- Email: [email protected]
251 WNT-PATHWAY ACTIVATION IN TWO MOLECULAR CLASSES OF HEPATOCELLULAR CARCINOMA AND EXPERIMENTAL MODULATION BY SORAFENIB
POSTERS 251 WNT-PATHWAY ACTIVATION IN TWO MOLECULAR CLASSES OF HEPATOCELLULAR CARCINOMA AND EXPERIMENTAL MODULATION BY SORAFENIB A. Lachenmayer1,2 , C. Alsinet2,3 , R. Savic2 , L. Cabellos2 , S. Toffanin2,4 , Y. Hoshida5 , A. Villanueva3 , B. Minguez2 , P. Newell2 , H.-W. Tsai2 , J. Barretina5 , S. Thung2 , S.C. Ward2 , J. Bruix3 , V. Mazzaferro4 , M. Schwartz2 , S.L. Friedman2 , J.M. Llovet2,3,6 . 1 Department of General-, Visceral- and Pediatric Surgery, University Hospital D¨ usseldorf, Dusseldorf, Germany; 2 Mount Sinai Liver Cancer Program (Divisions of Liver Diseases, Department of Medicine; Tisch Cancer Institute; Division of Surgical Oncology, Department of Surgery; Transplant Institute; Department of Pathology), Mount Sinai School of Medicine, New York, NY, USA; 3 Barcelona Clinic Liver Cancer Group (BCLC; Translational Research Laboratory and Liver Unit), Hospital Cl´ınic, IDIBAPS, Centro de Investigaci´ on Biomedica en Red de Enfermedades Hep´ aticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain; 4 Hepato-Oncology Group, Department of Surgery and Experimental Oncology, National Cancer Institute, Milan, Italy; 5 Cancer Program, Broad Institute of Harvard and MIT, Boston, MA, USA; 6 Instituci´ o Catalana de Recerca i Estudis Avancats ¸ (ICREA), Spain, Spain E-mail: [email protected] Purpose: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt-signaling in the molecular subclasses CTNNB1 and Wnt-TGFb. Underlying biological mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer. Experimental design: The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs) and CTNNB1mutation data (91 HCCs). Effects of sorafenib on Wnt-signaling were evaluated in 4 liver cancer cell lines with active Wnt signaling and a tumor xenograft model. Results: Wnt activation was demonstrated for 315 (49.1%) patients, classified as CTNNB1-class [138 (21.5%)] or Wnt-TGFb-class [177 (27.6%)]. CTNNB1-class was characterized by up-regulation of liverspecific Wnt-targets, nuclear b-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, while Wnt-TGFb-class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear b-catenin. Sorafenib decreased Wnt-signaling and b-catenin protein in HepG2 (CTNNB1-class), SNU387 (Wnt-TGFb-class), SNU398 (CTNNB1mutation) and Huh7 (Lithium-chloride-pathway activation) cell lines. Additionally, sorafenib attenuated expression of liver-specific Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1-class-specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals. Conclusions: Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFb), accounting for half of all HCC patients. Sorafenib modulates b-catenin/Wnt-signaling in experimental models that harbor the CTNNB1-class-signature.
252 INHIBITION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-a (PPAR-a) REDUCES PROLIFERATION OF PRENEOPLASTIC LIVER FOCI M.L. Casella1 , J.A. Monti1 , J.P. Parody1 , M.P. Ceballos1 , P.I. Ingaramo1 , D.E. Frances1 , M.T. Ronco1 , G.B. Pisani2 , C.E. Carnovale1 , M.C. Carrillo1 , M.L. Alvarez1 . 1 Institute of Experimental Physiology (IFISE), CONICET, National University of Rosario, 2 Faculty of Biochemistry, National University of Rosario, Rosario, Argentina E-mail: [email protected] PPAR-a is a ligand-activated transcriptional factors expressed in liver, kidney, small intestine, heart, and muscle, where it is involved in fatty acids and carbohydrates metabolism, wound healing, vascular homeostasis, and differentiation and cellular proliferation of normal and neoplastic tissues. The relationship between cancer and PPAR-a is not fully understood. Some studies postulate PPAR-a has carcinogenic properties, whereas others have demonstrated anti-neoplastic and anti-angiogenic activities of this nuclear receptor. The aim of this work was to study the effect of PPAR-a inhibition on the development of rat preneoplasic liver foci, in order to clarify its role in hepatocarcinogenesis. Adult male Wistar rats were subjected to iniciation-promotion model of hepatocarcinogenesis (IP). A group of IP rats was treated with quercetin (competitive inhibitor of PPAR-a activation). The doses of quercetin used were 10 (Q10) and 20 (Q20) mg/kg body weight. Western blot assays were performed to confirm PPAR-a inhibition by quercetin. Results showed a significant reduction of PPARa protein levels in Q20 group (IP: 100±12; Q10: 78±20; Q20: 29±07*). Preneoplastic foci were detected by immunofluorescence and confocal microscopy by using the antibody raised against the placental form of glutathione S-transferase. Immunodetection and quantification of preneoplastic foci showed a significant decrease in their number and size in Q20 group (number foci/mm2 : IP = 12.7±1.0; Q10 = 10.5±1.5; Q20 = 6.1±0.6*, % of liver occupied by foci: IP = 3.8±0.6; Q10 = 3.6±0.1; Q20 = 1.6±0.6*). The proliferative index (PI) in the foci was determined by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). PI was significantly reduced in Q20 (IP: 28.8±1.2; Q20: 21.9±0.9*). Moreover, the percentage of PCNA-positive preneoplastic hepatocytes in S and M phases of the cell cycle were lower in Q20 group (%S: IP: 2.0±0.2; Q20: 4.1±0.2*; %M: IP: 2.4±0.2; Q20: 1.2±0.2*) (*p < 0.05). In conclusion, PPAR-a inhibition by quercetin during the development of liver tumorigenesis prevents the development of preneoplastic lesions, with reduction of the proliferation rates into the foci. These results strongly suggest that blockage of PPARa activation could be useful for new preventive and therapeutic strategies against liver tumors. 253 EPCAM AND AFP AS POOR PROGNOSTIC MARKERS IN PATIENTS WITH HEPATOBLASTOMA S. Ragull1,2 , I. Serra1 , M. Garrido3 , R. Maibach4 , I. Io Ng5 , P. Czauderna4 , R. Bartoli1,2 , R. Planas1,2,6 , M. Sala2,6 , C. Armengol1,2 . 1 Health Sciences Research Institute Germans Trias i Pujol (IGTP), Badalona, 2 CIBERehd, 3 Pathology Service, Universitary Hospital Vall d’Hebron, Barcelona, Spain; 4 International Childhood Liver Tumours Strategy Group, Bern, Switzerland; 5 Department of Pathology, The University of Hong Kong, Hong Kong, Hong Kong S.A.R.; 6 Digestive Service, Universitary Hospital Germans Trias i Pujol (HGTiP), Badalona, Spain E-mail: [email protected] Background: Hepatoblastoma (HB) is the predominant malignant liver tumour in childhood. The aetiology of HB is unknown, but it differs from hepatocellular carcinoma by the absence of hepatitis
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252 INHIBITION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-a (PPAR-a) REDUCES PROLIFERATION OF PRENEOPLASTIC LIVER FOCI M.L. Casella1 , J.A. Monti1 , J.P. Parody1 , M.P. Ceballos1 , P.I. Ingaramo1 , D.E. Frances1 , M.T. Ronco1 , G.B. Pisani2 , C.E. Carnovale1 , M.C. Carrillo1 , M.L. Alvarez1 . 1 Institute of Experimental Physiology (IFISE), CONICET, National University of Rosario, 2 Faculty of Biochemistry, National University of Rosario, Rosario, Argentina E-mail: [email protected] PPAR-a is a ligand-activated transcriptional factors expressed in liver, kidney, small intestine, heart, and muscle, where it is involved in fatty acids and carbohydrates metabolism, wound healing, vascular homeostasis, and differentiation and cellular proliferation of normal and neoplastic tissues. The relationship between cancer and PPAR-a is not fully understood. Some studies postulate PPAR-a has carcinogenic properties, whereas others have demonstrated anti-neoplastic and anti-angiogenic activities of this nuclear receptor. The aim of this work was to study the effect of PPAR-a inhibition on the development of rat preneoplasic liver foci, in order to clarify its role in hepatocarcinogenesis. Adult male Wistar rats were subjected to iniciation-promotion model of hepatocarcinogenesis (IP). A group of IP rats was treated with quercetin (competitive inhibitor of PPAR-a activation). The doses of quercetin used were 10 (Q10) and 20 (Q20) mg/kg body weight. Western blot assays were performed to confirm PPAR-a inhibition by quercetin. Results showed a significant reduction of PPARa protein levels in Q20 group (IP: 100±12; Q10: 78±20; Q20: 29±07*). Preneoplastic foci were detected by immunofluorescence and confocal microscopy by using the antibody raised against the placental form of glutathione S-transferase. Immunodetection and quantification of preneoplastic foci showed a significant decrease in their number and size in Q20 group (number foci/mm2 : IP = 12.7±1.0; Q10 = 10.5±1.5; Q20 = 6.1±0.6*, % of liver occupied by foci: IP = 3.8±0.6; Q10 = 3.6±0.1; Q20 = 1.6±0.6*). The proliferative index (PI) in the foci was determined by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). PI was significantly reduced in Q20 (IP: 28.8±1.2; Q20: 21.9±0.9*). Moreover, the percentage of PCNA-positive preneoplastic hepatocytes in S and M phases of the cell cycle were lower in Q20 group (%S: IP: 2.0±0.2; Q20: 4.1±0.2*; %M: IP: 2.4±0.2; Q20: 1.2±0.2*) (*p < 0.05). In conclusion, PPAR-a inhibition by quercetin during the development of liver tumorigenesis prevents the development of preneoplastic lesions, with reduction of the proliferation rates into the foci. These results strongly suggest that blockage of PPARa activation could be useful for new preventive and therapeutic strategies against liver tumors. 253 EPCAM AND AFP AS POOR PROGNOSTIC MARKERS IN PATIENTS WITH HEPATOBLASTOMA S. Ragull1,2 , I. Serra1 , M. Garrido3 , R. Maibach4 , I. Io Ng5 , P. Czauderna4 , R. Bartoli1,2 , R. Planas1,2,6 , M. Sala2,6 , C. Armengol1,2 . 1 Health Sciences Research Institute Germans Trias i Pujol (IGTP), Badalona, 2 CIBERehd, 3 Pathology Service, Universitary Hospital Vall d’Hebron, Barcelona, Spain; 4 International Childhood Liver Tumours Strategy Group, Bern, Switzerland; 5 Department of Pathology, The University of Hong Kong, Hong Kong, Hong Kong S.A.R.; 6 Digestive Service, Universitary Hospital Germans Trias i Pujol (HGTiP), Badalona, Spain E-mail: [email protected] Background: Hepatoblastoma (HB) is the predominant malignant liver tumour in childhood. The aetiology of HB is unknown, but it differs from hepatocellular carcinoma by the absence of hepatitis
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