Unit: Liver cancer and Molecular virology

Director: PATERLINI -BRECHOT Patrizia

HBV and HCV infections (approximately 350 million people in the world are infected with the HBV and 300 million with the HCV) are characterized with high risk to go to chronicity (5% for adult (VHB), 60-80% in neonatal (HBV); 70-80% for the VHC) which implies a risk to develop a cirrhosis and liver cancer (HCC). The antiviral treatments indisputably progressed with the use of the interferon and of Lamivudine for the HBV and the association of the interferon to ribavirin for the HCV. The effectiveness of these treatments remains however insufficient, allowing a rate of prolonged response from 30 to 40% in average for the VHC. The hepatic carcinogenesis is related to the combination in the liver of a chronic patient carrying the HBV or HCV, chronic inflalamation, direct effects of viral proteins and (for the HBV) the integration of the viral genome: 1) the inflamation plays an essential role in formation of the fibrose and the deregulation of the hepatocyte proliferation. This point is illustrated by the development of the HCC in approximately 90% of the cases on cirrhotic liver or with active chronic hepatitis, the risk of HCC for cirrhotic patient being of approximately 30-50 % after ten years of follow-up. 2) proteins of the HBV and the HCV, as well as the insertion of the HBV DNA in the chromosomal DNA, exert direct effects on the hepatocyte proliferation and viability, on the interferon response and on cytokines production (TNFalpha). 3) recurrent genetic alteration (deletions and amplifications of some chromosomal loci), were recently identified according to the development of the genetic analyses applied to the HCC. (Reviews: Ozturk Seminars in liver diseases, 1999,19:235. Bréchot: Seminars in Biology Cancer, 2000;10:211-231; Buendia: Viral Hepatitis; Zuckerman and Thomas ED, Churchill Livingstone 1998, 179-200; Thorgeirsson SS, Grisham JW, Natural Genetics, 31, 339,2002).

In this context, the program of our group is focused on the following points:

Hepatocellular proliferation and adhesion

1) Equipe C. Desdouets - Ce groupe a initié une étude sur les mécanismes qui régulent la ploîdie hépatocytaire, à la fois au cours du développement hépatique et dans des conditions pathologiques comme au cours des infections virales et aux différents stades de la carcinogenèse hépatique. Les approches techniques permettant la quantification du contenu en ADN des noyaux hépatocytaires, l'étude des protéines ayant un rôle dans la cytodiérèse, et l'implication des inhibiteurs des kinases (P27 et P21) sont en train d'être mises au point ou sont en cours d'application. Au cours de l'année 2003 cette équipe a caractérisé la cinétique in vivo de la polyploïdisation hépatique et démontré que le blocage de la cytokinèse détermine la formation d'hépatocytes binuclées, une étape fondamentale dans le processus de polyploidisation.

2) Equipe L. Christa  - Protéine HIP, adhésion hépatocytaire et marquage de cellules "progénitrices" :Cette équipe a montré l' interaction de cette lectine de type C avec la sous-unité RIIa de la PKA. Le projet en cours vise la définition de la fonction de la protéine HIPPAP in vivo, dans des cellules souches embryonnaires et hépatiques, au cours de la régénération hépatique chez la souris transgénique, et in vitro, par la recherche d'intéractions protéine-protéine (méthode protein chip Ciphergen)

3) Equipe J. Faivre : L'étude de la caractérisation des transcrits du gène cycline A2 a pu mettre en évidence des nouveaux variants d'épissage de ce gène. La localisation subcellulaire et l'étude de la fonction des protéines correspondantes sont en cours de réalisation.

Persistence of the hepatites B (HBV) and C (HCV)

Research team D. Kremsdorf. a) 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 and the ratio of defective/ wild type viral particles is significantly higher in patients with sever fibrosis compared to patients with moderate fibrosis. In vitro, the expression of HBSP induces a moderate activation of the TGFα pathway. This suggests that HBSP protein and defective viral particles play a role in HBV pathogenesis and in particular hepatic fibrosis. b) We were also interested in the characterization of the biological properties of the HBx protein of the HBV, this in order to better define its implication in hepatic carcinogenesis. A transcriptome approach have showed, among the cellular genes with a modulated expression after the transduction of human hepatocytes with a lentivirus HBx, an increased expression of the receptor of the acid gamma-aminobutyric (GABA) of type A ß3 of approximately 3 fold as compared to the control. The impact of the overexpression of the receptor GABAAα 3 on the inhibition of cellular proliferation induced by the HBx protein will be evaluated. c) The team has established a murin model, transgenic mice for the urokinase plasminogen activator, suitable for human hepatocytes transplantation. The differentiated status of transplanted human hepatocytes was confirmed by their receptivity to HBV infection. This provides a new tool to study in vivo new antiviral molecules. Furthermore, this murin model was used to demonstrate the capacity of mouse adult liver cell lines to proliferate in a context of liver regeneration.

Hepatic carcinogenesis and viral infections (VHB, VHC)

1) Equipe N. Pavio, C. Bréchot - Cette équipe étudie l'effet biologique de séquences de capside du VHC isolées à partir du tissu tumoral et non tumoral de patients avec cancer du foie. Récemment, elle a mis en évidence que les variants de capside isolés à partir de tissus tumoraux inhibent de manière ciblée la voie du TGF-_ impliquée dans la fribrose et le contrôle de la prolifération cellulaire. L'étude du mécanisme moléculaire a mis en évidence une interaction directe entre les capsides tumeur avec Smad3, un des facteurs de transcription activé par le TGF-_ et un blocage de l'interaction du complexe Smad3/4 avec l'ADN. Ce processus pourrait contribuer au développement du CHC.

2) Equipe V. Thiers - F. Demaugre - Nous développons un projet ayant pour objectif d'étudier à partir de cellules hépatocytaires isolées par microdissection laser chez des sujets ayant développé un CHC sur cirrhose virale C : 1) la variabilité intra-hépatique de la capside du VHC afin d'identifier de nouveaux variants naturels de la protéine de Capside du VHC 2) l'étude comparative des protéomes d'hépatocytes tumoraux et non-tumoraux. L'approche repose sur l'association de l'électrophorèse bi-dimensionnelle (2D-PAGE) et de la spectrométrie de masse pour identifier les protéines différentiellement exprimées dans le CHC lié au VHC. L'objectif à terme de cette étude est d'utiliser les données obtenues dans cette investigation, pour définir de nouveaux outils diagnostiques et/ou thérapeutiques pour le traitement du cancer du foie. Parallèlement, le Centre de Référence pour les hépatites B et C contribue à la surveillance moléculaire et à l'identification des risques résiduels de transmission de ces virus.

3) Research team Patrizia Paterlini-Brechot-Recently we showed the impact of calcium signalling on the HCV core effects. Indeed, we showed that the HCV core determines the reduction of the ER calcium concentration, a mechanism by which the HCV core leads to apoptotic cell death in vitro and in vivo through a calcium dependent manner (Benali-Furet et al., 2005). These effects are very interesting in the contest of the hepatic carcinogenesis studies. A new research axe, based on the hypothesis that the modulation of the calcium concentration induced by the HCV core protein (Benali-Furet, 2005) could have a significant role in the HCV multiplication. The possibility to control the HCV multiplication constitutes an important role either in the public health and the hepatic carcinogenesis. Recent data from our group show that the action of three drugs able to reduce significantly the calcium concentration within the ER, determines, in a HCV Replicon model, a decrease of the HCV proteins (NS3, E2 and Core) accumulation, shown by western blot, and of the viral RNA quantity, shown by real time quantitative RT-PCR. The analysis of the calcium concentration modulation by the use of the recombinant aequorin calcium probe targeted to the ER revealed a significant decrease of the calcium concentration induced by these three drugs. Since ER is the targeted site of the HCV replication and of the HCV proteins maturation, the decrease of the calcium level within ER seems to be able to inhibit the HCV replication and the viral proteins expression.

New methodological approaches for HCC treatment

1) Research team J. Faivre - Gene transfer : A novel therapeutic approach to non-thyroid cancers is the transfer of the sodium iodide symporter (NIS) gene into tumour tissues, followed by radiotherapy with 131-Iodine, so that these tissues acquire the ability to take up radioiodine, and then be selectively destroyed. It has been shown by numerous teams that NIS expression was possible in vitro in transduced non-thyroid tumour cells (colon, prostate, breast, lung, myeloma), and that this expression endowed cells with the capacity to take up and concentrate iodine which, ideally, should cause their selective destruction by radioiodine therapy. However, a major limitation to this approach is the shortlived iodine uptake (the biological half-life of iodine being a few hours), with the presence of a massive efflux of radioisotope into the extracellular medium, hence a residence time for iodine which is too short to permit the deposit of a sufficiently high dose of radiation. To understand this statement, we should recall that the effects of 131-Iodine (physical half-life: 8.05 days) are proportional to the effective dose of radiation delivered to the tumour, which in turn depends on the effective half-life of the radioisotope in the tumour and the degree of iodine concentration in the cells. The latter parameter depends in turn upon the rates of uptake and efflux of 131-Iodine. To further our understanding of this efflux phenomenon, we have studied the biodistribution of iodine using repeated quantitative imaging in hepatocellular carcinoma (HCC) models developed in situ which, although restrictive, are closer to human HCC than the xenografted tumour. Rats treated with a chemical carcinogen (diethylnitrosamine, DEN) for two months developed dysplastic nodules and then multifocal HCC, between 1 and 2 months after withdrawal of the carcinogen. After transfer of a recombinant AdCMV-NIS adenovirus, administered via the portal vein, we showed that iodine uptake was marked (40% of the iodine administered in an infected liver, versus 2% in a non-infected liver), and prolonged over time (up to 11 days). After treatment with 131-Iodine, we observed a highly significant inhibition of tumour growth, a complete regression of micro-nodules and a prolongation of the lifespan in all treated animals. It should be noted that the anti-tumour effect achieved in this type of model is particularly significant, given the natural history of HCC in the DEN rat, with development from the start of multifocal tumour nodules, rapid tumour progression and the inevitable death of all animals (Faivre et al. Cancer Research, 2004).

2) Research team Patrizia Paterlini-Brechot-Metastatic cells: following our results based on the prognostic role of the ISET method for patients with liver cancer (Vona et al., 2004), we hypothesized that the specific and sensitive cytopathological identification of the CTC by the ISET method in patients with localized prostate cancer can predict recurrence after prostatectomy. We tested by ISET at diagnosis 109 patients with localized prostate adenocarcinoma undergoing prostatectomy for the number of circulating tumor cells. The early, sensitive and specific identification of CTC allows to identify patients with localized tumor who are at high risk of developing recurrence after prostatectomy. This information is highly relevant in order to address adjuvant treatments selectively to patients with invasive cancer.

Clinical research and therapeutics

We organized the constitution of DNA banks of patients infected with the HCV and the patients having an alcoholic cirrhosis to research of genetic polymorphisms associated to the various type of hepatic disease. Several pharmacological studies and one vaccinotherapy study (HBV) are ongoing to improve hepatitis B and C treatments. We are analyzing the impact of the HCV infection on the lipid metabolism and its relationship with the viremia, antiviral treatments and fibrates. We analyzed the evolutionary profiles of the biliary diseases of the liver and carried out the evaluation of the treatments currently suggested. A project focused on genetic factors intervention which implied the start and/or the severity of the CBP is under development.

1) Group Leader: Marie-Louise 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, we provided evidences for safety 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.

Using mice transgenic for human MHC-class I molecules epitopes derived from HBx and HBSP HBV proteins were identified (collaboration with Y-C Lone, I.P.). Based on these results, a clinical study in chronic HBV-infected patients is on going. Identification of HBV-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).

2) Research team Patrizia Paterlini-Brechot-Diagnostic of genetic diseases by the analysis of calcium concentration and circulating foetal cells-Two new complementary research axes started 1) the exploration of the mitochondrial calcium concentration in the context of the mitochondrial respiratory chain deseases (MCRM); 2) the study of the impact of the genetic analysis of the circulating foetal cells (CFC) on the prenatal diagnosis. 1) Our objective is to evaluate the subcellular calcium concentration of fibroblasts derived from patients with MCRM by using the aequorin calcium probes combined with the various approaches: structural (Organelle structure), functional (calcium signalling and ATP concentration), and genetics (ADNmit and ADNn mutations), to elucidate the real impact of the calcium concentration variation on the mitochondrial pathology development. 2) The role of the diagnosis by using the ISET (Insolation by Size of Epithelial Tumor/Trophoblastic cells) test was shown for women with the high risk to have a child with spinal amiotrophy (SMA). We recently validated the ISET-SMA test by the analysis of 10 CFC per maternal blood sample for 4 among the 16 women of the protocol. The feasibility of the ISET-mucoviscidosis test was proven (submitted article), and will be followed by the clinical validation. The development of the ISET-trisomy 21 test is ongoing.

Keywords: Hepatitis C Virus, Hepatitis B Virus, Calcium, Hepatocellular Carcinoma, Prenatal Diagnosis, circulating fœtal cell, genetic deseases


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