Epigenetic aberrations and therapeutic implications in gliomas

Abstract

Almost all cancer cells have multiple epigenetic abnormalities, which combine with genetic changes to affect many cellular processes, including cell proliferation and invasion, by silencing tumor-suppressor genes. In this review, we focus on the epigenetic mechanisms of DNA hypomethylation and CpG island hypermethylation in gliomas. Aberrant hypermethylation in promoter CpG islands has been recognized as a key mechanism involved in the silencing of cancer-associated genes and occurs at genes with diverse functions related to tumorigenesis and tumor progression. Such promoter hypermethylation can modulate the sensitivity of glioblastomas to drugs and radiotherapy. As an example, the methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter is a specific predictive biomarker of tumor responsiveness to chemotherapy with alkylating agents. Further, we reviewed reports on pyrosequencing - a simple technique for the accurate and quantitative analysis of DNA methylation. We believe that the quantification of MGMT methylation by pyrosequencing might enable the selection of patients who are most likely to benefit from chemotherapy. Finally, we also evaluated the potential of de novo NY-ESO-1, the most immunogenic cancer/testis antigen (CTA) discovered thus far, as an immunotherapy target. The use of potent epigenetics-based therapy for cancer cells might restore the abnormally regulated epigenomes to a more normal state through epigenetic reprogramming. Thus, epigenetic therapy may be a promising and potent treatment for human neoplasia.

Figure 1

Pyrosequencing technology. A sequencing primer is hybridized to a single‐stranded, PCR‐amplified DNA template and incubated with a DNA polymerase. Each event of incorporation of a dNTP is accompanied by the release of pyrophosphate (PPi) in a quantity equimolar to the amount of nucleotide incorporated. This PPi is quantitatively converted to ATP by ATP sulfurylase in the presence of adenosine 5′‐phosphosulfate. The ATP thus obtained drives the luciferase‐mediated conversion of luciferin to oxyluciferin, thus generating visible light in amounts proportional to the amount of ATP generated. The light produced in this luciferase‐catalyzed reaction is detected by a charge‐coupled device (CCD) camera and seen as a peak in a pyrogram. Apyrase – a nucleotide‐degrading enzyme – continuously degrades unincorporated dNTPs and excess ATP, during synthesis of the complementary DNA. Gray columns indicate quantified CpG sites.

Figure 2

Methylation status and mRNA expression in glioma cell lines and normal brain cells. Normal brain (NB) cells and the U251nu/nu glioma cells expressed MGMT, as determined by RT‐PCR (upper panel), and contained an unmethylated MGMT promoter, as determined by methylation‐specific PCR (MSP) (middle panel). Pyrosequencing revealed that the methylation levels in NB and U251nu/nu cells were 2% and 3%, respectively (lower panel). MGMT was not expressed in three other glioma cell lines (i.e. AO2, SKMG1, and U251SP), but the level of promoter methylation was very high in these cells (70–85%).

Figure 3

Intratumoral heterogeneity of MGMT expression and pyrosequencing. One part of the tumor showed high MGMT expression with 18% methylation in the nucleus (a,b), as determined by pyrosequencing, whereas a different lesion in the same tumor showed low MGMT expression with 53% methylation (c,d). However, with methylation‐specific PCR (MSP), both these areas were found to be unmethylated. (a,c) Hematoxylin & Eosin stained sections of two different lesions in a single tumor. (b,d) MGMT immunohistochemical stained sections.

Figure 4

A schematic representation of the proposed mechanism underlying histone modifications in the NY‐ESO‐1 promoter. (a) Methylation of lysine 9, moderate methylation of lysine 4, and hypermethylation of the CpG islands caused the formation of a folded chromatin structure, leading to NY‐ESO‐1 gene silencing. (b) 5‐aza‐2′‐deoxycytidine (DAC) dramatically decreases lysine 9 methylation, demethylates the CpG sites, and unfolds the chromatin, thus reactivating gene expression. (c) VPA increases lysine 9 acetylation but has no effect on the methylation of lysine 9 or lysine 4 and does not reverse transcriptional silencing. (d) The combination of DAC and VPA decreases lysine 9 methylation and increases lysine 9 acetylation, but does not affect lysine 4 methylation. This combination unfolds the chromatin to a considerable extent and reactivates gene expression with high efficiency.