- Email: [email protected]
325 EVIDENCE FOR BILIARY EPITHELIAL TO MESENCHYMAL TRANSITION IN HUMAN CHOLANGIOCARCINOMA
S128
POSTERS
genome-wide genetic alterations in CCAs by high-resolution microarraycomparative genomic hybridization (aCGH) and characterized novel candidate oncogenes in high level amplification region. Methods: Sixty intrahepatic cholangiocarcinomas (CCA) consisting of 7 adenomas [intraductal type (ID)], 53 CCAs [38 mass forming type (MF) and 15 ID] were screened for genome-wide genetic alterations using aCGH. The array consisted of 2,440 bacterial artificial chromosome (BAC) clones with 1.4 Mb resolution. We observed recurrent and characteristic genomic alternations among the tumor samples and also identified distinct chromosomal gains and losses associated with adenoma and malignant CCAs. The hot spots on chromosomes with high level amplification were defined amplicons boundaries. Fine mapping of amplicon boundaries and genes within the minimal amplicons were identified. The novel candidate genes were validated by quantitative real-time PCR and quantitative RTPCR in comparison to hepatocellular carcinoma (HCC). Results: The genome-wide DNA copy-number alterations in 60 biliary tumors showed variation of DNA copy-number gains and losses with multiple of recurrent amplifications and deletions. The genomic alteration profiles revealed distinct chromosomal gains and losses associated with adenoma and malignant CCAs as well as with MF and ID type. Those distinct alterations represented regions that are important for CCA progression and differentiation. The candidate genes on amplified regions showed high level DNA copy-number and mRNA expression. These putative novel candidate genes may be important for CCA pathogenesis. In addition, we also compared aCGH profiles of CCA and HCC and found that the two liver cancers exhibited different genetic profiles. Conclusion: aCGH is a powerful tool to pave the way for identification of cancer-related gene. The study provides the first aCGH of OV-associated CCAs. The putative oncogenes were identified and may be value in clinical applications such as patient prognosis, diagnosis markers and novel molecular targets for effective therapies. 324 MYC-ASSOCIATED ZINC-FINGER PROTEIN (MAZ) IS A POTENT REGULATOR OF PROX1-GENE-EXPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA J. Dudas1 , F. Haller2 , L. Fuezesi2 , G. Ramadori1 . 1 Department of Internal Medicine, Section of Gastroenterology, 2 Department of Gastroenteropathology, Georg-August-University, Goettingen, Germany E-mail: [email protected] Background and Aims: The Myc-associated Zinc-finger protein (MAZ) was originally found to specifically bind to the c-myc transcription initiation sites. MAZ-gene-expression was proved in human heart, brain, placenta, lung, liver, skeletal muscle, and pancreas. By investigating the regulation of Prox1-gene-expression, a homeobox transcription factor involved in the migration of embryonic hepatocytes, we identified the consensus binding sequences of MAZ in the Prox1-promoter. In this work the function of MAZ was investigated, in the regulation of Prox1. Methods: 54 liver surgical specimens (peritumoral cirrhotic and noncirrhotic liver samples, hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), 4 normal liver specimens, HCC (HepG2, Hep3B and HuH7) and CCC (Mz-Cha1, Mz-Cha2, SK-Cha1) cell lines were analysed for MAZ-gene-expression. The regulatory potential of MAZ on Prox1 was investigated by electrophoretic mobility shift assay (EMSA) and RNAinhibition. Results: MAZ-expression was detected in all analysed liver samples, in hepatocellular (HCC) and cholangiocellular carcinoma (CCC) cell lines. While normal and peritumoral non-cirrhotic liver, cirrhosis and CCC showed a similar MAZ expression, significantly increased expression could be found in HCC. Using EMSA, a significant specific binding of MAZ to the Prox1-promoter was found, in the cell nuclear extracts of HCC cell lines, HCC tissue samples, and of the Mz-Cha2 CCC cell line. Following siRNA transfection in HepG2 cells, the MAZ mRNA expression was reduced to 8.38% of the control, which subsequently caused a reduction of the Prox1-expression until 41.72% of the control.
Conclusions: In breast cancer MAZ was showed to be involved in cancer progression. Similarly, our results suggest, that MAZ is a potential oncogene, and a direct positive regulator of Prox1-gene-expression in HCC. This finding might have a therapeutic relevance. 325 EVIDENCE FOR BILIARY EPITHELIAL TO MESENCHYMAL TRANSITION IN HUMAN CHOLANGIOCARCINOMA L. Fabris1,2 , M. Cadamuro1,2 , C. Spirli1,3 , R. Fiorotto1,2,3 , A. Sonzogni1 , M. Colledan1 , S. Fagiuoli1 , L. Okolicsanyi2 , M. Strazzabosco1,3 . 1 Center for Liver Research (CeLiveR), Ospedali Riuniti di Bergamo, Bergamo, Italy; 2 Department of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy; 3 Liver Center, Yale University, New Haven, CT, USA E-mail: [email protected] Background and Aim: The incidence of cholangiocarcinoma (CC) is increasing, but the disease still carries an extremely poor prognosis and has eluded attempts at clarifying its molecular mechanisms. A peculiar feature of CC is that a rich desmoplastic reaction often accompanies the growth of the neoplastic bile ducts. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells loose some (or all) epithelial phenotype and acquire some mesenchymal phenotypic features. An emerging concept is that, during tumor progression, EMT occurs and provides carcinoma cells the ability to infiltrate surrounding tissue and to metastasize. The aim of this study was to evaluate morphological markers of EMT in a series of CC. Materials. The immunohistochemical expression of different phenotypic markers of EMT, such as S100A4 (mesenchymal Ca++-binding protein), vimentin (mesenchymal cystoskeletal protein), E-Cadherin, N-Cadherin, b-catenin (adherens junction proteins normally expressed by cholangiocytes), was studied in 16 paraffin human liver specimens of CC (both tumoral and extra-tumoral tissues) obtained from surgical resections performed between 2003 and 2007 at Ospedali Riuniti, Bergamo (M/F = 6/10; mean age = 68, range = 44−81 yrs; extra/intrahepatic = 5/11). Immunostained sections were evaluated by two blinded observers as negative or positive for each phenotypic marker. Results: Striking differences of the biliary immunophenotype were present between tumoral and extra-tumoral tissues. In extra-tumoral tissue, bile ducts were negative for S100A4 and positive for b-catenin, E- and N-Cadherin, in sharp contrast with tumoral structures which were extensively positive for S100A4 and negative for b-catenin and N-Cadherin. Membrane expression of E-cadherin was strongly down-regulated in CC cells only at the tumor-host interface. Vimentin was negative in tumoral as well as in non-tumoral bile ducts. Conclusions. Phenotypic changes consistent with EMT are present in the biliary epithelium of CC. Diffuse S100A4 expression (an early marker of EMT) indicates that CC cells are actively primed to develop EMT. Absence of vimentin (a late marker) indicates that CC cells do not fully acquire a mesenchymal phenotype. The selective loss of E-Cadherin at the tumor-host interface indicates impaired tumor cell adhesiveness, in keeping with a role of EMT in promoting tumor invasiveness. 326 LEPTIN IS A GROWTH FACTOR FOR CHOLANGIOCARCINOMA: AN IN VIVO EXPERIMENTAL STUDY G. Fava1 , G. Alpini2 , G. Svegliati-Baroni1 , C. Rychlicki1 , S. Saccomanno1 , L. Trozzi1 , C. Candelaresi1 , A. Di Sario1 , M. Marzioni1 , A. Benedetti1 . 1 Department of Gastroenterology, Universita’ Politecnica delle Marche, Ancona, Italy; 2 Scott & White Hospital, The Texas A&M University System and Central Texas Veterans HCS, Temple, TX, USA E-mail: [email protected] Background and Aims: Leptin, a protein hormone regulating caloric homeostasis, enhances development and growth of several cancers. It was
POSTERS
genome-wide genetic alterations in CCAs by high-resolution microarraycomparative genomic hybridization (aCGH) and characterized novel candidate oncogenes in high level amplification region. Methods: Sixty intrahepatic cholangiocarcinomas (CCA) consisting of 7 adenomas [intraductal type (ID)], 53 CCAs [38 mass forming type (MF) and 15 ID] were screened for genome-wide genetic alterations using aCGH. The array consisted of 2,440 bacterial artificial chromosome (BAC) clones with 1.4 Mb resolution. We observed recurrent and characteristic genomic alternations among the tumor samples and also identified distinct chromosomal gains and losses associated with adenoma and malignant CCAs. The hot spots on chromosomes with high level amplification were defined amplicons boundaries. Fine mapping of amplicon boundaries and genes within the minimal amplicons were identified. The novel candidate genes were validated by quantitative real-time PCR and quantitative RTPCR in comparison to hepatocellular carcinoma (HCC). Results: The genome-wide DNA copy-number alterations in 60 biliary tumors showed variation of DNA copy-number gains and losses with multiple of recurrent amplifications and deletions. The genomic alteration profiles revealed distinct chromosomal gains and losses associated with adenoma and malignant CCAs as well as with MF and ID type. Those distinct alterations represented regions that are important for CCA progression and differentiation. The candidate genes on amplified regions showed high level DNA copy-number and mRNA expression. These putative novel candidate genes may be important for CCA pathogenesis. In addition, we also compared aCGH profiles of CCA and HCC and found that the two liver cancers exhibited different genetic profiles. Conclusion: aCGH is a powerful tool to pave the way for identification of cancer-related gene. The study provides the first aCGH of OV-associated CCAs. The putative oncogenes were identified and may be value in clinical applications such as patient prognosis, diagnosis markers and novel molecular targets for effective therapies. 324 MYC-ASSOCIATED ZINC-FINGER PROTEIN (MAZ) IS A POTENT REGULATOR OF PROX1-GENE-EXPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA J. Dudas1 , F. Haller2 , L. Fuezesi2 , G. Ramadori1 . 1 Department of Internal Medicine, Section of Gastroenterology, 2 Department of Gastroenteropathology, Georg-August-University, Goettingen, Germany E-mail: [email protected] Background and Aims: The Myc-associated Zinc-finger protein (MAZ) was originally found to specifically bind to the c-myc transcription initiation sites. MAZ-gene-expression was proved in human heart, brain, placenta, lung, liver, skeletal muscle, and pancreas. By investigating the regulation of Prox1-gene-expression, a homeobox transcription factor involved in the migration of embryonic hepatocytes, we identified the consensus binding sequences of MAZ in the Prox1-promoter. In this work the function of MAZ was investigated, in the regulation of Prox1. Methods: 54 liver surgical specimens (peritumoral cirrhotic and noncirrhotic liver samples, hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), 4 normal liver specimens, HCC (HepG2, Hep3B and HuH7) and CCC (Mz-Cha1, Mz-Cha2, SK-Cha1) cell lines were analysed for MAZ-gene-expression. The regulatory potential of MAZ on Prox1 was investigated by electrophoretic mobility shift assay (EMSA) and RNAinhibition. Results: MAZ-expression was detected in all analysed liver samples, in hepatocellular (HCC) and cholangiocellular carcinoma (CCC) cell lines. While normal and peritumoral non-cirrhotic liver, cirrhosis and CCC showed a similar MAZ expression, significantly increased expression could be found in HCC. Using EMSA, a significant specific binding of MAZ to the Prox1-promoter was found, in the cell nuclear extracts of HCC cell lines, HCC tissue samples, and of the Mz-Cha2 CCC cell line. Following siRNA transfection in HepG2 cells, the MAZ mRNA expression was reduced to 8.38% of the control, which subsequently caused a reduction of the Prox1-expression until 41.72% of the control.
Conclusions: In breast cancer MAZ was showed to be involved in cancer progression. Similarly, our results suggest, that MAZ is a potential oncogene, and a direct positive regulator of Prox1-gene-expression in HCC. This finding might have a therapeutic relevance. 325 EVIDENCE FOR BILIARY EPITHELIAL TO MESENCHYMAL TRANSITION IN HUMAN CHOLANGIOCARCINOMA L. Fabris1,2 , M. Cadamuro1,2 , C. Spirli1,3 , R. Fiorotto1,2,3 , A. Sonzogni1 , M. Colledan1 , S. Fagiuoli1 , L. Okolicsanyi2 , M. Strazzabosco1,3 . 1 Center for Liver Research (CeLiveR), Ospedali Riuniti di Bergamo, Bergamo, Italy; 2 Department of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy; 3 Liver Center, Yale University, New Haven, CT, USA E-mail: [email protected] Background and Aim: The incidence of cholangiocarcinoma (CC) is increasing, but the disease still carries an extremely poor prognosis and has eluded attempts at clarifying its molecular mechanisms. A peculiar feature of CC is that a rich desmoplastic reaction often accompanies the growth of the neoplastic bile ducts. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells loose some (or all) epithelial phenotype and acquire some mesenchymal phenotypic features. An emerging concept is that, during tumor progression, EMT occurs and provides carcinoma cells the ability to infiltrate surrounding tissue and to metastasize. The aim of this study was to evaluate morphological markers of EMT in a series of CC. Materials. The immunohistochemical expression of different phenotypic markers of EMT, such as S100A4 (mesenchymal Ca++-binding protein), vimentin (mesenchymal cystoskeletal protein), E-Cadherin, N-Cadherin, b-catenin (adherens junction proteins normally expressed by cholangiocytes), was studied in 16 paraffin human liver specimens of CC (both tumoral and extra-tumoral tissues) obtained from surgical resections performed between 2003 and 2007 at Ospedali Riuniti, Bergamo (M/F = 6/10; mean age = 68, range = 44−81 yrs; extra/intrahepatic = 5/11). Immunostained sections were evaluated by two blinded observers as negative or positive for each phenotypic marker. Results: Striking differences of the biliary immunophenotype were present between tumoral and extra-tumoral tissues. In extra-tumoral tissue, bile ducts were negative for S100A4 and positive for b-catenin, E- and N-Cadherin, in sharp contrast with tumoral structures which were extensively positive for S100A4 and negative for b-catenin and N-Cadherin. Membrane expression of E-cadherin was strongly down-regulated in CC cells only at the tumor-host interface. Vimentin was negative in tumoral as well as in non-tumoral bile ducts. Conclusions. Phenotypic changes consistent with EMT are present in the biliary epithelium of CC. Diffuse S100A4 expression (an early marker of EMT) indicates that CC cells are actively primed to develop EMT. Absence of vimentin (a late marker) indicates that CC cells do not fully acquire a mesenchymal phenotype. The selective loss of E-Cadherin at the tumor-host interface indicates impaired tumor cell adhesiveness, in keeping with a role of EMT in promoting tumor invasiveness. 326 LEPTIN IS A GROWTH FACTOR FOR CHOLANGIOCARCINOMA: AN IN VIVO EXPERIMENTAL STUDY G. Fava1 , G. Alpini2 , G. Svegliati-Baroni1 , C. Rychlicki1 , S. Saccomanno1 , L. Trozzi1 , C. Candelaresi1 , A. Di Sario1 , M. Marzioni1 , A. Benedetti1 . 1 Department of Gastroenterology, Universita’ Politecnica delle Marche, Ancona, Italy; 2 Scott & White Hospital, The Texas A&M University System and Central Texas Veterans HCS, Temple, TX, USA E-mail: [email protected] Background and Aims: Leptin, a protein hormone regulating caloric homeostasis, enhances development and growth of several cancers. It was