- Email: [email protected]
Hepatocyte-Specific SMAD7 Deletion Accelerates Den Induced Mouse Hepatocellular Carcinoma via Activation of STAT3 Signaling
POSTER PRESENTATIONS FRI-071 TOLL-LIKE RECEPTOR 3: A POTENTIAL TUMOR SUPPRESSOR GENE IN HEPATOCELLULAR CARCINOMA? N. Fares1, M. Bonnin1, A. Garcia1, B. Testoni1, Lydie Lefrancois1, B. Bancel2, V. Hervieu2, T. Renno1, P. Merle1,3, S. Lebecque1. 1Innate Immune Signaling and Oncogenesis, Centre de recherche en cancérologie de LYON CRCL; 2Pathology; 3Hepatology Croix rousse hospital, Hospices civils de Lyon, Lyon, France E-mail: [email protected] Background and Aims: Toll-like receptor 3 (TLR3) detects viral or endogenous double-stranded RNA, and promotes in primary human hepatocytes (PHH) an inflammatory response notably via the interferon type I pathway. In cancer cells, TLR3 can also induce apoptosis by its ability to activate the extrinsic caspase pathway. Our aim was to investigate whether TLR3 has an antitumor effect in hepatocellular carcinoma (HCC). Methods: 62 resected human HCCs were used to evaluate the prognostic impact of TLR3 mRNA (iQPCR) and protein (Western blot) down-regulation on patients’ outcome. in vitro the involvement of TLR3 receptor was evaluated in HCC cell lines, as well as in PHH transformation model using the oncogenes SV40LT+ST and H-RASV12. The mechanisms underlying TLR3 invalidation in HCC cell lines were assessed at genetic (allelic loss) and epigenetic levels (repressive histone modification, micro RNA expression). In vivo, transgenic mice expressing SV40LT in the liver were crossed with TLR3-KO or TLR3 + / + mice to assess the impact of TLR3 invalidation on hepatocarcinogenesis. Results: In human resected HCCs, low levels of TLR3 mRNA in tumors was significantly associated with shorter time to recurrence (multivariate analysis HR = 2.16 p = 0.04). Western Blot analysis confirmed the down-regulation of TLR3 in about half of the HCCs. In vitro, TLR3 was poorly expressed in all HCC cell lines (Hep G2, Hep 3B, HuH7, PLC/PRF/5, SK-HEP1, Focus). TLR3 re-expression by lentiviral transduction in HCC lines led to apoptosis after activation with a TLR3 ligand ( poly I:C) and cancerous transformation of PHH by SV40 and H-RasV12 was associated with a drastic decrease of TLR3 expression at mRNA and protein levels. In the transgenic SV40 mouse model of HCC, SV40/TLR3-KO mice exhibit significantly more dysplasic and tumoral nodules than SV40/TLR3 +/+ at different ages (8, 10 and 12 weeks), for example at 12 weeks, mean number of pathological foci: SV40-TLR3KO = 0.88/mm2 vs. SV40-TLR3 +/+ = 0.31/mm2 p < 0.01. In TLR3-KO mice, histological staining for cleaved caspase 3 showed a significative decrease of hepatocyte apoptosis while the proliferation rate remained stable. Conclusions: These results suggest that TLR3 could represent the first receptor of innate immunity acting as a potential tumor suppressor gene during hepatocarcinogenesis. mRNA levels in HCC could be used as a biomarker for early relapse. In HCC cases with a persistent expression of TLR3, this protein could represent a potential therapeutic target. FRI-072 HEPATOCYTE-SPECIFIC SMAD7 DELETION ACCELERATES DEN INDUCED MOUSE HEPATOCELLULAR CARCINOMA VIA ACTIVATION OF STAT3 SIGNALING T. Feng1, J. Dzieran1, T. Maass2, A. Teufel2, S. Marhenke3, A. Vogel3, S. Dooley1, N. Meindl-Beinker1. 1Heidelberg University, Mannheim; 2 University Hospital Regensburg, Regensburg; 3Medical School Hannover, Hannover, Germany E-mail: [email protected] Background and Aims: Transforming growth factor-beta (TGF-β) signaling plays critical roles in different cell types in the liver, in multiple liver diseases as well as in HCC. Smad7 is one of the most important negative regulators of TGF-β signaling. We previously found that high Smad7 levels correlate with better clinical outcome in hepatocellular carcinoma (HCC) patients. However, the underlying
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molecular mechanisms, how Smad7 suppresses the development of HCC, are still not clear. Methods: Hepatocyte specific Smad7 transgenic mice were generated by crossing TTR-Cre mice with Alb-Smad7 transgenic mice. For the induction of HCC, two weeks old mice were injected intraperitoneally with 25 mg/kg DEN (Sigma). To induce Smad7 expression, 4 weeks old mice were injected with 1 mg/mice/day tamoxifen (Sigma, T5648) for 5 days. At 9 months of age, mice were sacrificed and livers were processed for further analysis. Human HCC cell line Huh7 was used for in vitro signaling analysis. Results: Conditional (TTR-Cre) hepatocyte specific (Alb-promotor) Smad7 knockout (ko) mice showed higher tumor numbers than wildtype (Wt) mice and Smad7 transgenic (Tg) mice 9 months after challenge with DEN, indicating a tumor suppressive role of Smad7. In line with our previous findings in human HCC patient samples, we found a significant correlation between Smad7 levels and tumor numbers in both tumor tissue and surrounding tissue, although Smad7 levels varied in individual mice. Smad7 ko mice exhibited increased p-Smad2/3 levels and decreased apoptosis in the tumor tissue. Increased tumor incidence in Smad7 ko mice was also accompanied by reduced p21 and induced c-MYC expression. Interestingly, activation of STAT3 signaling was found in Smad7 ko mice. Upregulation of STAT3 downstream targets such as VEGF and Mcl-1 were also observed in the tumors of Smad7 ko mice. In Huh7 cells, adenovirus mediated Smad7 overexpression reduced endogenous as well as IL-6 induced p-STAT3 levels, and subsequent downregulation of VEGF and Mcl-1 expression, while there was no effect on IL-6 production. Further, activation of JNK signaling was found in tumors of Smad7 ko mice. This is in line with a previous report showing that JNK controlled proliferation of mouse liver tumor cells by affecting p21 and c-Myc. Conclusions: Our results suggest that Smad7 has an inhibitory role in the initiation and development of DEN induced HCC in mice. Signaling analysis provided new mechanistic insights into the pathophysiological role of Smad7 in hepatocarcinogenesis. FRI-073 PROFILING OF INFILTRATING IMMUNE CELL IN TUMOR TISSUE OF HEPATOCELLULAR CARCINOMA PATIENTS. N. Rohr-Udilova1, M. Trauner1, M. Peck-Radosavljevic1. 1 Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Profiling of immune cells in hepatocellular tumors is of great interest for better understanding of the role of the immune system and for the proper choice of the upcoming therapies which target immune checkpoint inhibition. Microarray analysis of gene expression patterns allows insights into molecular pathways activated in HCC. Whole tumor tissue is commonly used for microarray analysis and contains not only tumor cells, but also infiltrating immune cells. Characteristic gene expression patterns can be determined for each type of immune cells. Thus, application of proper mathematical methods can enable immune cell profiling from the gene expression pattern of whole tissue. Methods: Recently developed mathematical methods allow quantification of the contribution of each type of immune cells to the microarray gene expression profile from whole tissue. This approach has been applied to re-analyze 387 samples of human HCC gene expression data published (Hoshida NEJM 2009). For each sample, 22 immune cell types were quantified. In a subset of 67 samples, a comparison between infiltrating immune cell profile in tumor tissue with the corresponding surrounding tissue as well as correlation with survival was performed. Results: Immune cell types found in HCC tumors are given in the Table 1. A clear difference in the immune cell composition between tumor and surrounding tissue was found. Presence of certain immune cell types significantly correlated with overall survival
Journal of Hepatology 2016 vol. 64 | S425–S630
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molecular mechanisms, how Smad7 suppresses the development of HCC, are still not clear. Methods: Hepatocyte specific Smad7 transgenic mice were generated by crossing TTR-Cre mice with Alb-Smad7 transgenic mice. For the induction of HCC, two weeks old mice were injected intraperitoneally with 25 mg/kg DEN (Sigma). To induce Smad7 expression, 4 weeks old mice were injected with 1 mg/mice/day tamoxifen (Sigma, T5648) for 5 days. At 9 months of age, mice were sacrificed and livers were processed for further analysis. Human HCC cell line Huh7 was used for in vitro signaling analysis. Results: Conditional (TTR-Cre) hepatocyte specific (Alb-promotor) Smad7 knockout (ko) mice showed higher tumor numbers than wildtype (Wt) mice and Smad7 transgenic (Tg) mice 9 months after challenge with DEN, indicating a tumor suppressive role of Smad7. In line with our previous findings in human HCC patient samples, we found a significant correlation between Smad7 levels and tumor numbers in both tumor tissue and surrounding tissue, although Smad7 levels varied in individual mice. Smad7 ko mice exhibited increased p-Smad2/3 levels and decreased apoptosis in the tumor tissue. Increased tumor incidence in Smad7 ko mice was also accompanied by reduced p21 and induced c-MYC expression. Interestingly, activation of STAT3 signaling was found in Smad7 ko mice. Upregulation of STAT3 downstream targets such as VEGF and Mcl-1 were also observed in the tumors of Smad7 ko mice. In Huh7 cells, adenovirus mediated Smad7 overexpression reduced endogenous as well as IL-6 induced p-STAT3 levels, and subsequent downregulation of VEGF and Mcl-1 expression, while there was no effect on IL-6 production. Further, activation of JNK signaling was found in tumors of Smad7 ko mice. This is in line with a previous report showing that JNK controlled proliferation of mouse liver tumor cells by affecting p21 and c-Myc. Conclusions: Our results suggest that Smad7 has an inhibitory role in the initiation and development of DEN induced HCC in mice. Signaling analysis provided new mechanistic insights into the pathophysiological role of Smad7 in hepatocarcinogenesis. FRI-073 PROFILING OF INFILTRATING IMMUNE CELL IN TUMOR TISSUE OF HEPATOCELLULAR CARCINOMA PATIENTS. N. Rohr-Udilova1, M. Trauner1, M. Peck-Radosavljevic1. 1 Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Profiling of immune cells in hepatocellular tumors is of great interest for better understanding of the role of the immune system and for the proper choice of the upcoming therapies which target immune checkpoint inhibition. Microarray analysis of gene expression patterns allows insights into molecular pathways activated in HCC. Whole tumor tissue is commonly used for microarray analysis and contains not only tumor cells, but also infiltrating immune cells. Characteristic gene expression patterns can be determined for each type of immune cells. Thus, application of proper mathematical methods can enable immune cell profiling from the gene expression pattern of whole tissue. Methods: Recently developed mathematical methods allow quantification of the contribution of each type of immune cells to the microarray gene expression profile from whole tissue. This approach has been applied to re-analyze 387 samples of human HCC gene expression data published (Hoshida NEJM 2009). For each sample, 22 immune cell types were quantified. In a subset of 67 samples, a comparison between infiltrating immune cell profile in tumor tissue with the corresponding surrounding tissue as well as correlation with survival was performed. Results: Immune cell types found in HCC tumors are given in the Table 1. A clear difference in the immune cell composition between tumor and surrounding tissue was found. Presence of certain immune cell types significantly correlated with overall survival
Journal of Hepatology 2016 vol. 64 | S425–S630