Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome

Abstract

Background: Gliomas are the most common human brain tumors. Glioblastoma, also known as glioblastoma multiform (GBM), is the most aggressive, malignant, and lethal glioma. The investigation of prognostic and diagnostic molecular biomarkers in glioma patients to provide direction on clinical practice is urgent. Recent studies demonstrated that abnormal DNA methylation states play a key role in the pathogenesis of this kind of tumor. In this study, we want to identify a novel biomarker related to glioma initiation and find the role of the glioma-related gene.

Methods: We performed a methylation-specific microarray on the promoter region to identify methylation gene(s) that may affect outcome of GBM patients. Normal and GBM tissues were collected from Tiantan Hospital. Genomic DNA was extracted from these tissues and analyzed with a DNA promoter methylation microarray. Testis derived transcript (TES) protein expression was analyzed by immunohistochemistry in paraffin-embedded patient tissues. Western blotting was used to detect TES protein expression in the GBM cell line U251 with or without 5-aza-dC treatment. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V/PI staining.

Results: We found that the TES promoter was hypermethylated in GBM compared to normal brain tissues under DNA promoter methylation microarray analysis. The GBM patients with TES hypermethylation had a short overall survival (P <0.05, log-rank test). Among GBM samples, reduced TES protein level was detected in 33 (89.2%) of 37 tumor tissues by immunohistochemical staining. Down regulation of TES was also correlated with worse patient outcome (P <0.05, log-rank test). Treatment on the GBM cell line U251 with 5-aza-dC can greatly increase TES expression, confirming the hypermethylation of TES promoter in GBM. Up-regulation of TES prompts U251 apoptosis significantly. This study demonstrated that both TES promoter hypermethylation and down-regulated protein expression significantly correlated with worse patient outcome. Treatment on the GBM cell line (U251) with 5-aza-dC can highly release TES expression resulting in significant apoptosis in these cells.

Conclusions: Our findings suggest that the TES gene is a novel tumor suppressor gene and might represent a valuable prognostic marker for glioblastoma, indicating a potential target for future GBM therapy.

Figure 1

TES promoter methylation status in glioblastoma (GBM, n = 42) and normal (n = 8) samples analyzed by DNA promoter methylation microarray. (A) Comparison of TES promoter methylation status in GBM and normal samples by microarray analysis (up) and independent t-test analysis (down). (B) Western blot of TES in U251 cell line before and after 5-aza-2-deoxycytidine treatment. Anti-tublin was used as a protein loading control.

Figure 2

TES expression in GBM. (A) Immunohistochemical staining of TES protein expression in normal tissue (left) and a GBM specimen (right) (magnification × 400). (B) Collective summary of TES staining intensity from all samples.

Figure 3

Cell apoptosis induced by treatment of 5-aza-2-deoxycytidine. The percentage of apoptotic cells was calculated by flow cytometry using the Annexin-V/PI double-staining assay. Control vs. 5AZA (*P <0.05). (A) (Control) Apoptosis of U251 cells in the group without 5-aza-dC treatment (8.75); (B) (5AZA) Apoptosis of U251 cells in the 5 mL 5-aza-2-dC group (15.25).

Figure 4

Survial analysis according to TES expression and methylation status. Kaplan-Meier survival estimates of overall survival according to the TES methylation status in 42 GBM patients (A) and according to the TES expression level in GBM patients (B).