Epigenetische mechanismen zijn essentieel voor de aanmaak en het behoud van adequate genexpressiepatronen in menselijke weefsels. Het is inmiddels duidelijk dat deze mechanismen bij vele kankers grondig verstoord zijn. De oorzaken en gevolgen van epigenetische veranderingen in tumorcellen moeten nog opgehelderd worden. Dit zou kunnen leiden tot de ontwikkeling van nieuwe epigenetische kankertherapieën.
Het behoud van genexpressieprogramma’s is noodzakelijk om de goede werking van de verschillende celtypes in ons lichaam te garanderen. Daarvoor hebben de cellen “epigenetische” regelmechanismen ontwikkeld, gebaseerd op het aanbrengen van chemische modificaties aan bepaalde genen. Hierbij wordt DNA-methylatie gebruikt om bepaalde genen inactief te maken.
Bij vele kankers is de verspreiding van methylatiemerkers sterk gewijzigd en er is bewijs dat dit bijdraagt tot de progressie van tumoren. De oorzaken en gevolgen van deze epigenetische verstoring zijn echter nog onduidelijk.
Ons team ontdekte dat veranderingen in DNA-methylatie vaak een bepaalde groep genen treft, die normaal uitsluitend tot expressie worden gebracht in kiembaancellen (de cellen die aan de oorsprong liggen van eicellen bij vrouwen en spermatozoïden bij mannen). Deze genen verliezen methylatie in vele tumoren en worden daardoor abnormaal actief. Op basis van hun bijzondere expressieprofiel werden dergelijke genen “kanker-kiemlijn” genoemd.
Ons werk is gericht op de identificatie van de mechanismen die leiden tot een verlies van methylatie en een activatie van kanker-kiemlijngenen in tumoren. Bovendien willen we begrijpen hoe de activatie van deze genen bijdraagt tot de tumorontwikkeling. Het uiteindelijke doel is een manier te vinden om de cellulaire dysfuncties te corrigeren die het gevolg zijn van DNA-methylatieverlies in tumoren.
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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EPIGENETISCHE VERANDERINGEN IN TUMOREN
