- Email: [email protected]
P97 NEW INSIGHTS IN HOXA13 AND HOTTIP ROLE AS PROGNOSTIC FACTORS IN HEPATOCELLULAR CARCINOMA
POSTERS for hepatocyte proliferation, was markedly downregulated. In a mouse model of HCC xenograft, ERK5 silencing or administration of XMD8–92 significantly decreased tumor volume. These effects were associated with reduced cell proliferation, as indicated by lower BrdU incorporation. Upon reduction of ERK5 levels of activity in vivo, levels of c-Rel and of c-Jun, a proto-oncogene essential for cell proliferation, were reduced. Expression of MEF2, a known target in the ERK5 pathway, was also inhibited. Conclusions: We found that ERK5 regulates cell proliferation in HCC in vivo and in vitro, affecting the expression of different oncogenic targets, including c-Rel, which we identify for the first time as a target of ERK5. P95 EXPRESSIONS OF MICRORNAS DIFFER BETWEEN CIRRHOTIC AND TUMOUR-SURROUNDING CIRRHOTIC LIVER AS WELL AS BETWEEN HEPATOCELLULAR CARCINOMA AND FOCAL NODULAR HYPERPLASIA K. Schlachter1 , B. Gyongy ¨ osi ¨ 1 , M. Gyugos1 , G. Lendvai1,2 , K. Baghy3 , 1,2 1 1 Z. Schaff , A. Kiss . 2nd Department of Pathology, 2 MTA-SE Tumor Progression Research Group, 3 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary E-mail: [email protected] Background and Aims: The altered expressions of microRNAs (miRNA) may contribute to liver diseases. In the present study, we determined the expressions of 12 miRNAs – suggested to be up- or downregulated in hepatocellular carcinoma (HCC) – in different tumorous and non-tumorous liver tissues. Methods: Isolated RNA samples of 75 patients were analysed including 29 HCC and 16 tumour-surrounding cirrhosis (TSCirrhosis), as well as 20 cirrhotic liver tissue (Cirrhosis), 10 focal nodular hyperplasia (FNH) and 15 normal liver tissue (Normal). The levels of miRNAs were determined using TaqMan MicroRNA Assays. Results: miR-17–5p and miR-210 expressions were reduced, whereas miR-34a and miR-224 levels were increased in the disease groups compared with Normal (p < 0.02). miR-122 was elevated in FNH (p < 0.3), although it is usually reduced in liver diseases. miR223 was decreased in HCC compared with Normal and Cirrhosis. Similarly, miR-214 was reduced in HCC as compared with TSCirrhosis (p < 0.03). The expressions of miR-221 and miR-222 were elevated in TS-Cirrhosis and HCC when compared with Cirrhosis (p < 0.02). The expressions of miR-18a, miR-21 and miR-222 were reduced in FNH as compared with HCC (p < 0.01). In HCC, miR-18a, miR-222 and miR-223 were found to be increased from low to high tumour grade (p < 0.04). Conclusions: miRNA expressional differences were revealed between FNH and HCC, Cirrhosis and TS-Cirrhosis. Increased miR221/222 expression characterized HCC compared with Cirrhosis, whereas miR-221/222 were reduced and miR-224 were elevated in Cirrhosis as compared with Normal. Acknowledgement: This work was supported by grants OTKA 101435 and K108548 from the National Scientific Research Fund.
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P96 ALDOSE REDUCTASE CONTRIBUTES TO HEPATOCARCINOGENESIS IN TRANSALDOLASE DEFICIENCY Z. Oaks1,2 , R. Hanczko1 , M. Beckford1 , S.K. Chung3 , S.K. Landas4 , J.M. Asara5 , A. Perl1,2,6 . 1 Medicine, 2 Biochemistry & Molecular Biology, SUNY – Upstate Medical University, Syracuse, NY, United States; 3 Department of Anatomy, The University of Hong Kong, Hong Kong, Hong Kong; 4 Department of Pathology, SUNY – Upstate Medical University, Syracuse, NY, 5 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, 6 Microbiology and Immunology, SUNY – Upstate Medical University, Syracuse, NY, United States E-mail: [email protected] Background and Aims: Transaldolase (TAL) is the rate limiting enzyme of the pentose phosphate pathway. TAL deficiency is a model for NAFLD, NASH, and hepatocellular carcinoma (HCC) and results in the accumulation of sugar phosphates, depletion of NADPH and glutathione, which underlie oxidative stress (J. Clin. Invest. 2009;119:1546–57). TAL-deficient mice exhibit the overexpression of the NADPH dependent enzyme aldose reductase (AR). Here, we investigated if the deletion of AR influences the depletion of NADPH and its consequences in TAL deficiency. Methods: Metabolites were extracted from TAL, AR, and TAL/AR double-knockout livers that were sacrificed with C57BL/6 controls, using 5 age-matched mice of each strain. 258 metabolites were detected by LC-MS/MS with selective reaction monitoring. Relative metabolite quantities were then analyzed using Metaboanalyst to identify metabolites and pathways that were significantly changed. 21 TAL/AR double-knockout mice were monitored up to 78 weeks of age for the development of NAFLD, NASH, and HCC. Results: 0/21 TAL/AR double-knockouts, 45/97 TAL knockouts, and 3/106 controls developed HCC. Double-knockouts were not significantly different from controls (p=1.0), but had significantly reduced HCC compared to TAL knockouts (p < 0.0001). AR knockout livers had minimal changes relative to control animals. TAL knockout livers showed accumulation of sedoheptulose 7-phosphate (S7P) and erythrose 4-phosphate (E4P) and depletion of NADPH. TAL/AR knockout livers exhibited increased S7P and E4P but NADPH levels were normalized. Conclusions: These data show that AR activation contributes to NADPH depletion and oxidative stress and its blockade prevents hepatocarcinogenesis in TAL deficiency. P97 NEW INSIGHTS IN HOXA13 AND HOTTIP ROLE AS PROGNOSTIC FACTORS IN HEPATOCELLULAR CARCINOMA L. Quagliata1 , M. Matter1 , S. Piscuglio1 , L. Tornillo1 , M. Heim2 , C. Cillo3 , L. Terracciano1 . 1 Molecular Pathology Unit, Institute of Pathology, University Hospital Basel, 2 Department of Biomedicine, Hepatology Laboratory, University of Basel, Basel, Switzerland; 3 Federico II University Medical School, Naples, Italy E-mail: [email protected] Background and Aims: Previously, we observed that among the transcriptional factors family of HOX-genes, HOXA13 is highly deregulated in hepatocellular carcinoma (HCC). HOTTIP, a recently described long non-coding RNA, is located at the 5’HOXA locus (in contiguity with HOXA13) and binds WDR5/MLL-complexes driving transcription along the entire HOXA locus. Lately, we reported HOTTIP/HOXA13 deregulation as a key feature in HCC development, controlling liver-cancer cells apoptosis. Finally, we outlined HOTTIP/HOXA13 as predictive markers of HCCpatients’ outcome and disease progression. Here, driven by ChromatinImmunoprecipitation (ChiP) data, we corroborate our findings by screening a large cohort of HCCs evaluating HOXA13 levels and selected metastasis-associated genes directly regulated by HOXA13.
Journal of Hepatology 2014 vol. 60 | S67–S214
POSTERS Methods: A TMA (tissue-microarray), comprises a total of n = 305 specimens, n = 82 from normal liver, n = 108 from cirrhotic patients and n = 115 from HCCs, has been immunohistochemically stained for HOXA13, CK-7, CK-19, E-Cad, Galectin-3,Prune and Nm23-H2. Staining scores have been correlated with clinical-pathological data. Results: HOXA13 is altered in 41% of analysed samples, confirming our previous reports. Corroborating our gene expression data, we observed that higher HOXA13 levels are associated with poorer patients’ outcome and grading (Edmondson and BCLC). HOXA13 expression is accompanied by high liver progenitor markers levels, CK-7 and CK19. Furthermore, high HOXA13 expression is coupled with low E-Cad and increased Gal-3, Prune and Nm23-H2, providing a molecular basis for its clinical association with metastasis formation. Conclusions: HOXA13 IHC-protein levels predict HCC disease outcome, confirming our gene-expression data and making HOXA13 a suitable marker for liver carcinogenesis evaluation. Additional experiments aiming to elucidate HOXA13 role in promoting metastasis are urgently needed. P98 CROSS TALK BETWEEN MULTIPOTENT STEM-LIKE CELLS AND CANCER CELLS IN HEPATOCELLULAR CARCINOMA C.H.C. Sukowati1,2 , B. Anfuso1 , S.I. Ie3 , L.S. Croce1,2 , C. Tiribelli1,2 . 1 Fondazione Italiana Fegato – Italian Liver Foundation, 2 Dept. of Medical Sciences, University of Trieste, Trieste, Italy; 3 Eijkman Institute for Molecular Biology, Jakarta, Indonesia E-mail: [email protected] Background and Aims: The presence of various tumor-supportingcell types in hepatocellular carcinoma (HCC) has become an important target in basic study and future therapy development. The stem cells (SC) might contribute in stromal and tumor cells cross-talk in microenvironment. This study elucidates the role of stromal cells of HCC and their association with cancer cells in hepatocarcinogenesis. Methods: Primary cells from paired tumoral and non-tumoral tissues of HCC (n = 9) and normal liver (n = 2) were isolated and characterized by flow cytometry, RT-PCR, anchorage-independent assay and trans-differentiation. Co-culture with HuH-7 and JHH-6 HCC cells was performed in a transwell system. In vivo xenograft assay was performed in immunodeficient mice. Results: Cells were CD90+CD44+CD34−CD45−CD117−CD133−, and expressed mesenchymal SC and pluripotency factors. In stimulated condition, they differentiated into pancreatic cell, adipocyte, and osteoblast. Co-culture between tumoral primary cells and HCC cell lines up-regulated mRNA expression of TGFb1 and FAP and downregulated CTGF in both HuH-7 and JHH-6 (p < 0.05). In contrast, non-tumoral primary cells did not induce TGFb1 and FAP of HCC cells, while HCC cells up-regulated CTGF, vimentin, COL1, and integrin b1 of the primary cells. Xenograft assay showed the cells capacity to enter into circulation resulting in a mixed population between human and mouse cells as confirmed by RT-PCR and DNA sequencing. Conclusions: Our data provides evidence of the plasticity of the SClike cells isolated from HCC in the process of hepatocarcinogenesis and metastasis. These cells mutually interacted with HCC cells. Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.
4A. MOLECULAR AND CELLULAR BIOLOGY: CELL CYCLE CONTROL/APOPTOSIS
P99 THE PROTECTIVE ROLES OF NERVE GROWTH FACTOR IN BILE DUCT LIGATION INDUCED LIVER INJURY M.-S. Tsai, Y.-H. Kao, P.-H. Lee. E-Da Hospital and I-Shou University, Kaohsiung, Taiwan E-mail: [email protected] Background and Aims: Nerve growth factor (NGF) was shown to vital for liver carcinogenesis, regeneration, and angiogenesis. However, it remains elusive regarding the role of NGF in liver injury induced by bile duct ligation (BDL). We aimed to investigate whether NGF can protect hepatocytes from BDL-induced injury in vivo and in vitro. Methods: Liver and serum levels of NGF were measured in BDL mice. We also explore the possible upstream molecule regulating NGF expression in vitro. In order to determine the protective role of NGF in BDL, animal received either recombinant mouse NGF peptide (rmNGF) or anti-NGF (aNGF) neutralizing Ab after BDL surgery. Liver pathology, serum liver function and apoptotic index were investigated in each group. Results: Biochemical and histopathological analysis confirmed that BDL effectively induced liver injury and fibrosis, whereas the NGF expression in both in plasma ad liver tissues was up-regulated and well correlated with the progression of liver fibrosis. Immunohistochemistry showed that the up-regulated NGF antigenicity was mainly localized in parenchymal hepatocytes in diseased livers. Suppression of inflammation in vivo decreased NGF production. TGF-b1 could upregulate NGF expression in hepatocytes. NGF overexpression protected TGF-b1 and oxidative stress-induced hepatocyte death in vitro. The possible mechanisms included up-regulated Akt phosphorylation, Bcl-2 expression and down-regulated Bax expression. In vivo, rmNGF therapy could decreased the activation of caspase 3 within liver tissue after BDL, whereas aNGF activated caspase 3. TUNEL staining further confirmed the protective role of NGF. Conclusions: The present study suggested the protective role and possible therapeutic applications of NGF in BDL-induced cholestatic liver injury. P100 Nur77 MEDIATES BILE ACID-INDUCED DEATH AND SURVIVAL IN LIVER AND COLON CELLS Y. Hu, T. Chau, H.-X. Liu, Y.-J.Y. Wan. Pathology, University of California Davis, Sacramento, CA, United States E-mail: [email protected] Background and Aims: Bile acids (BA) are endogenous agents known to cause cancer throughout the gastrointestinal tract. The current study aims to uncover novel mechanism by which BA exert their pathological effects. Methods: Cells of either human hepatocyte (Huh7, Hep3B) or colonic (HCT116, HT29) origin were treated with different BA at various concentrations and then assayed for apoptosis and survival. The role of nuclear receptor Nur77 in modulating cell death and survival was analyzed. Results: Hydrophobic deoxychollic acid (DCA) and lithocholic acid (LCA) significantly induced NF-úB, c-Fos, c-Jun, and Nur77 expression in the studied cell lines. In contrast, the same concentration hydrophilic cholic acid and chenodeoxycholic acid did not induce Nur77 expression. BA-induced cytoplasmic Nur77 expression coincided with the presence of cleaved caspase 3
Journal of Hepatology 2014 vol. 60 | S67–S214
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P96 ALDOSE REDUCTASE CONTRIBUTES TO HEPATOCARCINOGENESIS IN TRANSALDOLASE DEFICIENCY Z. Oaks1,2 , R. Hanczko1 , M. Beckford1 , S.K. Chung3 , S.K. Landas4 , J.M. Asara5 , A. Perl1,2,6 . 1 Medicine, 2 Biochemistry & Molecular Biology, SUNY – Upstate Medical University, Syracuse, NY, United States; 3 Department of Anatomy, The University of Hong Kong, Hong Kong, Hong Kong; 4 Department of Pathology, SUNY – Upstate Medical University, Syracuse, NY, 5 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, 6 Microbiology and Immunology, SUNY – Upstate Medical University, Syracuse, NY, United States E-mail: [email protected] Background and Aims: Transaldolase (TAL) is the rate limiting enzyme of the pentose phosphate pathway. TAL deficiency is a model for NAFLD, NASH, and hepatocellular carcinoma (HCC) and results in the accumulation of sugar phosphates, depletion of NADPH and glutathione, which underlie oxidative stress (J. Clin. Invest. 2009;119:1546–57). TAL-deficient mice exhibit the overexpression of the NADPH dependent enzyme aldose reductase (AR). Here, we investigated if the deletion of AR influences the depletion of NADPH and its consequences in TAL deficiency. Methods: Metabolites were extracted from TAL, AR, and TAL/AR double-knockout livers that were sacrificed with C57BL/6 controls, using 5 age-matched mice of each strain. 258 metabolites were detected by LC-MS/MS with selective reaction monitoring. Relative metabolite quantities were then analyzed using Metaboanalyst to identify metabolites and pathways that were significantly changed. 21 TAL/AR double-knockout mice were monitored up to 78 weeks of age for the development of NAFLD, NASH, and HCC. Results: 0/21 TAL/AR double-knockouts, 45/97 TAL knockouts, and 3/106 controls developed HCC. Double-knockouts were not significantly different from controls (p=1.0), but had significantly reduced HCC compared to TAL knockouts (p < 0.0001). AR knockout livers had minimal changes relative to control animals. TAL knockout livers showed accumulation of sedoheptulose 7-phosphate (S7P) and erythrose 4-phosphate (E4P) and depletion of NADPH. TAL/AR knockout livers exhibited increased S7P and E4P but NADPH levels were normalized. Conclusions: These data show that AR activation contributes to NADPH depletion and oxidative stress and its blockade prevents hepatocarcinogenesis in TAL deficiency. P97 NEW INSIGHTS IN HOXA13 AND HOTTIP ROLE AS PROGNOSTIC FACTORS IN HEPATOCELLULAR CARCINOMA L. Quagliata1 , M. Matter1 , S. Piscuglio1 , L. Tornillo1 , M. Heim2 , C. Cillo3 , L. Terracciano1 . 1 Molecular Pathology Unit, Institute of Pathology, University Hospital Basel, 2 Department of Biomedicine, Hepatology Laboratory, University of Basel, Basel, Switzerland; 3 Federico II University Medical School, Naples, Italy E-mail: [email protected] Background and Aims: Previously, we observed that among the transcriptional factors family of HOX-genes, HOXA13 is highly deregulated in hepatocellular carcinoma (HCC). HOTTIP, a recently described long non-coding RNA, is located at the 5’HOXA locus (in contiguity with HOXA13) and binds WDR5/MLL-complexes driving transcription along the entire HOXA locus. Lately, we reported HOTTIP/HOXA13 deregulation as a key feature in HCC development, controlling liver-cancer cells apoptosis. Finally, we outlined HOTTIP/HOXA13 as predictive markers of HCCpatients’ outcome and disease progression. Here, driven by ChromatinImmunoprecipitation (ChiP) data, we corroborate our findings by screening a large cohort of HCCs evaluating HOXA13 levels and selected metastasis-associated genes directly regulated by HOXA13.
Journal of Hepatology 2014 vol. 60 | S67–S214
POSTERS Methods: A TMA (tissue-microarray), comprises a total of n = 305 specimens, n = 82 from normal liver, n = 108 from cirrhotic patients and n = 115 from HCCs, has been immunohistochemically stained for HOXA13, CK-7, CK-19, E-Cad, Galectin-3,Prune and Nm23-H2. Staining scores have been correlated with clinical-pathological data. Results: HOXA13 is altered in 41% of analysed samples, confirming our previous reports. Corroborating our gene expression data, we observed that higher HOXA13 levels are associated with poorer patients’ outcome and grading (Edmondson and BCLC). HOXA13 expression is accompanied by high liver progenitor markers levels, CK-7 and CK19. Furthermore, high HOXA13 expression is coupled with low E-Cad and increased Gal-3, Prune and Nm23-H2, providing a molecular basis for its clinical association with metastasis formation. Conclusions: HOXA13 IHC-protein levels predict HCC disease outcome, confirming our gene-expression data and making HOXA13 a suitable marker for liver carcinogenesis evaluation. Additional experiments aiming to elucidate HOXA13 role in promoting metastasis are urgently needed. P98 CROSS TALK BETWEEN MULTIPOTENT STEM-LIKE CELLS AND CANCER CELLS IN HEPATOCELLULAR CARCINOMA C.H.C. Sukowati1,2 , B. Anfuso1 , S.I. Ie3 , L.S. Croce1,2 , C. Tiribelli1,2 . 1 Fondazione Italiana Fegato – Italian Liver Foundation, 2 Dept. of Medical Sciences, University of Trieste, Trieste, Italy; 3 Eijkman Institute for Molecular Biology, Jakarta, Indonesia E-mail: [email protected] Background and Aims: The presence of various tumor-supportingcell types in hepatocellular carcinoma (HCC) has become an important target in basic study and future therapy development. The stem cells (SC) might contribute in stromal and tumor cells cross-talk in microenvironment. This study elucidates the role of stromal cells of HCC and their association with cancer cells in hepatocarcinogenesis. Methods: Primary cells from paired tumoral and non-tumoral tissues of HCC (n = 9) and normal liver (n = 2) were isolated and characterized by flow cytometry, RT-PCR, anchorage-independent assay and trans-differentiation. Co-culture with HuH-7 and JHH-6 HCC cells was performed in a transwell system. In vivo xenograft assay was performed in immunodeficient mice. Results: Cells were CD90+CD44+CD34−CD45−CD117−CD133−, and expressed mesenchymal SC and pluripotency factors. In stimulated condition, they differentiated into pancreatic cell, adipocyte, and osteoblast. Co-culture between tumoral primary cells and HCC cell lines up-regulated mRNA expression of TGFb1 and FAP and downregulated CTGF in both HuH-7 and JHH-6 (p < 0.05). In contrast, non-tumoral primary cells did not induce TGFb1 and FAP of HCC cells, while HCC cells up-regulated CTGF, vimentin, COL1, and integrin b1 of the primary cells. Xenograft assay showed the cells capacity to enter into circulation resulting in a mixed population between human and mouse cells as confirmed by RT-PCR and DNA sequencing. Conclusions: Our data provides evidence of the plasticity of the SClike cells isolated from HCC in the process of hepatocarcinogenesis and metastasis. These cells mutually interacted with HCC cells. Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.
4A. MOLECULAR AND CELLULAR BIOLOGY: CELL CYCLE CONTROL/APOPTOSIS
P99 THE PROTECTIVE ROLES OF NERVE GROWTH FACTOR IN BILE DUCT LIGATION INDUCED LIVER INJURY M.-S. Tsai, Y.-H. Kao, P.-H. Lee. E-Da Hospital and I-Shou University, Kaohsiung, Taiwan E-mail: [email protected] Background and Aims: Nerve growth factor (NGF) was shown to vital for liver carcinogenesis, regeneration, and angiogenesis. However, it remains elusive regarding the role of NGF in liver injury induced by bile duct ligation (BDL). We aimed to investigate whether NGF can protect hepatocytes from BDL-induced injury in vivo and in vitro. Methods: Liver and serum levels of NGF were measured in BDL mice. We also explore the possible upstream molecule regulating NGF expression in vitro. In order to determine the protective role of NGF in BDL, animal received either recombinant mouse NGF peptide (rmNGF) or anti-NGF (aNGF) neutralizing Ab after BDL surgery. Liver pathology, serum liver function and apoptotic index were investigated in each group. Results: Biochemical and histopathological analysis confirmed that BDL effectively induced liver injury and fibrosis, whereas the NGF expression in both in plasma ad liver tissues was up-regulated and well correlated with the progression of liver fibrosis. Immunohistochemistry showed that the up-regulated NGF antigenicity was mainly localized in parenchymal hepatocytes in diseased livers. Suppression of inflammation in vivo decreased NGF production. TGF-b1 could upregulate NGF expression in hepatocytes. NGF overexpression protected TGF-b1 and oxidative stress-induced hepatocyte death in vitro. The possible mechanisms included up-regulated Akt phosphorylation, Bcl-2 expression and down-regulated Bax expression. In vivo, rmNGF therapy could decreased the activation of caspase 3 within liver tissue after BDL, whereas aNGF activated caspase 3. TUNEL staining further confirmed the protective role of NGF. Conclusions: The present study suggested the protective role and possible therapeutic applications of NGF in BDL-induced cholestatic liver injury. P100 Nur77 MEDIATES BILE ACID-INDUCED DEATH AND SURVIVAL IN LIVER AND COLON CELLS Y. Hu, T. Chau, H.-X. Liu, Y.-J.Y. Wan. Pathology, University of California Davis, Sacramento, CA, United States E-mail: [email protected] Background and Aims: Bile acids (BA) are endogenous agents known to cause cancer throughout the gastrointestinal tract. The current study aims to uncover novel mechanism by which BA exert their pathological effects. Methods: Cells of either human hepatocyte (Huh7, Hep3B) or colonic (HCT116, HT29) origin were treated with different BA at various concentrations and then assayed for apoptosis and survival. The role of nuclear receptor Nur77 in modulating cell death and survival was analyzed. Results: Hydrophobic deoxychollic acid (DCA) and lithocholic acid (LCA) significantly induced NF-úB, c-Fos, c-Jun, and Nur77 expression in the studied cell lines. In contrast, the same concentration hydrophilic cholic acid and chenodeoxycholic acid did not induce Nur77 expression. BA-induced cytoplasmic Nur77 expression coincided with the presence of cleaved caspase 3
Journal of Hepatology 2014 vol. 60 | S67–S214
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