Hepatitis B Virus and Hepatocellular CarcinomaGroup Leader: Marie Annick BUENDIA
The molecular basis of liver carcinogenesis in patients infected with hepatitis B virus remains incompletely understood. The viral regulatory protein HBX has been implicated in the tumoral process by its ability to activate transcription from a broad range of cellular promoters. We analyze the interactions of HBX with the co-activators CBP, p300 and PCAF in transcriptional activation of cellular and viral promoters, and the activity of wild-type and mutated HBX in cell cycle progression and apoptosis. Current studies are aimed at identifying cellular target genes of HBX by transcriptome analysis. To recapitulate the hepatic context in HBV-infected patients, most studies use primary human hepatocytes or mouse liver.
Molecular Mechanisms of Liver OncogenesisGroup Leader: Marie Annick BUENDIA
Our recent studies of the molecular steps involved in liver cell transformation have shown that activation of Wnt signaling by oncogenic mutations of ß-catenin plays an important role in the development of hepatocellular carcinoma and hepatoblastoma. The impact of ß-catenin on gene expression profiles and cell cycle progression is currently analyzed in primary human hepatocytes and fibroblasts. We are also studying the functional interactions of ß-catenin with a recently identified partner protein. Our studies in transgenic mice suggest cross-talks between the Wnt/ß-catenin and p53 pathways.
Comprehensive allelotyping of human and murine hepatocarcinomas with microsatellite markers has revealed frequently altered chromosomal regions that contain candidate tumor suppressor genes. In human tumors, we have evaluated the implication of E-cadherin by genetic and expression studies combined with fine mapping of chromosome 16. In murine tumors induced by c-Myc, specific chromosomal alterations suggest disruption of the INK4/RB pathway as an important step in liver oncogenesis.
Genetics of primary liver cancerGroup Leader: Anne DEJEAN
One of our main research interest focuses on the genetic basis of hepatocellular carcinoma (HCC). Our first goal is to identify novel tumor suppressor genes involved in the development of HCC. To this aim, we searched for allelic deletions affecting tumor DNA through a genome-wide scanning for LOH. Such approach is of great value for locating candidate genes implicated in cancer development. The chromosomes most frequently affected by deletions were 1p, 4q, 6q, 8p, 13q and 16q. Since the highest percentage of deletions was found for a locus in 8p23, this particular region is now being studied in details. A second extensive allelotyping performed on chromosomes 8p has revealed the presence of three distinct deleted regions suggesting the existence of several tumor suppressor genes on 8p. To delineate smaller regions, further allelotyping on a large number of additional HCC tumors is ongoing in the lab. To this aim, we have also created a collection of 60 hepatoma-derived cell lines and are looking for double deletions. It should narrow down the regions of interest and allow the search for mutations in potentially transcribed regions. In parallel, we used comparative genomic hybridization (CGH) to detect amplified regions in the human genome susceptible to harbor oncogenes. We focus now on chromosome 1q which we found to be amplified in more than 60% of liver tumors. The powerful CGH-array technique will be applied to narrow down the regions of interest. Finally, a transcriptome study of HCC has been recently undertaken in collaboration with MA Buendia's group.
Functional compartmentalization of the nucleus and acute promyelocytic leukemiaGroup Leader: Anne DEJEAN
A major aspect of Acute Promyelocytic Leukemia (APL) pathogenesis involves (i) a chromosomal translocation affecting the gene encoding the retinoic acid receptor a (RARa) and the PML gene (giving rise to the fusion PML-RARa
oncoprotein) as well as (ii) a retinoic acid- reversible disruption of a particular subnuclear structure, the PML Nuclear Body (NB). This latter observation, that provides a striking parallelel to the therapeutic effect of retinoic acid in this type of leukemia, implies a new type of nuclear 'organelle' in a human disease. The interest of our lab is focused on the role of these PML Nuclear Bodies both in 'normal' and 'pathological' cells. We will mainly concentrate on the relationship between these particular substructures and the chromatin compartment for which we recently established a link. In an attempt to investigate the signals that regulate the dynamics of the NBs, we recently found that both PML and SP100, the two major components of the NBs, were modified by covalent linkage with the ubiquitin-related SUMO-1 modifier. By contrast to ubiquitination, sumoylation' does not target protein for degradation but is rather involved in subcellular localization. Notably, sumoylation of PML is necessary for its proper targeting to the NBs and for the maintenance of the structural integrity of these structures. An important effort will now be devoted to elucidate the role of this new type of post-translational modification in vivo.
HBs antigen and VaccinationGroup Leader: Marie-Louise MICHEL
The goal of our studies is to develop therapeutic vaccines that can control persistent HBV infection. We are studying the immunogenicity of selected HBV envelope-based vaccine formulations for the induction or the broadening of T and B cell responses that are deficient in HBV chronic carriers. Using transgenic mice that constitutively express the HBsAg in the liver as a model of asymptomatic chronic HBV carriers, we have shown that immunization can overcome functional tolerance to HBsAg by inducing a specific immune response. These findings provided the rationale for a clinical study, in which we have demonstrated that specific vaccine therapy by standard anti-HBV vaccination can reduce or circumvent HBV replication in some chronic carrier subjects. We have recently developed a new approach of vaccination for hepatitis B infections based on intramuscular injection of plasmid DNA encoding hepatitis B virus antigen. Immunization of mice, that are transgenic for the human HLA A2.1 molecule with a vector encoding the middle and the small HBV envelope proteins shows that epitopes presented after in vivo processing in HLA A2.1 Tg mice are very similar to those generated in human during HBV infection. In the HBsAg-transgenic mice, immunization with HBsAg and CpG-containing oligodeoxynucleotides induced the clearance of HBsAg circulating in the sera with a concomitant appearance of specific antibodies and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Our results in animal models prompt us to apply for a phase I clinical trial in HBV-chronically infected patients. In this trial conducted by clinicians from the Necker Hospital (Pr. C. Brechot, Pr. Pol), we will assess the immune response before and during the vaccine therapy.
We have previously reported that fusion of foreign viral B cell epitopes within the preS2 domain of the HBV middle protein leads to the synthesis of hybrid proteins which self-assemble together with small HBV envelope proteins to form virus like particles (VLP). In a pilot experiment in macaques, we have assessed the protective efficacy of our DNA-based vectors encoding HIV/HBs VLP against a SHIV challenge.
Relation between hepatitis B (HBV) and C (HCV) viruses and their hostGroup Leader: Catherine TRANSY
Our studies aim at characterizing the molecular interactions taking place during the life cycle of HBV and HCV in order to identify new therapeutic targets.
In the case of HBV, we focus on the regulatory X protein (HBx) because this protein is essential for productive infection although its precise function is still unknown. We have shown that interaction of X protein with the host protein UV-DDB is crucial not only for in vitro X-mediated activities but also in vivo for productive infection. We have obtained a peptidic compound which includes the minimal interaction domain of X with UV-DDB. We will study the ability of this molecule to inhibit X-mediated activities as well as its possible anti-viral properties.
The nature and the function of interactions arising between the mature viral proteins produced from the single HCV polyprotein precursor are largely unknown, due to the absence of an efficient system to propagate HCV in vitro. This also holds for interactions between the virus and the host proteomes. We have designed tools for expression in human cells of random sub-regions from the viral polyprotein. We will use these expression systems, (i) to further study the interactions between viral proteins previously explored using the yeast two-hybrid system, (ii) identify interactions between viral and host proteins, (iii) identify B and T viral epitopes.