Abstract
BackgroundGlioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity.MethodsMGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.ResultsWe show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels.ConclusionsThese in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting.
Highlights
Glioblastoma (GBM), a WHO grade IV astrocytoma, is the most common [1] and most aggressive type of primary brain tumour
TMZ does not affect steady state levels of methylguanine-DNA methyltransferase (MGMT) protein or protein synthesis in T98G cells TMZ treatment causes damage to DNA, creating O6methylguanine lesions, which are repaired by MGMT [17,18]
As TMZ is the standard treatment for patients with Glioblastoma multiforme (GBM) [2], we investigated its effect on mTORC1 signalling and MGMT levels in the T98G GBM cell line
Summary
Glioblastoma (GBM), a WHO grade IV astrocytoma, is the most common [1] and most aggressive type of primary brain tumour. TMZ is a methylating agent that creates lesions in DNA, the most cytotoxic of which is O6-methylguanine [3] This multimodal treatment regimen is rigorous, yet prognosis remains poor and TMZ chemotherapy improves survival in a subset of patients, 75% die within 2 years and the vast majority of patients experience disease recurrence [4]. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity
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