). However, the specific contribution of mTOR complex components as either potential oncogenes or targets for therapies is unknown. Aims: 1. To evaluate the role of mTOR complex 2 (RICTOR) in human hepatocarcinogenesis and 2. to assess the impact of selective RICTOR blockade in cellular models of HCC. Methods: DNA copy numbers (Affymetrix 238K Sty-arrays) and mRNA levels of RICTOR (Affymetrix U133 Plus 2.0 arrays and qRTPCR using Taqman Probes) were analyzed in 102 human HCV-related HCC (fresh-frozen samples). RICTOR over-expression was correlated with both signaling pathway activation (Wnt-bCatenin, IGF/AKT/MTOR and RAS/MAPK), and with the existing molecular classification of HCC (Chiang et al. Cancer Res 2008). Data were validated in a separate cohort of 164 HCC patients (paraffin-embedded samples), profiled using DASL (Illumina). Molecular data were correlated with clinico-pathological features and patient prognosis using Kaplan–Meier and Cox Proportional Hazard Test. For in vitro studies, Huh-7 cells were transfected with siRNARICTOR, and western blots done to evaluate downstream signals (p-AKT, p-RPS6 and p-ERK). Cell viability and proliferation were assessed by MTT and [H3 ]-Thymidine incorporation assay respectively, whereas FACS was used to evaluate cell cycle. Results: 25% (25/100) of HCV-related HCC samples had DNA gains in the RICTOR locus. Among them, 12% (11/87) had high mRNA levels of RICTOR (P = 0.03). These patients had significantly worse survival (P = 0.001) and higher rates of early recurrence (P < 0.001). Similarly, patients in the validation set with highest mRNA levels of RICTOR (11.6%, 19/163) had significantly higher rates of early recurrence (P = 0.01). In cultured cells after 48 hours of transfection with siRNA-RICTOR, cell viability was decreased by 20% (P = 0.03) and proliferation by 30% (P = 0.03), in addition to a mild but significant G1 cell cycle arrest (p = 0.005), without evidence of increased apoptosis. AKT phosphorylation was reduced by siRNA-RICTOR, without interfering with MTOR complex 1 activation (phospho-RPS6). Conclusion: Over-expression of RICTOR identifies a subgroup of HCC patients (10%) with poor prognosis. Selective blockade of RICTOR has anti-neoplastic effects in cellular models of HCC, and thus further evaluation of RICTOR as a potential oncogene in animal models is warranted.
109 DEPLETION OF CASPASE-8 IN MICE MODULATES TNF-INDUCED COMPLEX FORMATION AND CELL CYCLE SIGNALING IN HEPATOCYTES FOLLOWING PARTIAL HEPATECTOMY J. Freimuth1 , D. Riethmacher2 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, Germany; 2 Division of Human Genetics, University of Southampton School of Medicine, Southampton, UK E-mail: [email protected] Background and Aims: Binding of the cytokine TNF-a to TNF-Receptor 1 mediates pleiotropic effects via two different signaling complexes. Complex I triggers activation of NF-úB and activation of c-Jun N-terminal kinases (JNK) leading to inflammation and/or cell proliferation, whereas complex II induces apoptosis through caspase-8. TNF signaling is crucial for the priming phase of liver regeneration following partial hepatectomy (PH) which directly contributes to gene activation of cyclin D via NF-úB, c-jun/AP1 and E2F1. The aim of this study was to investigate the consequences of caspase-8 depletion for TNF signaling following PH. Methods: Hepatocyte-specific conditional knockout mice for caspase-8 (Casp8Dhep ) and wildtype (WT) animals were subjected to PH and investigated for cell cycle progression and TNF complex formation. Results: Following PH, Casp8Dhep mice showed accelerated liver regeneration and hepatocyte proliferation due to earlier activation of cyclin D, which is regulated by a signaling cascade involving transcription factors c-Jun/AP1 and E2F. Consistently, in Casp8Dhep mice expression of E2F1 was amplified at all timepoints between 24−60 hours (h) post PH and phosphorylation and nuclear translocation of c-jun was strongly accelerated compared to controls. In WT controls, maximal but transient JNK recruitment to complex I occurred 2 h after PH, whereas in Casp8Dhep mice strong interaction of JNK with complex I was already detectable 0.5 h after surgery for up to 6 h post PH. Interestingly, in Casp8Dhep mice we also detected sustained and prolonged NF-úB activation following PH. Accordingly, cFLIP, a NF-úB target gene and caspase-8 antagonist, was up to 6fold higher expressed in Casp8Dhep mice compared to WT littermates. The adaptor kinase RIP1 is a component of complex I and was shown to be crucial for activating NF-úB and JNK. Control mice showed a transient expression and up-regulation of RIP1 2 h post PH. In
S45
contrast, depletion of caspase-8 resulted in constitutive expression of RIP1 with further, prolonged up-regulation immediately after surgery (0.5 h post PH). Conclusion: Caspase-8 performs a novel, non-apoptotic function in affecting TNF-receptor complex formation and subsequent regulation of NF-úB and c-Jun. Therefore, accelerated liver regeneration in Casp8Dhep mice is a unique consequence of dysregulated NF-úB and JNK activation. 110 MOLECULAR AND CLINICAL CHARACTERIZATION OF RICTOR (MTORC2) AS A CANDIDATE ONCOGENE IN HEPATOCELLULAR CARCINOMA A. Villanueva1 , H. Cornella1 , V. Tovar1 , C. Alsinet1 , D. Chiang2 , J. Peix1 , B. Minguez3 , T. Sara3 , R. Savic3 , Y. Hoshida2 , A. Lachenmayer3 , S. Thung4 , S. Roayaie5 , M. Schwartz5 , B. Jordi1 , V. Mazzaferro6 , S. Friedman3 , J. Llovet1,3 . 1 BCLC-Group, HCC Translational Research Lab, IDIBAPS, CIBEREHD, Hospital Clinic, Barcelona, Spain; 2 Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, 3 Division of Liver Diseases, 4 Pathology, 5 Surgical Oncology, Mount Sinai School of Medicine, New York, New York, USA; 6 Gastrointestinal Surgery and Liver Transplantation Unit, National Cancer Institute, Milan, Italy E-mail: [email protected] Introduction: Recent data implicate mTOR signaling in human hepatocellular carcinoma (HCC). However, the specific contribution of mTOR complex components as either potential oncogenes or targets for therapies is unknown. Aims: 1. To evaluate the role of mTOR complex 2 (RICTOR) in human hepatocarcinogenesis and 2. to assess the impact of selective RICTOR blockade in cellular models of HCC. Methods: DNA copy numbers (Affymetrix 238K Sty-arrays) and mRNA levels of RICTOR (Affymetrix U133 Plus 2.0 arrays and qRTPCR using Taqman Probes) were analyzed in 102 human HCV-related HCC (fresh-frozen samples). RICTOR over-expression was correlated with both signaling pathway activation (Wnt-bCatenin, IGF/AKT/MTOR and RAS/MAPK), and with the existing molecular classification of HCC (Chiang et al. Cancer Res 2008). Data were validated in a separate cohort of 164 HCC patients (paraffin-embedded samples), profiled using DASL (Illumina). Molecular data were correlated with clinico-pathological features and patient prognosis using Kaplan–Meier and Cox Proportional Hazard Test. For in vitro studies, Huh-7 cells were transfected with siRNARICTOR, and western blots done to evaluate downstream signals (p-AKT, p-RPS6 and p-ERK). Cell viability and proliferation were assessed by MTT and [H3 ]-Thymidine incorporation assay respectively, whereas FACS was used to evaluate cell cycle. Results: 25% (25/100) of HCV-related HCC samples had DNA gains in the RICTOR locus. Among them, 12% (11/87) had high mRNA levels of RICTOR (P = 0.03). These patients had significantly worse survival (P = 0.001) and higher rates of early recurrence (P < 0.001). Similarly, patients in the validation set with highest mRNA levels of RICTOR (11.6%, 19/163) had significantly higher rates of early recurrence (P = 0.01). In cultured cells after 48 hours of transfection with siRNA-RICTOR, cell viability was decreased by 20% (P = 0.03) and proliferation by 30% (P = 0.03), in addition to a mild but significant G1 cell cycle arrest (p = 0.005), without evidence of increased apoptosis. AKT phosphorylation was reduced by siRNA-RICTOR, without interfering with MTOR complex 1 activation (phospho-RPS6). Conclusion: Over-expression of RICTOR identifies a subgroup of HCC patients (10%) with poor prognosis. Selective blockade of RICTOR has anti-neoplastic effects in cellular models of HCC, and thus further evaluation of RICTOR as a potential oncogene in animal models is warranted.