Unit: Nuclear Organization and Oncogenesis - INSERM U.579
Director: Anne DEJEAN
The research of our laboratory is aimed at investigating the molecular and cellular mechanisms underlying cellular transformation in humans. Apart from its clinical implications, the study of these pathological mechanisms will also shed important light on fundamental physiological processes in normal cellular contexts. Our work takes two complementary research paths. The first seeks to establish the genetic basis underlying hepatocarcinogenesis, a particular effort will be devoted to the study of alterations in epigenetic modification profiles, while the second aims to elucidate the role of the PML Nuclear Bodies involved in acute promyelocytic leukemia in the general context of the functional organization of the cell nucleus and its relationship with the chromatin compartment. An effort toward the elucidation of the SUMO modification pathway will be developed to this aim.
Genetics and epigenentics of primary liver cancer (leading scientist: Pascal Pineau)
One of our main research interest focuses on the genetic basis of hepatocellular carcinoma. Our first goal is to identify novel tumor suppressor genes involved in the development of primary liver cancer. To this aim, we searched for allelic deletions affecting tumor DNA through a genome-wide scanning for LOH coupled to a comparative genomic hybridization (CGH) study. Such approaches are 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 were found at chromosomes 8p and 4q ( >50%), these two regions have been studied in details. The study of tumors and tumor derived cell lines using LOH and reduction to homozygosity has pointed to three regions on 8p that likely harbor hepatic tumor suppressor genes. The role of these candidate genes in liver tumorigenesis will be studied by mutational and expression analysis, including tissue arrays'. Putative suppressor genes at 4q will be identified by positional cloning using a novel approach, the CGH array. Moreover, a search for homozygous deletions in a collection of a hundred tumor cell lines, including sixty of hepatobiliary origin, revealed five candidate tumor suppressor genes not previously associated with liver cancer. We will carry out mutational analysis of these genes in different etiological contexts (infection with HBV and HCV, alcohol use, metabolic disorders). The mutational analysis will be complemented by functional studies. In addition, an extensive transcriptome analysis of hepatocellular carcinoma was undertook with the aim of defining the major expression profiles of this heterogenous class of tumors. This should hopefully lead to the establishment of a tumor classification system of significant prognostic and therapeutic value for primary liver cancer. Finally we develop a project aimed at studying alterations (by immunohistochemistry, Western and QRT-PCR) in patterns of histone modification (acetylation, methylation, ubiquitination, sumoylation) in liver cancer. This should hopefully lead to the identification of novel targets based on the use of drugs regulating gene activity through chromatin remodelling and validate the so-called transcriptional therapy' concept.
Functional compartmentalization of the nucleus and SUMO modification (leading scientists : Jacob Seeler, Oliver Bischof)
A major aspect of acute promyelocytic leukemia pathogenesis involves, at the cellular level, the expression of the PML-RARα hybrid protein and, at the cellular level, the retinoic acid (RA)-reversible disaggregation of the PML nuclear bodies (NBs). This latter observation, that provides a striking physical parallel to the therapeutic effect of RA in this leukemia, has also implicated, for the first time, a novel subnuclear organelle' in a human disease. Study of the signals that may regulate PML NB dynamics led us to identify a novel ubiquitin-like post-translational modification system, the SUMO pathway, as playing a major role in NB dynamics. Unlike ubiquitination, this modification does not lead to the degradation of its target proteins but raher plays important roles in their subcellular localization. Our laboratory and others have identified several novel SUMO susbtrates, a number of which are associated with the PML NBs (see Figure). The SUMO pathway uses an E1 activation enzyme (Uba2/Aos1 heterodimer), an E2 conjugation enzyme (Ubc9) and three families of E3 ligases (RanBP2, PIAS and Pc2) that are believed to confer substrate specificity. Demodification is achived by SUMO hydrolases (SENPs), of which seven isoforms exist in mammals. Given the intimate link between sumoylation and NB dynamics, a significant effort is dedicated to the study of this pathway and its role in the general organization of the nucleus both in normal and pathological cells. A parallel research path seeks to elucidate the role of the PML protein, notably in the cellular senescence process.
To dissect the mechanisms implicated in PML-induced senescence, we used the HPV oncoproteins E6 and E7 that target respectively the p53 and Rb tumor suppressor pathways and found that both pathways are activated and necessary. More interestingly we found that E7 inhibits PML function through physical interaction thus identifying PML as a novel target for E7. A collaboration with R. Bernard's lab (NKI, Amsterdam) within the FP6 EEC Intact' is aimed at elucidating the signalling pathways involved in PML-induced senescence. The findings obtained will then be applied to the study of the other PML-associated processes such as apoptosis, cell differentiation an tumor suppression.
To address the biological function of the SUMO modification pathway in mammals, we inactivated the murine Ubc9 gene that encodes the unique SUMO E2 conjugating enzyme. Disruption of the SUMO pathway causes early embryonic death as well as major defects in protein localization and nuclear architecture (Nacerddine et al., manuscript in preparation). In parallel, we performed a conditional KO of the Ubc9 gene to study the consequences of a lack of sumoylation in a given tissue (myeloid cells, skin, brain ), at a given time point (Cre-ER/tamoxifen system) as well at the cellular level through infection of floxed fibroblasts with a Cre-adeno virus. This study should help in clarifying the role of the SUMO pathway in major fundamental cellular processes. In a study performed in collaboration with the lab of B. Arcangioli (Institut Pasteur), we have found that, in S. pombe, the Pli1p protein functions as a SUMO E3 ligase. Cells deleted for Pli1p exhibit enhanced minichromosome loss, a particular sensitivity to the TBZ drug and consistent telomere length increase, suggesting a role of Pli1p, and by implication of the SUMO pathway, in protecting heterochromatic repeated sequences (i.e. centromeres and telomeres) from illegitimate recombination. These data open the way to identifying the SUMO targets involved as well as the mechanisms by which sumoylation controls their activity.
Keywords: Oncogenesis, PML nuclear bodies, SUMO, cellular senescence, chromatin, epigenetic, leukemia, liver cancer