- Email: [email protected]
[69] IDENTIFICATION OF FIBROBLAST GROWTH FACTOR RECEPTOR 2IIIB (FGFR2IIIB) AS TUMOR SUPPRESSOR IN HEPATOCANCEROGENESIS
Parallel Session 8: MOLECULAR BASIS OF LIVER CANCER in these sequences we aimed to understand how inherent mutability might affect the clinical application of anti-HCV inhibitors. Methods: Structural study: we employed the program ESCET [ I ] to define objectively regions of the NS5B protein that are conformationally variant and invariant. Mutational analysis: the CONSURF server [2] was used to derive a probability score for the mutability of each residue in NSSB based on quasispecies or euHCVdb sequences. Results: NS5B is quite rigid and the slight domain movements show little correlation with inhibitor binding. Polymerases from genotypes l b and 2a have similar conformations although, significantly, flexibility between the thumb and palm domains was discerned. For genotype-lb structures, the open conformation is seen only for the truncated Delta55 form. Crucially, for genotype 2a the same is observed for the Delta2 I truncation. Furthermore, the distribution of mutations is neither identical between genotypes, nor between quasispecies and database sequences. Some known inhibitor-binding sites have an inherent tendency to mutate. Conclusions: The C-terminal region plays a key role in NS5B regulation. The crystallised forms from NSSB genotypes show subtle yet significant differences. This, together with the fact that mutations are distributed differentially between genotypes, suggests the need for alternative clinical strategies for the treatment of different HCV strains. References [ I ] Schneider TR. (2004) Acta Cryst. D60:2269-2275. [2] Landau M. et al. (2005) Nucl. Acids Res. 33: W299-W302
INSIGHTS IN HCV PERSISTENCE: IDENTIFICATION OF VIRAL AND CELLULAR FACTORS THAT DESENSITISE THE NEUTRALISING ANTIBODY RESPONSE AGAINST HCV AND FAVOUR ITS IMMUNE ESCAPE M. Dreux, B. Bartosch, C. Granier, D. Lavillette, EL. Cosset. Hunian Virology Department, INSERM U758 ENS Lyon, Lyon, France E-mail: [email protected] ~
Background and Aims: HCV has developed several mechanisms to escape from the host immune responses. Our previous results demonstrated that the neutralisation response plays a role in the early phase ofthe disease and that the emergence of neutralising antibodies correlates with decrease of high initial viremia. Moreover, high-titre, broadly cross-neutralising antibodies are readily detected in chronically-infected patients. Here, we clarify factors that mitigate the impact of the neutralising antibody response. Methods and Results: We demonstrate that high-density lipoprotein (HDL) is a key serum factor that desensitises neutralisation by monoclonal and HCV patient-derived polyclonal antibodies. Over 10-fold higher antibody concentrations are required to neutralise HCV in the presence of HDL or human serum (HS) and only a limited portion of virus is neutralised at saturating antibody concentrations, in contrast to complete inhibition of infectivity in HS-free conditions. We show that HDL-interaction with the scavenger receptor B1 (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating cholesterol-uptake from HDL, strongly reduces neutralisation of both HCVpp and HCVcc. We characterise compounds that, by inhibiting the lipid transfer function of SR-BI, fully restore neutralisation by antibodies in HS. We demonstrate that the hypervariable region-1 (HVRl) of the HCV-E2 glycoprotein, an immunodominant epitope under strong evolution pressure during disease outcome, coordinates the HDL/SR-BT interplay. Deletion or mutation of HVR 1 strongly increased sensitivity of HCVpp to neutralising antibodies present in these sera, assigning to HVRl a critical role in modulation of neutralisation effectiveness and clar ng its previously proposed role in viral persistence and immune escape. Furthermore, we demonstrate that this HVR 1 IHDLISR-BT interplay only desensitises the antibodies that block HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Consistently, we show that
S3 1
antibodies targeted to HCV-El efficiently neutralise HCV in the presence of HS or HDL.. Conclusions: These results have several implications for the design of therapeutic strategies against HCV. Drugs that inhibit HDL/SR-BT interaction will stimulate the neutralising activity of antibodies inoculated in patients or naturally induced by HCV Alternatively, our data provides guidance for designing vaccines that induce efficient antibodies in vivo.
Parallel Session 8: MOLECULAR BASIS OF LIVER CANCER
IDENTIFICATION OF FIBROBLAST GROWTH FACTOR RECEPTOR 2111B (FGFRZIIIB) AS TUMOR SUPPRESSOR IN HEPATOCANCEROGENESlS T. Amanu’, T. Spruss’, F. Bataille3, C. Liedtke4, M. Miihlbauerl, C. Trautwein4, A.K. Bosserhoff3, C. Hellerbrand’ . ’Department of Internal Medicine I, Uniuersity of Regenshurg, Regenshurg; ’Institute of
Pharmucj~,Uniuersih~of Regenshurg, Regenshurg; ‘Institute of Puthologj~, Uniuer~xityof Regenshurg, Regenshurg; ‘Department of Internal Medicine Ill, RWTH Aachen, Aarhen, Germany E-mail: [email protected] The b isoform of Fibroblast Growth Factor Receptor 2 (FGFR2TTTb) is highly expressed in hepatocytes and has been shown to play an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2lllb in hepatocellular carcinoma (HCC). Methods and Results: FGFR2TTTb mRNA expression was downregulated in HCC cell lines compared to primary human hepatocytes as determined by quantitative PCR. Similarly, HCC tissues from patients and animal models (c-myc and IgEGF transgenic mice) showed reduced or lost FGFR2lllb mRNA expression compared to corresponding non-cancerous liver tissue. Tmmunohistochemical analysis of HCC tissues applying a newly generated polyclonal antibody that specifically recognizes the lllb variant of the FGFR2 receptor confirmed these findings on the protein level. The S’-region of the FGFR2-gene contains a CpG island, but 5-azacytidine treatment did not affect FGFR2lllb expression in HCC cells, excluding promotor methylation as potential cause for the FGFR2TTTb downregulation. Furthermore, sequencing revealed no genetic changes suggesting that negative regulation is controlled at the transcriptional level. To gain insight into the functional role ofFGFR2TTTb downregulation in HCC, FGFR2TTTb was re-expressed in HCC cell lines by stable transfection, and the generated cell clones were analyzed in vitro and in vivo. FGFR2TTTb clones exhibited a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential as compared to mock transfected and control cells. In accordance, FGFR2lllb clones grew significantly slower in nude mice and Tune1 staining of FGFR2lllb tumors revealed higher apoptosis rates compared to controls. Moreover and interestingly, FGFR2lllb reexpression induced E-Cadherin expression in HCC cells in vitro, and in vivo in human HCC tissue FGFR2lllb and E-Cadherin expression revealed a significant correlation. Further, higher FGFR2TTTb and E-cadherin expression were found in differentiated HCC cells. Summary and Conclusion: Data presented indicate FGFR2TTTb as tumor suppressor of HCC. In the absence of FGFR2lllb dedifferentiation processes may be activated resulting in the loss of E-Cadherin, providing a potential mechanism of FGFR2TTTb tumor suppression. Identification and therapeutic targeting of the molecular mechanism responsible for FGFR2lllb suppression in HCC may be a viable method of inhibiting the progression of HCC.
INSIGHTS IN HCV PERSISTENCE: IDENTIFICATION OF VIRAL AND CELLULAR FACTORS THAT DESENSITISE THE NEUTRALISING ANTIBODY RESPONSE AGAINST HCV AND FAVOUR ITS IMMUNE ESCAPE M. Dreux, B. Bartosch, C. Granier, D. Lavillette, EL. Cosset. Hunian Virology Department, INSERM U758 ENS Lyon, Lyon, France E-mail: [email protected] ~
Background and Aims: HCV has developed several mechanisms to escape from the host immune responses. Our previous results demonstrated that the neutralisation response plays a role in the early phase ofthe disease and that the emergence of neutralising antibodies correlates with decrease of high initial viremia. Moreover, high-titre, broadly cross-neutralising antibodies are readily detected in chronically-infected patients. Here, we clarify factors that mitigate the impact of the neutralising antibody response. Methods and Results: We demonstrate that high-density lipoprotein (HDL) is a key serum factor that desensitises neutralisation by monoclonal and HCV patient-derived polyclonal antibodies. Over 10-fold higher antibody concentrations are required to neutralise HCV in the presence of HDL or human serum (HS) and only a limited portion of virus is neutralised at saturating antibody concentrations, in contrast to complete inhibition of infectivity in HS-free conditions. We show that HDL-interaction with the scavenger receptor B1 (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating cholesterol-uptake from HDL, strongly reduces neutralisation of both HCVpp and HCVcc. We characterise compounds that, by inhibiting the lipid transfer function of SR-BI, fully restore neutralisation by antibodies in HS. We demonstrate that the hypervariable region-1 (HVRl) of the HCV-E2 glycoprotein, an immunodominant epitope under strong evolution pressure during disease outcome, coordinates the HDL/SR-BT interplay. Deletion or mutation of HVR 1 strongly increased sensitivity of HCVpp to neutralising antibodies present in these sera, assigning to HVRl a critical role in modulation of neutralisation effectiveness and clar ng its previously proposed role in viral persistence and immune escape. Furthermore, we demonstrate that this HVR 1 IHDLISR-BT interplay only desensitises the antibodies that block HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Consistently, we show that
S3 1
antibodies targeted to HCV-El efficiently neutralise HCV in the presence of HS or HDL.. Conclusions: These results have several implications for the design of therapeutic strategies against HCV. Drugs that inhibit HDL/SR-BT interaction will stimulate the neutralising activity of antibodies inoculated in patients or naturally induced by HCV Alternatively, our data provides guidance for designing vaccines that induce efficient antibodies in vivo.
Parallel Session 8: MOLECULAR BASIS OF LIVER CANCER
IDENTIFICATION OF FIBROBLAST GROWTH FACTOR RECEPTOR 2111B (FGFRZIIIB) AS TUMOR SUPPRESSOR IN HEPATOCANCEROGENESlS T. Amanu’, T. Spruss’, F. Bataille3, C. Liedtke4, M. Miihlbauerl, C. Trautwein4, A.K. Bosserhoff3, C. Hellerbrand’ . ’Department of Internal Medicine I, Uniuersity of Regenshurg, Regenshurg; ’Institute of
Pharmucj~,Uniuersih~of Regenshurg, Regenshurg; ‘Institute of Puthologj~, Uniuer~xityof Regenshurg, Regenshurg; ‘Department of Internal Medicine Ill, RWTH Aachen, Aarhen, Germany E-mail: [email protected] The b isoform of Fibroblast Growth Factor Receptor 2 (FGFR2TTTb) is highly expressed in hepatocytes and has been shown to play an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2lllb in hepatocellular carcinoma (HCC). Methods and Results: FGFR2TTTb mRNA expression was downregulated in HCC cell lines compared to primary human hepatocytes as determined by quantitative PCR. Similarly, HCC tissues from patients and animal models (c-myc and IgEGF transgenic mice) showed reduced or lost FGFR2lllb mRNA expression compared to corresponding non-cancerous liver tissue. Tmmunohistochemical analysis of HCC tissues applying a newly generated polyclonal antibody that specifically recognizes the lllb variant of the FGFR2 receptor confirmed these findings on the protein level. The S’-region of the FGFR2-gene contains a CpG island, but 5-azacytidine treatment did not affect FGFR2lllb expression in HCC cells, excluding promotor methylation as potential cause for the FGFR2TTTb downregulation. Furthermore, sequencing revealed no genetic changes suggesting that negative regulation is controlled at the transcriptional level. To gain insight into the functional role ofFGFR2TTTb downregulation in HCC, FGFR2TTTb was re-expressed in HCC cell lines by stable transfection, and the generated cell clones were analyzed in vitro and in vivo. FGFR2TTTb clones exhibited a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential as compared to mock transfected and control cells. In accordance, FGFR2lllb clones grew significantly slower in nude mice and Tune1 staining of FGFR2lllb tumors revealed higher apoptosis rates compared to controls. Moreover and interestingly, FGFR2lllb reexpression induced E-Cadherin expression in HCC cells in vitro, and in vivo in human HCC tissue FGFR2lllb and E-Cadherin expression revealed a significant correlation. Further, higher FGFR2TTTb and E-cadherin expression were found in differentiated HCC cells. Summary and Conclusion: Data presented indicate FGFR2TTTb as tumor suppressor of HCC. In the absence of FGFR2lllb dedifferentiation processes may be activated resulting in the loss of E-Cadherin, providing a potential mechanism of FGFR2TTTb tumor suppression. Identification and therapeutic targeting of the molecular mechanism responsible for FGFR2lllb suppression in HCC may be a viable method of inhibiting the progression of HCC.