Differential Epigenetic Inactivation of Genes in Gliomas

Normal epigenetic modifications such as DNA methylation or histone modifications regulate normal cell differentiation while abnormal epigenetic changes could lead to oncogenic transformation of normal cells. Aberrant or altered DNA methylation cause genomic stability, which is often linked to various pathologies including cancer. Specifically, in glioma epigenetic dysfunctions are often shown to drive oncogenic transformation of low grade glioma to high-grade glioblastoma multiforme (GBM). While few epigenetic events are linked to glioma transformation, more transformation events remain to be identified and validated as viable targets for reversing the epigenetic gene silencing. Reversing the epigenetic silencing or resetting these genes hold a promise in preventing or arresting the progression of low grade glioma to GBM. We treated glioma cells with demethylating agent 5-aza (DNA methyltransferase inhibitor) and Trichostatin A (TSA, Histone deacetylase inhibitor) and whole genome expression array was performed to identify genes that were reactivated by treatment. Array results were confirmed by RT-PCR analysis using glioma cells treated with 5-aza and TSA. Genes were filtered by the following criteria: 2-fold reactivated by treatment as determined by microarray; CpG island in their promoter region; reactivated by treatment in majority of the glioma cell lines, as determined by RT-PCR. Methylation specific PCR (MSP) was carried out to determine methylation status of the promoter region using low grade and high-grade glioma cell lines.

Our results indicate that there is a significant difference in the expression of genes between low and high-grade glioma cells, when treated with 5-aza plus TSA. Induction of CLDN6, NDN, OSMR, ADFP, CDCP1, ANGPT2, ZFP42, BTG4 and MYEOV was seen in most of the glioma cells treated with 5-aza and TSA. MSP-DNA methylation analysis revealed that ADFP, CDCP1 and ZFP42 that were uniquely silenced in high-grade glioma cells lines were reactivated after 5-AZA treatment. This result suggests that studying the methylation status of ADFP, CDCP1 and ZFP42 in brain tumor biopsies may indicate the potential aggressive nature of glioma that helps doctors in accurate and diagnosis and clinical treatment decisions .

Introduction

Gliomas are the most common primary tumors that arise within the central nervous system in adults accounting for 78% of malignant brain tumors. In recent years increasing use of genetic analysis in primary tumors resulted in identification of molecular events and pathways involved in the etiology of brain tumors. However, our understanding of molecular basis of most brain tumor cases remains poor. DNA methylation plays an important role in various cellular functions such as transcriptional silencing, X-chromosome inactivation, genomic imprinting, and genomic stability. Aberrant or altered DNA methylation is linked to various pathologies, including cancer [1]. Tumor cells exhibit global hypomethylation of the genome accompanied by region-specific hypermethylation events. Global hypomethylation occurs mainly in the repetitive sequences leading to genomic instability and tumor formation [2]. Aberrant hypermethylation occurs at CpG islands found in the promoter region of genes and is usually associated with the transcriptional silencing of that gene [3]. Another form of epigenetic gene silencing is the covalent modification of histone proteins. These are post-translational modifications that occur at the amino-terminal tail and include acetylation, methylation, phosphorylation, and ubiquitination. These forms of epigenetic modifications are closely linked to each other. Recent studies suggest that DNA methylation might be dependent on histone modifications and acts to preserve the silenced state rather than initiate silencing [3]. Increasing evidence suggests that epigenetic modifications, in addition to genetic changes, play an important role carcinogenesis [4-6]. DNA methylation changes, particularly CpG island hypermethylation are frequent, early, and common events (as common as mutations) in many types of cancers leading to the inactivation of tumor suppressor genes [7] and potentially aiding the transformation of low grade tumors to higher grades.

Several genetic changes have been identified in astrocytic gliomas and glioblastomas involving heterozygous deletion of 19q13, inactivation/deletion of tumor suppressor genes namely p16INK4A [8], p14ARF [9], RB1 [10], PTEN and p53 gene [11] and amplification of EGF receptor gene (EGFR) [12]. Investigations of the role of epigenetics in glioma pathogenesis revealed several of these genes to be epigenetically silenced by promoter CpG island hypermethylation, e.g., cell cycle regulatory proteins RB1 [13], p16INK4A [14-17], myelin related gene EMP3 [1], DNA repair protein MGMT and matrix metalloproteinases inhibitor TIMP3 [2]. Comprehensive whole-genome microarray studies using inhibitors of epigenetic modification identified several genes like CST6 (putative metastatic suppressor), BIK (apoptosis inducer), TSPYL5 (unknown function), BEX1, and BEX2 (uncharacterized function) as putative tumor suppressors that are frequently methylated in primary gliomas [18,19]. Another genome-wide study using restriction landmark genomic scanning identified as many as 1500 CpG islands to be aberrantly methylated in low grade gliomas [16], highlighting a role for DNA methylation in gliomagenesis.

DNA methylation status not only serves as a diagnostic or prognostic marker but is a potential therapeutic target because of the reversible nature of methylation. Since the primary DNA sequence of epigenetically modified genes remains intact, it is possible to reactivate genes using inhibitors of DNA methylation or histone modifications [20,21]. Clinical trials are being carried out using DNA methylation and histone deacetylase inhibitors to reactivate silenced genes in cancers. Some of the DNA methyltransferases inhibitors, 5-azacytidine (Vidaza), and 5-aza-2’-deoxycytidine (Decitabine), have been used with reasonable success in the treatment of hematologic malignancies [22]. Combination of HDAC inhibitors with DNA methyltransferases inhibitors seems to have a synergistic effect in inducing expression of silenced genes [6]. We set out to study whether there are unique hypomethylation events during the genotypic transformation of low to high-grade gliomas. A better understanding of the molecular basis of glioma progression, particularly identifying tumor suppressor genes that trigger glioma transformation to an aggressive high-grade will provide an opportunity to design novel therapeutic strategies to control an otherwise hard-to-treat brain tumor