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Mo1639 Hypoxia Induces Caspase-1 Activation and Promotes Hepatocellular Carcinoma Invasiveness
arise before the development of severe fibrosis from NAFLD, suggesting the involvement of alternative molecular pathways. The aim of this study was to investigate gene expression in paired tumor (T) and non-tumor (NT) HCC samples to identify target genes and molecular pathways of liver carcinogenesis related to fibrosis stage of NAFLD. Methods: Frozen human T and adjacent NT tissue was obtained at the time of surgical resection for HCC. Total RNA was extracted and hybridized to Affymetrix HG133A arrays and microarray data analyzed for 28 samples (14 pairs). Gene expression difference was calculated as the log ratio (log2(T)log2(NT)=log2(T/NT)) between T and NT for every gene probe by patient. Kendall's tau was used to measure the correlation between the expression difference and the fibrosis stage (mild fibrosis: F0-2; severe fibrosis: F3-4) for each gene probe. Results: In this small cohort, the majority of patients were men (93%) and most had diabetes (85.7%). Median age was 69 years and the median body mass index was 28.7 kg/m2. In 10 (90%), HCC resulted in the background of mild fibrosis. There were no significant differences in demographic or histologic characteristics between HCC arising from mild or severe fibrosis. Median tumor size was 8cm and most were well-differentiated (78.6%). The top genes where the expression difference was positively associated with fibrosis severity included ARL4A, RPS6KB1, ACTG2, and VEGFA. Pathway analysis associated the top 500 significant genes with cell signaling, motility and cell growth. Conversely, when the expression difference between T and NT was negatively associated with fibrosis severity (i.e.,T expression more up-regulated compared to NT with mild fibrosis), the top genes were involved in metabolism, regulation of transcription and immune function such as PPP1CA, APOE, MAX, and IL6. Conclusion: Our results suggest that different molecular pathways are active in the development of HCC in patients with NAFLD and that these may vary depending on the underlying severity of fibrosis. Top positively and negatively associated genes between tumor and non-tumor gene expression difference and fibrosis severity in NAFLD patients with HCC
Mo1640 Barx2 May Play a Role in Suppressing the Human Primary Hepatocellular Carcinoma Senlin Zhu, Yi Zhang
AASLD Abstracts
Abstract Background: The homeobox gene Barx2 was recently identified as a regulator of ovarian and breast cancer; however, the expression level of BARX2 and its significance in hepatocellular carcinoma (HCC) remain unknown. Methods: Protein and mRNA expression levels of Barx2 were examined using Western blotting and real-time PCR respectively, in paired HCC tissue and matched adjacent noncancerous tissue from 12 patients. The expression levels of epithelial-mesenchymal transition (EMT) markers were also detected in relation to BARX2 expression. Lastly, immunohistochemistry for BARX2 was also performed on a tissue microarray containing 231 HCC tissue samples. Results: We observed that BARX2 expression was lower in HCC tissues compared to matching adjacent noncancerous tissue. The low expression level of BARX2 was significantly correlated with metrics of tumor size, tumor differentiation, clinical stage, metastasis and relapse. Furthermore, the patients with low BARX2 expression had adverse survival outcomes. Importantly, multivariate cox regression analysis revealed that low BARX2 expression was an independent marker for lower overall survival (p = 0.007). Moreover, a significant negative relationship was observed between the expression of BARX2 and markers of EMT. Conclusions: These findings provide evidence that the low expression level of BARX2 in HCC is significantly correlated with tumor metastasis, and that BARX2 may be an independent prognostic biomarker for patients with HCC. Key word: BARX2, Hepatocellular carcinoma, Prognosis, EMT, Metastasis Cox multivariate analyses of prognostic factors on overall survival
Mo1639 Hypoxia Induces Caspase-1 Activation and Promotes Hepatocellular Carcinoma Invasiveness Wei Yan, Ping Han, Dean Tian, Yu Fu Background: Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which caspase-1 activation under hypoxic stress can promote invasion in hepatocellular carcinoma (HCC) cells. Methods: The expression of caspase-1 in HCC was examined by RT-PCR and western blot. Confocal microscopy was used to show the colocalization of NALP3 with ASC or NALP3 with caspase1. The Hepa1-6 and HepG2 cells were cultured in 21% O2 (normoxia) or 1% O2 (hypoxia) for 24 h. Expression of cleaved caspase-1 from hypoxic cells was examined by western blot and immunoflurensence. Colorimetric assays were used to assay caspase-1 activation. Cell invasion induced by hypoxia was analyzed by transwell chamber. Caspase-1 inhibitor ZWEHD-FMK and pan-caspase inhibitor Z-VAD-FMK were used to determine inhibition of hypoxia-induced invasion. Cytokine IL-6 and IL-8 mRNA levels were assessed by real-time PCR. Results: The expression of caspase-1 was upregulated in HCC tissue and cell lines. Colocalization of NALP3 with ASC or NALP3 with caspase-1 was increased in HCC compared to nontumorous liver. The expression of cleaved caspase-1 was increased in a time-dependent manner after hypoxia in both cell lines. After hypoxia, caspase-1 activity was significantly increased in both Hepa1-6 cells and HepG2 cells compared to normoxic control by colorimetric assays. Cleaved caspase-1 significantly increased in cytoplasm diffusely in both cell lines subjected to hypoxia. Invasion of Hepa1-6 cells and HepG2 was increased 1.89 ± 0.14 fold and 2.03 ± 0.14 fold, respectively following incubation with 1% O2. Both Caspase-1 inhibitor Z-WEHD-FMK and pan-caspase inhibitor Z-VAD-FMK blocked this hypoxia-induced invasion. Lastly, mRNA of IL-6 and IL-8 were also increased during hypoxia and decreased by caspase-1 inhibitor treatment. Conclusions: These results suggest that in hypoxic HCC cells, caspase-1 activation induces production of multiple inflammatory mediators which in turn promotes cancer invasion. (This study is supported by the National Natural Science Foundation of China. NO.81000928 and NO. 81000159)
Mo1641 The Role of FAK in Hepatocarcinogenesis Na Shang, Maribel Arteaga, Wei Qiu Background: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and causes more than 12,000 deaths each year in United States. Advanced HCC is hard to be treated, which results in the overall survival of patients with HCC less than 10%. Therefore, it is urgent to develop new and effective therapeutic strategies and agents to treat HCC. Focal Adhesion Kinase (FAK) is overexpressed in HCC specimens, offering a potential target to treat HCC. However, the role of FAK in HCC remains elusive. Studying the role of FAK in hepatocarcinogenesis by in vivo models is critical to provide preclinical proof to determine whether it is suitable to target FAK to treat HCC. We therefore intent to investigate the role of FAK in hepatocarcinogenesis by a mouse model. Methods: We first generated the mice with hepatocyte-specific deletion of FAK (AlbcreFAKflox/flox). We then hydrodynamically injected age- and gender-matched FAKflox/flox (wild type mice with expression of FAK, referred to as HepWT ) and AlbcreFAKflox/flox mice (HepFAK KO), with c-MET and constitutively active β-catenin by the Sleeping Beauty Transposon system to induce HCC. Results: Tumor incidence in HepFAK KO mice was significantly decreased compared with HepWT mice. In addition, HepFAK KO mice survived significantly longer compared to
S-999
AASLD Abstracts
Mo1640 Barx2 May Play a Role in Suppressing the Human Primary Hepatocellular Carcinoma Senlin Zhu, Yi Zhang
AASLD Abstracts
Abstract Background: The homeobox gene Barx2 was recently identified as a regulator of ovarian and breast cancer; however, the expression level of BARX2 and its significance in hepatocellular carcinoma (HCC) remain unknown. Methods: Protein and mRNA expression levels of Barx2 were examined using Western blotting and real-time PCR respectively, in paired HCC tissue and matched adjacent noncancerous tissue from 12 patients. The expression levels of epithelial-mesenchymal transition (EMT) markers were also detected in relation to BARX2 expression. Lastly, immunohistochemistry for BARX2 was also performed on a tissue microarray containing 231 HCC tissue samples. Results: We observed that BARX2 expression was lower in HCC tissues compared to matching adjacent noncancerous tissue. The low expression level of BARX2 was significantly correlated with metrics of tumor size, tumor differentiation, clinical stage, metastasis and relapse. Furthermore, the patients with low BARX2 expression had adverse survival outcomes. Importantly, multivariate cox regression analysis revealed that low BARX2 expression was an independent marker for lower overall survival (p = 0.007). Moreover, a significant negative relationship was observed between the expression of BARX2 and markers of EMT. Conclusions: These findings provide evidence that the low expression level of BARX2 in HCC is significantly correlated with tumor metastasis, and that BARX2 may be an independent prognostic biomarker for patients with HCC. Key word: BARX2, Hepatocellular carcinoma, Prognosis, EMT, Metastasis Cox multivariate analyses of prognostic factors on overall survival
Mo1639 Hypoxia Induces Caspase-1 Activation and Promotes Hepatocellular Carcinoma Invasiveness Wei Yan, Ping Han, Dean Tian, Yu Fu Background: Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which caspase-1 activation under hypoxic stress can promote invasion in hepatocellular carcinoma (HCC) cells. Methods: The expression of caspase-1 in HCC was examined by RT-PCR and western blot. Confocal microscopy was used to show the colocalization of NALP3 with ASC or NALP3 with caspase1. The Hepa1-6 and HepG2 cells were cultured in 21% O2 (normoxia) or 1% O2 (hypoxia) for 24 h. Expression of cleaved caspase-1 from hypoxic cells was examined by western blot and immunoflurensence. Colorimetric assays were used to assay caspase-1 activation. Cell invasion induced by hypoxia was analyzed by transwell chamber. Caspase-1 inhibitor ZWEHD-FMK and pan-caspase inhibitor Z-VAD-FMK were used to determine inhibition of hypoxia-induced invasion. Cytokine IL-6 and IL-8 mRNA levels were assessed by real-time PCR. Results: The expression of caspase-1 was upregulated in HCC tissue and cell lines. Colocalization of NALP3 with ASC or NALP3 with caspase-1 was increased in HCC compared to nontumorous liver. The expression of cleaved caspase-1 was increased in a time-dependent manner after hypoxia in both cell lines. After hypoxia, caspase-1 activity was significantly increased in both Hepa1-6 cells and HepG2 cells compared to normoxic control by colorimetric assays. Cleaved caspase-1 significantly increased in cytoplasm diffusely in both cell lines subjected to hypoxia. Invasion of Hepa1-6 cells and HepG2 was increased 1.89 ± 0.14 fold and 2.03 ± 0.14 fold, respectively following incubation with 1% O2. Both Caspase-1 inhibitor Z-WEHD-FMK and pan-caspase inhibitor Z-VAD-FMK blocked this hypoxia-induced invasion. Lastly, mRNA of IL-6 and IL-8 were also increased during hypoxia and decreased by caspase-1 inhibitor treatment. Conclusions: These results suggest that in hypoxic HCC cells, caspase-1 activation induces production of multiple inflammatory mediators which in turn promotes cancer invasion. (This study is supported by the National Natural Science Foundation of China. NO.81000928 and NO. 81000159)
Mo1641 The Role of FAK in Hepatocarcinogenesis Na Shang, Maribel Arteaga, Wei Qiu Background: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and causes more than 12,000 deaths each year in United States. Advanced HCC is hard to be treated, which results in the overall survival of patients with HCC less than 10%. Therefore, it is urgent to develop new and effective therapeutic strategies and agents to treat HCC. Focal Adhesion Kinase (FAK) is overexpressed in HCC specimens, offering a potential target to treat HCC. However, the role of FAK in HCC remains elusive. Studying the role of FAK in hepatocarcinogenesis by in vivo models is critical to provide preclinical proof to determine whether it is suitable to target FAK to treat HCC. We therefore intent to investigate the role of FAK in hepatocarcinogenesis by a mouse model. Methods: We first generated the mice with hepatocyte-specific deletion of FAK (AlbcreFAKflox/flox). We then hydrodynamically injected age- and gender-matched FAKflox/flox (wild type mice with expression of FAK, referred to as HepWT ) and AlbcreFAKflox/flox mice (HepFAK KO), with c-MET and constitutively active β-catenin by the Sleeping Beauty Transposon system to induce HCC. Results: Tumor incidence in HepFAK KO mice was significantly decreased compared with HepWT mice. In addition, HepFAK KO mice survived significantly longer compared to
S-999
AASLD Abstracts