Altered Expression of MGMT in High-Grade Gliomas Results from the Combined Effect of Epigenetic and Genetic Aberrations

João Ramalho-Carvalho,Mário Resende,Inês Graça,Joaquina Maurício,Mrinalini Honavar,Carmen Jerónimo,Joana Savva-Bordalo,Rui Henrique,João Diogo Barros-Silva,Patrícia Rocha,Manuel R Teixeira,Malini Pires,Susana Lisboa,Javier S Castresana

doi:10.1371/journal.pone.0058206

Abstract

MGMT downregulation in high-grade gliomas (HGG) has been mostly attributed to aberrant promoter methylation and is associated with increased sensitivity to alkylating agent-based chemotherapy. However, HGG harboring 10q deletions also benefit from treatment with alkylating agents. Because the MGMT gene is mapped at 10q26, we hypothesized that both epigenetic and genetic alterations might affect its expression and predict response to chemotherapy. To test this hypothesis, promoter methylation and mRNA levels of MGMT were determined by quantitative methylation-specific PCR (qMSP) or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) and quantitative RT-PCR, respectively, in a retrospective series of 61 HGG. MGMT/chromosome 10 copy number variations were determined by FISH or MS-MLPA analysis. Molecular findings were correlated with clinical parameters to assess their predictive value. Overall, MGMT methylation ratios assessed by qMSP and MS-MLPA were inversely correlated with mRNA expression levels (best coefficient value obtained with MS-MLPA). By FISH analysis in 68.3% of the cases there was loss of 10q26.1 and in 15% of the cases polysomy was demonstrated; the latter displayed the highest levels of transcript. When genetic and epigenetic data were combined, cases with MGMT promoter methylation and MGMT loss depicted the lowest transcript levels, although an impact in response to alkylating agent chemotherapy was not apparent. Cooperation between epigenetic (promoter methylation) and genetic (monosomy, locus deletion) changes affecting MGMT in HGG is required for effective MGMT silencing. Hence, evaluation of copy number alterations might add relevant prognostic and predictive information concerning response to alkylating agent-based chemotherapy.

Highlights

O6-methylguanine-deparaffinized and rehydrated using xylene (DNA) methyltransferase (MGMT) is a DNA repair enzyme that catalyzes the transfer of mutagenic and cytotoxic adducts from O6-guanine in DNA [1]
Therapeutic response to alkylating agents, such as temozolomide, is variable, but tumors with methylguanine-DNA methyltransferase (MGMT) promoter methylation have been found to have an increased response rate [31]. Because this epigenetic alteration is associated with gene silencing, we hypothesized that alternative genetic mechanisms, including chromosome 10 monosomy and MGMT locus deletion, might lead to MGMT downregulation and predict improved response to chemotherapy
We attempted to identify the CpG sites that could serve as surrogate markers and determine which method is more suitable for that purpose

Summary

Introduction

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that catalyzes the transfer of mutagenic and cytotoxic adducts from O6-guanine in DNA [1]. Following the incorporation of the alkyl group by MGMT, the enzyme is irreversibly inactivated and targeted for degradation, requiring de novo protein synthesis to sustain the enzyme activity. Through removal of alkyl groups from guanines, MGMT safeguards the cells against mutagenesis and malignant transformation [3]. MGMT deregulation may play an important role in carcinogenesis because tumors frequently display lower expression levels than their tissues of origin [2]. MGMT downregulation is associated with increased risk of tumorigenesis and with improved sensitivity to alkylating chemotherapeutics [3]

Methods

Results

Conclusion