Unit: Nuclear Organization and Oncogenesis

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 of primary liver cancer

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 acute promyelocytic leukemia

A major aspect of Acute Promyelocytic Leukemia (APL) pathogenesis involves (i) a chromosomal translocation affecting the gene encoding the retinoic acid receptor α (RARα ) and the PML gene (giving rise to the fusion PML-RARα 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 retinoids in this type of leukemia, implies a new type of nuclear 'organelle' in a human disease. While it is generally accepted that the eukaryotic cell nucleus is highly organized, its functional compartmentalization still remains poorly understood. Our work seeks to identify and characterize the signals that regulate the dynamics, and hence function, of the PML NBs both in normal and pathological contexts.

We will mainly concentrate on the relationship between these particular substructures and the chromatin compartment for which we recently established a link. Our goal is to clarify the role of the PML NBs in regulating chromatin dynamics by biochemical and localization studies with the aim of integrating transcriptional regulatory mechanisms into the framework of subnuclear architecture. Moreover we have shown that PML plays a major role in inducing premature cellular senescence by activation of the two p53 and RB pathways. Molecular and cellular mechanisms involved in this process will be analyzed through the identification of interacting proteins and target genes whose expression is regulated by PML during senescence. Finally, we showed that 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. Given the tight link between sumoylation and NB dynamics, a significant effort will be dedicated to the study of this new type of post-translational modification and its role in the general organization of the nucleus both in normal and leukemic cells.

Photo legend :

PML Nuclear Bodies and SUMO Modification

Keywords: Oncogenesis, PML nuclear bodies, SUMO, cellular senescence, chromatin, epigenetic, leukemia, liver cancer

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