Unit: Liver cancer and Molecular virology
Director: Patrizia Paterlini-Bréchot
Three hundred and fifty million people are infected by the hepatitis B Virus (HBV) and 300 million people by Hepatitis C Virus (HCV), worldwide.These viral infections are characterized by a high risk of evolution toward chronic infection (5% for HBV in adult life, and 60-80% in the neonatal life; 70-80% for HCV). Chronic infection by HBV and/or HCV may evolve to cirrhosis (10-20%) and to liver cancer (5% of cirrhoses evolve to HCC per year). Antiviral treatments, including Interferon, Lamivudine for HBV and combination of Interferon and Ribavirin for HCV, have a limited efficacy since they determine long-term responses in around 30-40% of patients.
Liver carcinogenesis is related to chronic liver inflammation due to HBV and/or HCV, to the direct effects of viral proteins and, for HBV, to the integration of the HBV genome into the cellular genome of liver cells. 1) Chronic inflammation plays a major role by the modification of the control of processes like liver fibrosis and liver cell proliferation. Actually, 90% of liver cancers develop on a cirrhotic liver and 30-50% of patients with cirrhosis develop a liver cancer over a period of 10 years. 2) Certain HBV and HCV viral proteins, as well as the integration of the HBV DNA into the cellular DNA can affect the control of liver cell proliferation and apoptosis and the response of liver cells to interferon and to cytokines (TNFalpha, etc ); 3) recurrent genetic abnormalities (deletions and amplifications of certain chromosomal loci) have been identified thanks to genetic studies performed in HCCs (reviewed in:Ozturk Seminars in liver diseases;1999,19:235. Bréchot:Seminars in Cancer biology, 2000;10:211-231, Buendia: Viral Hepatitis; Zuckerman et Thomas Ed, Churchill Livingstone 1998, 179-200;Thorgeirsson SS, Grisham JW, Nature Genetics,31,339,2002).
In this context, the research program of our Unit is focused on the following issues :
Liver cell adhesion and proliferation :
1) Research team C. Desdouets. This team studies the mechanisms regulating the liver cell ploidy, during liver development and during pathological conditions like viral infections and liver carcinogenesis. This study involves: the quantification of the DNA of liver cell nuclei, the study of proteins implicated in the cytodieresis and of kinases inhibitors (P27 and P21). In 2003, this team has characterised the in vivo kinetic of liver polyploidisation and has demonstrated that the inhibition of cytokinesis determines the formation of binucleated hepatocytes, a major step in the process of polyploidisation.
2) Research team L. Christa. This team has shown the interaction of a type C lectin with the sub-unit RIIa of PKA. The ongoing project studies the in vivo function of the HIP-PAP protein, in embryo and liver stem cells and during liver cell regeneration in transgenic mice, by looking for protein-protein interactions (protein chip Ciphergen method).
3) Research team J. Faivre. This team has demonstrated new splicing variants of the Cyclin A2 gene. Studies on cell localisation and function of the corresponding proteins are ongoing.
Persistence of hepatitis B and hepatitis C viruses :
1) Research team D. Kremsdorf. a) This team studies the MxA protein and has confirmed its antiviral effect in vivo. Transgenic mice for HBV and the MxA gene have been crossed and the expression level of HBV has been studied. In double transgenic female mice the team has observed a decrease of HBV replication. This demonstrates the implication of the MxA protein in the antiviral effect of interferon against HBV ; b) The team works on the in vivo expression of the HBSP protein, encoded by one of the spliced HBV transcripts. Spliced HBV transcripts are also encapsided, reverse transcripted and secreted as defective viral particles. HBSP expression correlates with the viral replication as well as with the severity of liver fibrosis. In addition, the ratio of defective/ wild type viral particles is significantly higher in patients with sever fibrosis compared to patients with moderate fibrosis. This suggests that HBSP protein and defective viral particles play a role in HBV pathogenesis and in particular hepatic fibrosis c) The team has established a murin model, transgenic mice for the urokinase plasminogen activator, suitable for human hepatocytes transplantation. Depletion of non adaptative defenses dramatically improved the survival of human hepatocytes. Furthermore, the highly differentiated status of transplanted human hepatocytes was confirmed by their receptivity to Plasmodium falciparum in the liver stage development and to HBV infection. This provides a new tool to study in vivo the biology of infection of hepatotropic viruses. Furthermore, this murin model was used to demonstrate, in vivo, the capacity of mouse embryonic liver cell lines to differentiate in hepatocytes or in biliary duct.
Liver carcinogenesis and viral infections (HBV, HCV) :
1) Research team N. Pavio, C. Bréchot: 1. This team studies the biological effect of core sequences isolated from tumor and non-tumor tissues in patients with liver cancer. It has shown that core proteins, derived from tumor, inhibit the TGF-β pathway, involved in fibrosis and anti-proliferative effects. The precise molecular mechanism involves a direct interaction of core from tumor with the TGF-β-induced transcription factor Smad3 that prevents the DNA binding of the Smad3/4 complex to its target sequence. Direct inhibition of this pathway by HCV core variants may favor cell proliferation and tumorigesis.
2) Research team V. Thiers - F. Demaugre. We are developing an approach, based on analysis of enriched population of hepatocytes, isolated from heterogenous liver sections, using Laser microdissection. Our projects aimed at analysing in matched pairs of tumorous (T) and non tumorous (NT) groups of hepatocytes selected from the liver of HCV-infected patients with HCC:
- The heterogeneity of HCV Core protein " In situ " : We will characterize full core HCV sequences in T and distinct NT areas. The impact (if any) of amino acid variations on the secondary structure of the protein will be studied using bioinformatic tools. The phenotype effects of the mutations identified in vivo will be also examined.
Protein fingerprinting of HCC tissues so as to clarify the mechanisms of carcinogenesis in HCV-infected patients and identify novel protein markers useful for diagnosis and prognosis. This study will combine microdissection with two-dimensional electrophoresis (2DE). Protein fingerprinting of matched pairs of HCC and non-HCC tissues will be compared and the differentially expressed proteins identified by mass spectrometry (MALDI-TOF-MS).
In parallel, the Reference Centre on hepatitis B and C viruses ensures the molecular characterization of specific B and C viral isolates, and investigates the residual risks of transmission of these viruses.
3) Research team P. Paterlini-Brechot. This team has shown that the HBV genome integrates into a cellular gene in more than 50% of HBV related HCCs. The genes targeted by HBV DNA integration are key genes in cell signalling pathways (MAPK, calcium signalling genes, transcription factors etc..). Moreover, recurrent integrations have been found in the telomerase gene and in the IP3R (inositol tri-phosphate receptor gene). This result demonstrates for the first time the impact of insertional mutagenesis in a human cancer and shows its key role in liver carcinogenesis.
4) Research team P. Paterlini-Brechot. This team has shown the impact of calcium signalling in the biological effect of the X protein of HBV and of the core protein of HCV, two proteins which have been involved in the process of liver carcinogenesis. This result opens a new research domain aiming to study the impact of calcium signalling in liver carcinogensis.
New approaches for the treatment of liver cancer
1) Research team J. Faivre. Radioiodine (131I) therapy is a recognized, well-tolerated approach in the treatment of human thyroid cancer, thanks to the ability of thyroidal cells to trap and retain iodine through the activity of a specific protein, the Natrium Iodide Symporter (NIS). The goal of this team is to define an efficient specific procedure of viral vector-mediated NIS gene delivery in the liver followed by 131I radiotherapy in order to destroy the tumoral hepatocytes. Using an adenoviral CMV-NIS vector, the team found strong and sustained NIS-related radioiodine uptake in tumor-affected livers of DEN rats, and a strong inhibition of tumor growth, the complete regression of small nodules, and prolonged survival in this aggressive model of HCC.
2) Research team P. Paterlini-Brechot. This team has shown the impact of detection and counting, by the ISET technique, circulating tumor cells (CTC) in patients with liver cancer. In fact, the study has shown that the presence and number of CTC significantly correlates with a reduced survival in 44 patients with HCC followed for a mean period of one year. CTC have also been individually microdissected and molecular analyses have been performed to look for beta-catenin mutations (which are found in around 20% of HCCs). The results show that beta-catenin mutations are rarely found in CTC (3 out of 60) demonstrating that this mutation is not required for the first step of tumor cells invasion.
ISET has also been applied to isolate circulating fetal cells from mothers at risk of having a baby with spinal amiotrophy. This study has shown that ISET may be considered a promising approach to develop a new approach of non invasive prenatal diagnosis.
Clinical research projects :
Our Unit has set up a bank of DNA isolated from patients infected by HCV, and patients with alcoholic cirrhosis to search for genetic polymorphism associated with liver diseases. Several pharmacological studies are ongoing targeting the treatment of HBV and HCV chronic infections. The clinical teams are studying the impact of the HCV infection on lipid metabolism and its relationship with viremia and anti-viral treatments. They also study the evolution of patients with biliary cholestatic liver diseases and the effect of the treatments on these diseases. A project dealing with the role of genetic susceptibility in the development and/or the severity of PBC is in process.
1) Team A. Budkowska (this team has left the laboratory on september. This team has recently shown that HCV core protein and HCV nucleocapsids have the capacity to bind non-immune IgG via their Fcgregions.. The FcgR-like site of the core protein is conformational, and requires the presence of amino acid sequence (3-75) for optimal activity. Several human viruses (such CMV, HSV-1 a VZV and EBV) encode receptors that bind the IgG Fcg domain non-immune IgG. Virus proteins with functional properties of FcgRs may enable the virus to evade immune surveillance by avoiding the effector consequences of antibody binding. The FcgR-like function of the core protein, expressed on HCV nucleocapsids or on infected cells, may offer a survival advantage to HCV by interfering with immunological defence mechanisms mediated by the Fcg region of anti-core antibodies, and with functions of FcRn.
2) Team M L Michel.
Preventive and therapeutic vaccines against chronic viral infections
The aim of this team is to develop preventive and therapeutic vaccines against persisting hepatitis B virus (HBV) or human immunodeficiency virus (HIV) infections. Based on results obtained in animal models, a phase I clinical trial of DNA-vaccine therapy was conducted in collaboration with clinicians from Necker Hospital (Pr. Brechot, Pr. Pol and Dr. H. Fontaine) and mainly served to assess safety. In this study, they provided evidences for the immunological efficacy of HBV-DNA vaccination and demonstrated that DNA vaccination can induce or activate T cell responses in chronic HBV-carriers. A phase II trial is planed to assess clinical efficacy of this new therapeutic approach. In addition, they search for new viral targets of the immune response in HBV-infected patients. Identification of HBx protein-specific T cells could give new insights on the role of these T cells in either disease resolution or evolution to chronicity.
In the macaque model of simian human immunodeficiency virus (SHIV) infection the team investigated the fate of vaccine-induced T-cell responses after SHIV challenge. It appeared that most of vaccine-induced CD8+ T cell responses were recalled during infection whereas vaccine-specific CD4+ T cells were preferentially impaired. These results may have important implications for the design both preventive and therapeutic vaccine against AIDS (collaborative work with Y. Rivière, I.P. , R. LeGrand, CEA and B. Moss, NIH).
Keywords: hepatitis B, immunotherapy, DNA vaccine, HBsAg, HBx, SHIV, clinical trial