Although distinct DNA hypermethylation patterns have been recognized for some time in human cancer, only recently have mechanisms emerged that might explain the targeting of specific groups of genes for aberrant methylation. The clear differentiation of secondary from primary GBM by methylation profiles confirmed our a priori
hypothesis based on studies of individual genes and indicates the potential clinical value of methylation profiles in distinguishing the two types of GBM. A recent study proposed using EMP3
methylation alone as a marker to differentiate secondary from primary GBM6
(Supplementary Material, Table S3
). In addition to EMP3
, the present study identified 7 other genes differentially methylated in secondary and primary GBMs. Combining our results with those of earlier reports7,8
could provide a highly discriminating panel for classifying these epigenetic subtypes of GBM (Supplementary Material, Table S3
). In one case, both low- and high-grade glioma tissues were available, and we found that the class 1 pattern was retained in the high-grade lesion. Another potential application of such an approach is suggested by our results in primary GBM in which 12% of cases displayed a methylation profile highly similar to known secondary GBMs that progressed from lower-grade tumors. Younger aged primary GBM cases were overrepresented in this group, and we speculate that they may represent a distinct epigenetic subtype that arises from clinically unrecognized lower-grade lesions. Epidemiologic studies have suggested the existence of long-latency, high-grade glioma based on the significantly higher incidence of seizure disorders preceding GBM diagnosis by 8–11 years.59
Molecular features such as mutations of TP53 and amplification of EGFR suggest that younger aged primary GBM comprises different genetic subtypes,2
but the distinctions noted here based on methylation profile provide an even more robust segregation.
Our 12-gene panel contained two genes, HOXA9 and SLIT2, that are recognized as PRC targets in embryonic stem cells, whereas the others are not known targets. A significant result of our study is that glioma tumor types were segregated by DNA methylation profile according to the PRC target status of hypermethylated genes. The clustering of gene methylation in 37% of primary GBMs was driven by the hypermethylation of PRC targets HOXA9 and SLIT2, whereas the coordinate methylation of 8 non-PRC targets characterized secondary GBMs and astrocytoma stage II, anaplastic astrocytoma III, oligodendroglioma II, and oligoastroglioma II.
An additional aim of this study was to characterize patterns of aberrant DNA methylation in glioma and explore the potential roles of PRC and PI3K/Akt in these events by assessing the overexpression of EZH2
mRNA, respectively. We assessed EZH2
in primary glioma of different grades and found that EZH2
was overexpressed in most astrocytic and oligodendroglial tumors, but even more highly expressed in the higher-grade GBM tumors. Thus, our results follow the trend seen in other cancer types of increasing PRC pathway activation and aggressive tumor characteristics. We assessed three different qRT-PCR methods for EZH2
mRNA expression and found that both the short and long EZH2
isoforms showed very similar associations with methylation class (data not shown).The long EZH2
isoform was the predominant form in glioma, as has been reported previously in normal tissues.60
Previous studies postulated that gene amplification may be a mechanism for the induction of EZH2
in non-glial tumors.32
We found no evidence of increased EZH2
gene copy number in our study, which makes amplification an unlikely mechanism for EZH2
overexpression in glioma.
, like EZH2
, was highly overexpressed in GBM but in contrast to EZH2
was not detectable among lower-grade astrocytic and oligodendroglial tumors (with the exception of two grade III astrocytomas). However, IGFBP2
levels varied considerably within primary GBMs, and lower IGFBP2
levels were observed among tumors with a distinct methylation profile and earlier age at onset. Primary GBMs without IGFBP2
overexpression exhibited a phenotype defined by methylation of 8 non-PRC targets. In contrast, PRC targets HOXA9
were methylated in GBMs containing both IGFBP2
overexpression. Although our observation that IGFBP2
expression is associated with DNA methylation phenotype is novel, our study does not provide evidence that this relationship is causal with respect to PI3K/Akt. Nonetheless, any connection between IGFBP2
expression and PI3K/Akt pathway activation rests on the validity of IGFBP2
as a marker of this pathway. Several studies support this assumption42–45
; however, there may be other phenomena such as tissue hypoxia61
that could be associated with both IGFBP2
expression and DNA methylation. Furthermore, our hypothesis that Akt activation could indirectly affect DNA methylation by phosphorylating EZH241
must be regarded as speculative, although the proposed pivotal role of H3K27me3 and EZH2 in controlling methylation provides a strong rationale for future studies to explore DNA methylation in cells with and without Akt activation. A recent study62
questioned the proposal that H3K27me3 is universally linked with DNA hypermethylation of PRC gene targets. The convergence of H3K27me3 with DNA methylation mechanisms was posited to be dependent on cell type.62
Our study provides a clue that PRC targeting for methylation may be modified by the activation status of the PI3K/Akt pathway. Supporting this idea further is our observation that a GBM progressing from an earlier astrocytoma II retained its class I methylation and low PRC target methylation and that both first tumor and second tumor were negative for IGFBP2 expression.
The finding that a pattern of gene hypermethylation correlated with LINE1 methylation indicates that a generalized mechanism operates in some subtypes of glioma, acting on specific genes associated with CpG sites as well as nongenic repetitive DNA regions. In addition, this evidence of epigenetic dysregulation was strongly linked with patient survival and low expression of the IGFBP2 gene. Taken together, these studies suggest a convergence of pathways that may offer new approaches for improving patient prognostication and therapeutic targets.
Our study has some notable strengths and limitations. One strength is our application of a quantitative MS-PCR method that has been shown to be superior to conventional MS-PCR for detecting hypermethylation patterns in human tumors. We also applied a novel unsupervised clustering methodology to create methylation classes that has advantages over conventional methods.58
A limitation of our work is the relatively small number of genes examined, which prevents us from knowing how generalizable our findings might be regarding PRC target and non–target gene methylation in different types of glioma. Future studies that expand the set of target genes interrogated will further inform PRC target versus nontarget methylation in these diseases. Our findings provide new insights into the associations of DNA hypermethylation profiles with 2 expression biomarkers that have strong connections to tumor progression and cancer survival.
Conflict of interest statement. None declared.