Recent evidence shows that mitochondrial DNA (mtDNA) content is responsible for radiation resistance in various cancers, but not evaluated in glioblastoma(GBM).Hence,we studied the role of mtDNA content in GBM pathogenesis and treatment response.
Archived FFPE tissues of newly diagnosed GBM(n=130), recurrent GBM (n=32 pairs) and non-neoplastic control brain(n=30) with available clinical details were utilized for the study. Immunohistochemistry, Sanger’s sequencing, methylation specific PCR and fluorescent in-situ hybridization were used to study IDH, ATRX and TERT promoter mutations, MGMT promoter methylation and EGFR amplification. mtDNA content was analyzed using quantitative real time PCR (relative quantification) and was calculated using the formula 2 - ΔΔCTx100. Malignant glioma cell lines U87 and LN229 were used to study the difference in mtDNA content following radiation exposure.LN229 cell line was subjected to mtDNA depletion by incubation with ethidium bromide for 4 days.The parent and mtDNA depleted LN229 cell lines were then assessed for sensitivity to radiation and Temozolomide(TMZ) therapy using MTT assay.
mtDNA content was lower than control brain tissue(mean mtDNA content 19.6) in all cases studied,with significantly lower content in older patients(p=0.04).Lower mean mtDNA content was seen in IDH wild type, MGMT unmethylated and EGFR amplified tumors when compared to their counterparts(p=1.06).Survival analysis using Cox regression showed that lower mtDNA copy number is associated with higher risk and hence poorer prognosis(p=0.047).Paired tumor analysis was performed in 32 patients with recurrence of whom only 19 had received radiation therapy (RT).The mean mtDNA content was higher at recurrence as compared to the primary tumor in those who received RT(mean at diagnosis 20.1; mean at recurrence 49.3, p=0.02) while no significant difference was observed in those who did not receive RT. U87 and LN229 cell lines exposed to radiation (0, 2, 4 and 6Gy) showed an increase of 8% and 25% in mtDNA content, respectively, after 6Gy radiation exposure.LN229 parent cells showed a radiation dose dependent decline in cell viability(86% at 2Gy, 68% at 4Gy and 50% at 6Gy).The mtDNA depleted LN229 cells were 100% viable at 0, 2 and 4 Gy and 82% viable at 6Gy.The IC50 of TMZ in parent LN229 cells was 69.3 µM while in the mtDNA depleted cells,it was 100.8 µM. Conclusion:Our study shows that lower mtDNA content is associated with poorer survival in GBM.RT increased the mtDNA content in both patient samples and malignant glioma cell lines.mtDNA depleted LN229 lines are more radio-chemo resistant than parent LN229 lines,thus showing that lower mtDNA content leads to treatment resistance.Hence,we establish the significant role of mtDNA content in the pathogenesis and treatment resistance of GBM.