- Email: [email protected]
318 GLUT1 EXPRESSION IS INCREASED IN HEPATOCELLULAR CARCINOMA AND PROMOTES TUMORIGENESIS
S126
POSTERS
requires further adjustment and ascites appears to be the defining event for the prognosis of the patient. Depending on the respective stage, MELD and HVPG are the independent predictors of survival. Stage
Sub-stage
n
Median survival (mos)
IQ range
Compensated
Stage Stage Stage Stage
55 73 201 129
88 74* 23 35
63–110 47−98 6−63 7−73
Decompensated
1 2 3 4
03A. LIVER TUMORS – A) EXPERIMENTAL
318 GLUT1 EXPRESSION IS INCREASED IN HEPATOCELLULAR CARCINOMA AND PROMOTES TUMORIGENESIS T. Amann1 , U. Maegdefrau2 , M. Kreuz3 , A. Hartmann4 , J. Schoelmerich1 , A.K. Bosserhoff2 , C. Hellerbrand1 . 1 Department of Internal Medicine I, University of Regensburg, Germany; 2 Institute of Pathology, University of Regensburg, Germany; 3 Department of Hematology and Oncology, University of Regensburg, Germany; 4 Institute of Pathology, University of Erlangen, Germany E-mail: [email protected] Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) is a key rate-limiting factor in the transport of glucose into cancer cells. Previous studies regarding GLUT1 expression in hepatocellular carcinoma (HCC) were inconclusive and the biological significance of GLUT1 expression in HCC was unknown. Therefore, we aimed to analyze the expression and function of GLUT1 in HCC. Methods and Results: In HCC tissues and cell lines GLUT1 expression was significantly higher than in primary human hepatocytes (PHH) and corresponding non-tumorous tissue, respectively. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding noncancerous liver tissue of 85 patients revealed that GLUT1 positive HCC tissue showed a significantly higher Ki67 labeling index, more advanced tumor stages and less differentiated tumor grading. In accordance, suppression of GLUT1 expression in HCC cells by siRNA significantly impaired attachment dependent and independent growth and the migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression inhibited both glucose uptake and lactate secretion, indicating impaired anaeobic glycolysis. Pharmacological induction of hypoxia further increased HIF1alpha and GLUT1 expression in HCC cells and further increased the tumorigenicity of the HCC cells. This induction was dependent on the activation of the transcription factor hypoxia-inducible factor (HIF)1alpha since transfection of HCC-cells with a dominant negative form of HIF-1alpha abolished the hypoxia induced GLUT1 induction. Summary and Conclusion: GLUT1 expression in HCC cells and functionally affects tumorigenicity of HCC cells via induction of anaerobic glycolysis, and these mechanism is further increased during hypoxia via induction of HIF-1alpha. These findings indicate GLUT1 as a prognostic marker for HCC progression and as an innovative therapeutic target for this highly aggressive tumor.
319 CELECOXIB-INDUCED GENE EXPRESSION PROFILING MODULATION IS RELATED TO THE RESTORATION OF PHOSPHORYLATION AND TO THE NUCLEAR TRANSLOCATION OF STAT-5 IN EARLY RAT HEPATOCARCINOGENESIS J. Arellanes-Robledo1 , M. Salcido-Neyoy1 , R. Garc´ıa-Rom´an1 , V. Le Berre2 , S. Sokol2 , J.M. Fran¸cois2 , S. Villa-Trevi˜no1 . 1 Departamento de Biolog´ıa Celular, Centro de Investigaci´on y Estudios Avanzados del Instituto Polit´ecnico Nacional, Mexico City, Mexico, 2 TranscriptomeBiochips Platform of Genopole of Toulouse Midi-Pyrenees, Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, UMR-INRA 792, Institut National des Sciences Appliquees F-31077, Toulouse, France E-mail: [email protected] Background and Aims: Celecoxib participation like chemoprotector agent involves the proliferation inhibition, cell cycle arrest, apoptosis activation and as consequence it inhibits both the angiogenesis and metastasis. We had demonstrated that celecoxib prevents the formation and regresses the existing preneoplastic lesions in rat liver through antiproliferative processes. However, most of celecoxib effect evaluations have been only at the level of discrete number of protein expression and there are not reports of an integral study at the gene transcription level in response to chemopreventive agents. Here, we evaluate celecoxib effect on gene expression profiles in early rat hepatocarcinogenesis. Methods: Animals were fed with a diet containing 1500 ppm of celecoxib. Rat livers were evaluated by DNA microarray, quantitative-PCR and western blot analysis. Results: Celecoxib administration resulted in a modulation of 46 genes probes, of which 17 were known rat genes, 3 mouse orthologue genes and 26 tag sequences. A gene group that encode alpha2-microglobulin (a2mG) variants which was suggested that its decrement favors cell growth, was highly downregulated by carcinogenic treatment (CT) but restored to normal expression by celecoxib treatment as well as at protein expression level; in the same way, the gene expression of CYP2b1 and CYP3a1, detoxification enzymes, downregulated by CT, was upregulated by celecoxib treatment; all these events confirmed by quantitative-PCR (p < 0.05). A search for the upstream mechanism revealed an inactivation and inhibition of nuclear translocation of signal transducer and activator of transcription-5 (Stat-5), which is constitutively active in normal rat liver; its level was attenuated to 13% by CT (p < 0.05). This phenomenon was restored by celecoxib because cytosolic Stat-5 phosphorylated had an accelerated nuclear translocation. Conclusion: These findings reveal that chemopreventive celecoxib potential can be exerted in part through the modulation of genetic changes induced by CT; moreover, it let us to present a2mG and Stat-5 as new targets that respond to celecoxib treatment in early rat hepatocarcinogenesis and convert it in a clinically important agent to treat this malignance. Acknowledgements: This work was supported by grant 39525-M from CONACYT-M´exico and a fellowship from CONACYT to JAR; 173787 (2004–2008). 320 HUMAN CD4+ T CELLS RECOGNIZE AN EPITOPE WITHIN ALPHA-FETOPROTEIN SEQUENCE AND DEVELOP INTO TH3 CELLS A. Alisa, S. Boswell, R. Williams, S. Behboudi. The Institute of Hepatology, London, UK E-mail: [email protected] Background: Th3 cells (TGF-beta producing CD4+ T cells) play an important role in modulation of anti-tumor immunity. However, there is limited information on the influence of tumor growth on the expansion of tumor-specific Th3 cells in man. We had observed that CD4+ T cells from some hepatocellular carcinoma (HCC) patients produce large amounts of
POSTERS
requires further adjustment and ascites appears to be the defining event for the prognosis of the patient. Depending on the respective stage, MELD and HVPG are the independent predictors of survival. Stage
Sub-stage
n
Median survival (mos)
IQ range
Compensated
Stage Stage Stage Stage
55 73 201 129
88 74* 23 35
63–110 47−98 6−63 7−73
Decompensated
1 2 3 4
03A. LIVER TUMORS – A) EXPERIMENTAL
318 GLUT1 EXPRESSION IS INCREASED IN HEPATOCELLULAR CARCINOMA AND PROMOTES TUMORIGENESIS T. Amann1 , U. Maegdefrau2 , M. Kreuz3 , A. Hartmann4 , J. Schoelmerich1 , A.K. Bosserhoff2 , C. Hellerbrand1 . 1 Department of Internal Medicine I, University of Regensburg, Germany; 2 Institute of Pathology, University of Regensburg, Germany; 3 Department of Hematology and Oncology, University of Regensburg, Germany; 4 Institute of Pathology, University of Erlangen, Germany E-mail: [email protected] Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) is a key rate-limiting factor in the transport of glucose into cancer cells. Previous studies regarding GLUT1 expression in hepatocellular carcinoma (HCC) were inconclusive and the biological significance of GLUT1 expression in HCC was unknown. Therefore, we aimed to analyze the expression and function of GLUT1 in HCC. Methods and Results: In HCC tissues and cell lines GLUT1 expression was significantly higher than in primary human hepatocytes (PHH) and corresponding non-tumorous tissue, respectively. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding noncancerous liver tissue of 85 patients revealed that GLUT1 positive HCC tissue showed a significantly higher Ki67 labeling index, more advanced tumor stages and less differentiated tumor grading. In accordance, suppression of GLUT1 expression in HCC cells by siRNA significantly impaired attachment dependent and independent growth and the migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression inhibited both glucose uptake and lactate secretion, indicating impaired anaeobic glycolysis. Pharmacological induction of hypoxia further increased HIF1alpha and GLUT1 expression in HCC cells and further increased the tumorigenicity of the HCC cells. This induction was dependent on the activation of the transcription factor hypoxia-inducible factor (HIF)1alpha since transfection of HCC-cells with a dominant negative form of HIF-1alpha abolished the hypoxia induced GLUT1 induction. Summary and Conclusion: GLUT1 expression in HCC cells and functionally affects tumorigenicity of HCC cells via induction of anaerobic glycolysis, and these mechanism is further increased during hypoxia via induction of HIF-1alpha. These findings indicate GLUT1 as a prognostic marker for HCC progression and as an innovative therapeutic target for this highly aggressive tumor.
319 CELECOXIB-INDUCED GENE EXPRESSION PROFILING MODULATION IS RELATED TO THE RESTORATION OF PHOSPHORYLATION AND TO THE NUCLEAR TRANSLOCATION OF STAT-5 IN EARLY RAT HEPATOCARCINOGENESIS J. Arellanes-Robledo1 , M. Salcido-Neyoy1 , R. Garc´ıa-Rom´an1 , V. Le Berre2 , S. Sokol2 , J.M. Fran¸cois2 , S. Villa-Trevi˜no1 . 1 Departamento de Biolog´ıa Celular, Centro de Investigaci´on y Estudios Avanzados del Instituto Polit´ecnico Nacional, Mexico City, Mexico, 2 TranscriptomeBiochips Platform of Genopole of Toulouse Midi-Pyrenees, Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, UMR-INRA 792, Institut National des Sciences Appliquees F-31077, Toulouse, France E-mail: [email protected] Background and Aims: Celecoxib participation like chemoprotector agent involves the proliferation inhibition, cell cycle arrest, apoptosis activation and as consequence it inhibits both the angiogenesis and metastasis. We had demonstrated that celecoxib prevents the formation and regresses the existing preneoplastic lesions in rat liver through antiproliferative processes. However, most of celecoxib effect evaluations have been only at the level of discrete number of protein expression and there are not reports of an integral study at the gene transcription level in response to chemopreventive agents. Here, we evaluate celecoxib effect on gene expression profiles in early rat hepatocarcinogenesis. Methods: Animals were fed with a diet containing 1500 ppm of celecoxib. Rat livers were evaluated by DNA microarray, quantitative-PCR and western blot analysis. Results: Celecoxib administration resulted in a modulation of 46 genes probes, of which 17 were known rat genes, 3 mouse orthologue genes and 26 tag sequences. A gene group that encode alpha2-microglobulin (a2mG) variants which was suggested that its decrement favors cell growth, was highly downregulated by carcinogenic treatment (CT) but restored to normal expression by celecoxib treatment as well as at protein expression level; in the same way, the gene expression of CYP2b1 and CYP3a1, detoxification enzymes, downregulated by CT, was upregulated by celecoxib treatment; all these events confirmed by quantitative-PCR (p < 0.05). A search for the upstream mechanism revealed an inactivation and inhibition of nuclear translocation of signal transducer and activator of transcription-5 (Stat-5), which is constitutively active in normal rat liver; its level was attenuated to 13% by CT (p < 0.05). This phenomenon was restored by celecoxib because cytosolic Stat-5 phosphorylated had an accelerated nuclear translocation. Conclusion: These findings reveal that chemopreventive celecoxib potential can be exerted in part through the modulation of genetic changes induced by CT; moreover, it let us to present a2mG and Stat-5 as new targets that respond to celecoxib treatment in early rat hepatocarcinogenesis and convert it in a clinically important agent to treat this malignance. Acknowledgements: This work was supported by grant 39525-M from CONACYT-M´exico and a fellowship from CONACYT to JAR; 173787 (2004–2008). 320 HUMAN CD4+ T CELLS RECOGNIZE AN EPITOPE WITHIN ALPHA-FETOPROTEIN SEQUENCE AND DEVELOP INTO TH3 CELLS A. Alisa, S. Boswell, R. Williams, S. Behboudi. The Institute of Hepatology, London, UK E-mail: [email protected] Background: Th3 cells (TGF-beta producing CD4+ T cells) play an important role in modulation of anti-tumor immunity. However, there is limited information on the influence of tumor growth on the expansion of tumor-specific Th3 cells in man. We had observed that CD4+ T cells from some hepatocellular carcinoma (HCC) patients produce large amounts of