TERT Promoter Methylation in Glioblastoma and its Paradoxical Association with Upregulated Gene Expression

To evaluate telomerase reverse transcriptase (TERT) promoter hyper-methylation as a potential causative epigenetic alteration of elevated mRNA expression in glioblastoma (GBM).

The hyper-methylation and mRNA expression were evaluated by methylation-sensitive high-resolution melting analysis (MS-HRM) and qRT-PCR, respectively. Bioinformatic analysis of The Cancer Genome Atlas (TCGA) database was also performed to obtain similar data. The mechanistic link between the hyper-methylation and mRNA expression was analyzed in vitro in U87MG and LN228 GBM cell lines. A cross-sectional study was conducted. A total of 50 cases of adult hemispheric GBMs (IDH wild type), and five cases of high-grade astrocytoma (IDH mutant, grade 4) (HGA) were included for the study. Eight normal brains (NB) from the autopsy of nonneurological diseases were used as control.

The TERT promoter methylation was significantly higher in GBM than NB (Median -19.5% vs 6.8%; P value 0.003). Considering 10% methylation as cutoff, hyper-methylation was detected in 65% of GBM, 60% of HGA, and only in a single case of normal brain. There was a significant positive correlation between the methylation level and mRNA expression (correlation coefficient 0.40; P value 0.002) in the study cohort. In the TCGA database, the methylation status of the probes covering the selected promoter region showed a similar association with gene expression. In vitro treatment of the GBM cell lines with demethylase agent Azacitidine led to a significant reduction in the mRNA level and cell proliferation. Hyper-methylation or mRNA expression did not correlate with overall survival.

The present study is the first to assess the TERT promoter methylation status in GBM in an Indian cohort. TERT hyper-methylation is crucially implicated in the pathobiology of GBM by enhancing the gene expression. This epigenetic alteration may be an important therapeutic target, especially with the use of a specific demethylating agent.