Les mécanismes épigénétiques sont essentiels pour l’établissement et le maintien des programmes d’expression géniques dans les tissus. Il est maintenant évident que ces mécanismes sont profondément perturbés dans la majorité des cancers. Les causes et les conséquences des altérations épigénétiques dans les tumeurs doivent être investiguées. Ceci pourrait aboutir au développement de nouvelles thérapies épigénétiques du cancer.
Le maintien des programmes d’expression génique est essentiel pour assurer le bon fonctionnement de l’ensemble des cellules qui composent l’organisme. Les cellules disposent à cette fin de mécanismes de régulation dits “épigénétiques“, basés sur l’ajout de modifications chimiques au niveau des gènes. Parmi celles-ci, la méthylation de l’ADN a pour rôle d’assurer l’inactivation de certains gènes.
Dans de nombreuses tumeurs, la répartition des marques de méthylation est profondément altérée, et l’on peut supposer que cela contribue de façon importante au développement tumoral. Cependant, les causes et les conséquences de ce dérèglement épigénétique dans les tumeurs demeurent largement incomprises.
Notre équipe a découvert que les altérations de méthylation dans les tumeurs touchent fréquemment un groupe particulier de gènes. Il s’agit de gènes qui sont normalement exprimés exclusivement dans les cellules germinales (les cellules qui produisent les ovocytes chez la femme et les spermatozoïdes chez l’homme). Ces gènes perdent leur méthylation dans de nombreuses tumeurs et y sont par conséquent activés de manière aberrante. En raison de leur profil d’expression, ces gènes ont été appelés gènes "cancer-lignée germinale" (CG).
Nos travaux ont pour but d’identifier les mécanismes qui conduisent à la perte de méthylation et à l’activation des gènes CG dans les tumeurs. Par ailleurs, nous cherchons à comprendre comment l’activation de ces gènes peut contribuer à la progression tumorale. L’objectif à terme est de trouver un moyen de corriger les disfonctionnements cellulaires induits par la perte de méthylation dans les tumeurs.
DNA hypomethylation and aberrant gene activation in cancer
Genomic DNA in multiple species is modified by the addition of a methyl group to cytosines in CpG dinucleotides. This heritable epigenetic modification is associated with transcriptional repression. Cell-type specific DNA methylation patterns are established during embryonic development, and are usually maintained in adult somatic cells.
DNA methylation patterns often become altered in cancer cells. Alterations include hypermethylation of selected promoters, leading to silencing of critical genes such as tumor suppressor genes, and hypomethylation of numerous other DNA sequences. We have shown that genome hypomethylation in tumors results in the activation of a group of germline-specific genes, which use primarily DNA methylation for repression in somatic tissues (De Smet 1999). These genes, which were originally discovered because their activation in tumors leads to the expression of tumor-specific antigens, were named cancer-germline (CG) genes. To date, ~50 CG genes or gene families have been identified. Several of these were isolated in our group (Martelange 2000, Loriot, 2003).
The process leading to hypomethylation of DNA sequences in tumors remains obscure. We undertook to address this issue by using MAGEA1, the founding member of the CG group of genes, as a model. Detailed methylation analyses of the MAGEA1 genomic locus in expressing tumor cells, revealed preferential hypomethylation within the 5’ region of the gene. Furthermore, transfection experiments with in vitro methylated MAGEA1 constructs, indicated that this site-specific hypomethylation relies on a historical event of DNA demethylation, and on the presence of appropriate transcription factors to protect the region against subsequent remethylation (De Smet & Loriot 2010). The factors that are responsible for the initial DNA demethylation process and for maintaining CG gene promoters unmethylated remain to be identified.
Histone modifications associated with CG gene demethylation in tumors
Histone modifications have been shown in some cases to dictate DNA methylation states, for instance by regulating access of DNA methyltransferases. We therefore searched to determine if MAGEA1 demethylation and activation in tumor cells is associated with changes in histone marks. Chromatin immunoprecipitation experiments revealed that DNA demethylation and transcriptional activation of MAGEA1 is accompanied by increases in histone H3 acetylation (H3ac) and H3 lysine 4 methylation (H3K4me), and by a decrease in H3 lysine 9 methylation (H3K9me). However, our experiments demonstrate that changes at the histone level within the MAGEA1 promoter are a consequence, not a cause, of DNA demethylation. Consistently, epigenetic drugs that target histone modifications were unable to induce DNA demethylation and stable activation of the MAGEA1 gene. Altogether, these observations confirm that DNA methylation has a dominant role in the epigenetic hierarchy that governs MAGEA1 silencing (Cannuyer 2013).
DNA hypomethylation and activation of CG-type miRNAs in tumors
The role of DNA hypomethylation and CG gene activation on tumor development is only partially understood (De Smet & Loriot 2013). To further explore the impact of DNA hypomethylation on tumorigenesis, we decided to find out if this epigenetic alteration also leads to the activation of CG-type microRNAs (miRNAs). It has indeed become clear that this type of small non-coding RNAs exerts important regulatory functions, by controlling the expression of targeted protein-coding genes at the post-transcriptional level. Dysregulated expression of miRNAs is a hallmark of many cancers, where it appears to contribute to several important steps of tumor development. To identify CG-type miRNAs that would become activated in hypomethylated tumors, we initially conducted an in silico selection of miRNAs displaying specific expression in the germline, and subsequently determined the miRNAs that are susceptible to induction upon treatment with a DNA demethylating agent. This led to the identification of several CG-type miRNAs, which we found to be aberrantly activated in a significant proportion of tumors. Studies aiming at understanding the cellular functions of these miRNAs and their role in tumor development are currently being pursued.
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ALTERATIONS EPIGENETIQUES DANS LES TUMEURS
