mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells
© Smalley et al.; licensee BioMed Central Ltd. 2014
Received: 3 March 2014
Accepted: 30 May 2014
Published: 8 June 2014
Glioblastoma 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.
MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.
We 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.
These 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.
KeywordsMGMT Stability Translation mTOR Initiation of translation KU0063794 Glioblastoma TMZ resistance
Glioblastoma (GBM), a WHO grade IV astrocytoma, is the most common  and most aggressive type of primary brain tumour. Current treatment consists of tumour resection (where possible), followed by ionising radiotherapy combined with concomitant and adjuvant temozolomide (TMZ) chemotherapy . TMZ is a methylating agent that creates lesions in DNA, the most cytotoxic of which is O6-methylguanine . This multimodal treatment regimen is rigorous, yet prognosis remains poor and although TMZ chemotherapy improves survival in a subset of patients, 75% die within 2 years and the vast majority of patients experience disease recurrence .
O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein, which removes O6-methylguanine adducts from damaged DNA . This reaction is irreversible and once bound to the alkyl group MGMT is ubiquitinated and destroyed by the proteasome . The cytotoxic effect of TMZ chemotherapy is therefore influenced by the ability of tumour cells to re-synthesise MGMT and maintain steady state levels of the protein. Previous work has shown that MGMT promoter methylation resulting in gene silencing and resultant low levels of MGMT protein, increases sensitivity to TMZ and is associated with improved patient survival. Unfortunately direct inhibition of MGMT using small molecule inhibitors such as lomeguatrib has not been successful as a clinical application because it also increases haematological toxicity [2, 7, 8]. However, downregulation of MGMT remains an attractive therapeutic strategy for patients with tumours exhibiting unmethylated MGMT promoters if it could be achieved in a tumour specific manner.
Common mutations in GBM cells include genetic changes that result in a loss of PTEN function and EGFR amplification , both of which can generate hyperactive phosphoinositide 3-kinase (PI3K)/mTOR signalling. mTOR is a serine/threonine kinase that belongs to the PI3K-related kinase family and interacts with proteins to form two distinct complexes in mammals, mTORC1 and mTORC2 . mTORC1, when active, regulates protein translation through the phosphorylation of 4EBP1. Phosphorylation of 4E-BP1 prevents it binding to eIF4E, which enables eIF4E to participate in the formation of the eIF4F complex on the m7 GTP cap structure of mRNA and mediate small ribosomal subunit binding and subsequent protein translation . The hyperactivity of this pathway therefore results in increased protein synthesis, promoting cell growth and proliferation.
Because of this there has been interest in the use of mTOR inhibitors in combination with radiation and TMZ in the treatment of GBM. The rationale for combining mTOR inhibitors with TMZ treatment is based on the reasoning that the lesions in DNA caused by TMZ will result in a depletion of cellular MGMT protein levels. When coupled with mTOR inhibition, not only would there be a decrease in MGMT levels, but the tumour cell would be compromised in its ability to synthesise new protein, thus sensitising the cells to further TMZ treatment. In addition to this, tumour cells should be specifically targeted with this course of treatment, due to the tumour cells oncogenic addiction to the PI3K/mTOR signalling pathway. This would avoid the current drawbacks faced during direct inhibition of MGMT, as it would avoid MGMT depletion in healthy cells, and therefore avoid undesirable cytotoxicity.
In this work, we have used Western blotting to examine the effect of inhibiting mTORC1/2 signalling on steady state MGMT protein levels in T98G GBM cells, a cell line which exhibits relatively high MGMT expression compared to other glioblastoma cell lines [12, 13]. KU0063794 is a specific mTORC1 and mTORC2 inhibitor that does not display significant activity against similar kinases such as PI3K, ERK1/2, or p38MAPK .
The findings described in this paper are of both biochemical and potential clinical interest. The research highlights how important it is to identify how DNA repair proteins are translated and maintained as proteins, which is an important consideration, especially when manipulating them for clinical benefit.
Materials and methods
T98G (ECACC) cells were cultured to confluency in minimum essential medium (MEM) supplemented with 5% non-essential amino acids (Invitrogen, UK), 10% foetal calf serum (Biosera, UK) and 5% GlutaMax (Invitrogen, UK). U87 (ECACC) cells were cultured in the same manner but with Hyclone foetal calf serum.
Concentrations of drug treatments
Cells were treated with the following concentrations of drugs: 10 μM KU0063794, 10 μM TMZ and 10 μM emetine.
Preparation of cell extracts
Following treatment, cells were isolated in a cooled microfuge and washed briefly with 0.5 ml ice-cold PBS. Pellets were resuspended in 100 μl ice-cold lysis buffer (20 mM MOPS-KOH, pH 7.2, 20 mM sodium fluoride, 1 μM microcystin LR, 75 mM KCl, 2 mM MgCl2, 2 mM benzamidine, 2 mM Na3VO4, complete protease inhibitor mix (−EDTA (Roche, UK), 0.1% (v/v) SDS), with the addition of 0.5% (v/v) Igepal and 0.5% (w/v) deoxycholate (DOC) with vortexing. Cell debris was removed by centrifugation in a microfuge for 5 min at 4°C and the resultant supernatants recovered.
Samples containing equal amounts of protein (10 μg) were resolved by polyacrylamide gel electrophoresis (SDS-PAGE) and processed as described previously [15, 16]. Briefly, membranes were blocked using TBS-Tween containing 3% (w/v) BSA for 1 hour and incubated with antisera diluted in the same overnight at 4°C. Following washing in TBS-Tween, membranes were incubated with horseradish peroxidase-conjugated secondary antibody and signals developed using ECL reagent. The antiserum used were raised against: eIF4A, phospho-eIF4E, eIF4G, eIF4E [15, 16]; MGMT, phospho-eIF2α, (Abcam, UK); 4E-BP1, phospho-4E-BP S65, phospho-4E-BP1 T70, phospho-p70 S6K T389, phospho- ERK1/2, phospho-p38 MAPK, phospho-rpS6 S240/44, phospho-Akt T308, p21cip1 (Cell Signaling, UK).
[35S]-methionine labelling of cellular protein
One hour prior to harvesting, T98G cells were pulse-labelled with [35S]-methionine (MP Biomedicals, UK; 10 μCi/ml) and cell extracts prepared as above and 5 μl of extract was spotted onto Whatman filter papers. Filters were transferred to 10% (v/v) trichloroacetic acid (TCA) containing 5 mM unlabelled methionine for 15 minutes then boiled in 5% (v/v) TCA. Once cooled, the filters were washed in ethanol, then acetone, dried and subjected to liquid scintillation counting. Protein synthesis, expressed as cpm/ μg protein, is shown as a % of the rate obtained in cells incubated in the absence of drugs.
Cells were cultured in a 96 well plate in 100 μl of complete medium. Following incubation with drugs, 20 μl of MTS/PMS solution (Promega, UK) was added, and left for 2 hours at 37°C. The colour reaction, reflecting metabolic activity was quantified by measuring absorbance at A490 using the control wells for comparison. All assays were carried out in triplicate.
Following incubation of cells as described in the figure legends, cells were washed with warm PBS then trypsinised and removed from culture dishes. Cells were washed in PBS and fixed in 70% ethanol/30% PBS at 4°C overnight. Immediately before analysis, the cells were washed in PBS and re-suspended in 500 μl PBS containing 0.1 mg/ml RNase A (Sigma, UK) and 30 μg/ml propidium iodide (Invitrogen, UK) and analysed by a FACSCanto™ flow cytometer (BD Biosciences) using BD FACSDiva™ software. Data shown are representative of those obtained in at least three separate experiments.
RNA was extracted from cell extracts using an RNA easy mini-kit (Qiagen, UK) as per manufacturer’s instructions. RNA concentration was then quantified using a Nanodrop and 1 μg of RNA was used for cDNA synthesis using the Promega ImpromII kit. The SYBR real-time PCR system (Kapa Biosystems, UK) was used to quantify transcript abundance for genes of interest and18S mRNA was used as a control. Template equivalent to 5 ng of RNA in cDNA library per reaction was added to each 20 μl reaction with a final primer concentration of 200 nM per reaction. Crossing thresholds were determined using MxPro software (Agilent), and fold-difference in RNA quantity was calculated using the relative quantification method (2-ΔΔct).
Cells were plated on glass coverslips 8 hours before treatment. Cells were then fixed with 4% (w/v) paraformaldehyde for 20 minutes at room temperature and permeabilised for 5 minutes in 0.1% Triton X-100/PBS. Cells were blocked for 20 minutes with 5% (w/v) gelatin/PBS in a humidified chamber. Cells were incubated with anti-MGMT (Abcam, UK) for 1 hour at room temperature also in a humidified chamber. Alexa Fluor 555-conjugated anti-rabbit secondary antibody was added for 1 hour at room temperature. Alexa Fluor 488 phalloidin was also used in the secondary antibody incubation. Following further extensive washing, nuclei were stained with DAPI (4’, 6-diamidino-2-phenylindole) for 5 minutes. After a further two washes, coverslips were mounted on microscope slides with Mowiol mounting solution (0.2 M Tris/HCl (pH 8.5), 33% (w/v) glycerol, 13% (w/v) Mowiol and 2.5% (w/v) DABCO (1, 4-diazobicyol -octane)) and sealed with clear nail polish. Images were collected on a Zeiss Axiovert LSM510 scanning confocal microscope using a × 100 objective. Single stain, bleed-though controls and antibody cross-reaction controls were prepared for each sample (results not shown).
m7GTP-Sepharose affinity chromatography
To isolate eIF4E and associated proteins [15, 16] cells were lysed as described and aliquots containing 100 μg were incubated with 30 μl (50% v/v in 1 mg/ml cytochrome c) m7GTP-Sepharose resin for 15 mins on ice, with mixing. The resin was washed twice in 200 μl buffer (20 mM MOPS/KOH pH 7.2, 75 mM KCl, 2 mM benzamidine, 7 mM 2-mercaptoethanol, 2 mM MgCl2, 0.1 mM GTP, complete protease inhibitor mix (−EDTA), 25 mM NaF). To visualise eIF4E and associated proteins, the resin was washed twice with 200 μl buffer then eluted in SDS-PAGE sample buffer containing 10% w/v β-mercaptoethanol prior to Western blotting.
Cells were treated with 100 μg/ml of cycloheximide (Sigma, UK) for 3 mins before harvesting. The cells were washed twice in PBS containing 100 μg/ml cycloheximide and lysed in hypertonic lysis buffer (10 mM Tris/HCl pH7.5, 10 mM KCl, 15 mM MgCl2, 1%(v/v) Igepal, 0.5%(v/v) DOC, 40 mM β-glycerophosphate, complete protease inhibitor mix (−EDTA), 2 mM DTT, 100 μg/ml cycloheximide, 10 U/ml RNase inhibitor) for 15 minutes on ice and homogenised every 5 mins. The cell lysate was centrifuged at 14,000 rpm for 3 mins and the supernatant was separated on a 11.2 ml sucrose gradient (10-60% (w/v) in hypertonic lysis buffer) for 130 mins at 38, 000 rpm using a Beckman SW40 rotor and fractionated using an ISCO, monitoring A260 nm through a flow cell. For analysis of GAPDH and MGMT mRNA, the mRNA from pooled fractions was extracted and quantitative RT-PCR was used to determine mRNA distribution across each fraction.
TMZ does not affect steady state levels of MGMT protein or protein synthesis in T98G cells
Concomitant TMZ treatment with KU0063794 causes an increase in MGMT protein levels
KU0063794 treatment is responsible for the dramatic rise in MGMT protein levels although global translation remains reduced
mTORC1, when active, regulates protein translation through the phosphorylation of 4EBP1. Phosphorylation of 4E-BP1 prevents it binding to eIF4E, which enables eIF4E to participate in the formation of the eIF4F complex on the m7 GTP cap structure of mRNA and mediate small ribosomal subunit binding and subsequent protein translation . As described above, cells were incubated for 72 hours and the binding of eIF4E and associated proteins with the 5’ end of mRNA was measured using Sepharose beads coupled with m7 GTP. Bound protein was recovered and visualised using SDS-PAGE and Western blotting alongside eIF4E and associated proteins isolated from untreated cells. As expected, inhibition of mTORC1/2 signalling pathway resulted in an increase in the binding of hypophosphorylated 4E-BP1 to recovered eIF4E and a moderate decrease in eIF4G associated with eIF4E (Figure 3B). Unsurprisingly this observation was coupled with a reduction in protein synthesis rates by around 70% upon treatment of KU0063794 for 48 hours (Figure 2C) and at 72 hours, the KU0063794 treated cells were synthesising protein at less than 10% of the rate of the untreated control.
The levels of MGMT protein were also monitored in cells treated with KU0063794, as this information was necessary to identify if the rise in MGMT levels during the combined treatment of KU0063794 and TMZ was solely caused by KU0063794 or if the effect observed was synergistic. MGMT protein levels were not only maintained during this treatment (Figure 3C, lanes 2–5 vs. lane 1), but in a similar fashion to cells treated with both KU0063794 and TMZ, protein levels increased over a 72 hour period, despite clear inhibition of mTORC1 (as shown by dephosphorylation of 4E-BP1, Figure 3C). Quantification of these data from three separate experiments show that, relative to the alpha tubulin protein loading control that MGMT protein levels were increased by statistically significant levels at 48 and 72 hours (p = 0.08 and 0.2, respectively) in cells treated with KU0063794 alone compared with untreated controls (Figure 3D).
This observation clearly shows that the rise in MGMT protein is caused by KU0063794. Yet it was not clear if the rise in MGMT protein was protein specific or if mTORC1/2 inhibition brought about a general inhibition of protein degradation. To investigate this, we analysed the steady state level of p21cip1, which is known to have a short half-life (approximately 100 minutes ). The disappearance of p21cip1 protein after 12 hours indicates that KU0063794 treatment does not cause a general inhibition of protein degradation (Figure 3C), suggesting that the increase in MGMT protein levels was indeed specific.
Cell viability (Figure 3E) and cell cycle progression in T98G cells (Additional file 1: Figure S6) was also investigated. KU0063794 had little effect on metabolic activity (Figure 3E) but did cause a reduction in the S phase population and a small increase in the sub-G1 population (Additional file 1: Figure S6), indicative of an inhibition of the cell cycle and some apoptosis at later incubation times.
To ensure that the effects observed during treatment of all of the compounds were not cell specific, we also used U87-MG cells. This glioma cell line expresses very low levels of MGMT, which is confirmed by the absence of MGMT protein when visualised by Western blotting (Additional file 1: Figures S7, S8 and Additional file 2: Figure S9). Both KU0063794 and TMZ generally had a similar effect on the U87-MG cell line compared to the T98G cell line. However, there were some differences in responses observed here. The most obvious differences were a less robust cessation of cell cycle progression in U87-MG cells in response to KU0063794 and subtle differences on mTORC1/2 and eIF2 alpha signalling pathways. In U87-MG cells incubated with TMZ, there was no change in 4E-BP1 or T308 phosphorylation but there was an increase in eIF2 alpha phosphorylation, suggesting that inhibition of translation under these conditions in U87-MG cells may have been caused by eIF2 alpha phosphorylation.
mTOR1/2 inhibition results in selective translation of MGMT during reduced protein synthesis rates
We also confirmed that total mRNA expression did not change on KU0063794 treatment using qRT-PCR (Additional file 2: Figure S10). MGMT mRNA levels remained unchanged at 24 hours compared to untreated cells under all incubation conditions tested (relative to 18S rRNA).
Inhibition of mTOR signalling does not affect the half-life of MGMT protein in T98G cells but stabilises MGMT protein in the presence of TMZ
GBM is the most aggressive adult brain cancer and TMZ, an oral methylating chemotherapeutic agent, is part of the standard treatment, often accompanied by radiotherapy [2–4]. TMZ exerts its antitumour effect by methylating guanine at a number of sites . The N7 guanine and N3 adenine adducts caused by TMZ are repaired by the base excision repair pathway, but the O6-methyl-guanine (O6MeG) lesion cannot be repaired in this fashion, making it the most potently cytotoxic adduct . O6MeG pairs with thymine and leads to GC-AT transitions, beginning a cycle of futile DNA mismatch repair which causes DNA double-stranded breaks and, ultimately causes tumour cell death [6, 22, 23].
MGMT is a DNA repair protein that repairs the O6MeG lesion created by TMZ by a process of irreversible binding and subsequent degradation of the protein [5, 23]. When O6MeG is repaired by MGMT, the majority of cells become resistant to O6MeG-triggered apoptosis [4–6]. Hence the level of MGMT activity correlates with resistance of tumour cells to TMZ . As mutations in the mTOR signalling pathway in GBM frequently result in hyperactive PI3-K/mTOR signalling, promoting cell survival and protein synthesis [9, 10, 20], we investigated whether mTOR kinase activity has a role in regulating levels of MGMT protein expression in human glioblastoma cells.
As predicted, we show that TMZ does not reduce MGMT protein levels in T98G cells, so the large amount of MGMT in the cells is effectively repairing any lesions created by TMZ. This suggests that, in agreement with previous studies [25–27], on its own TMZ is not sufficiently effective in cellular environments containing MGMT overexpression.
In contrast to cells treated with TMZ alone, when protein loading was accounted for in three separate experiments, our data show that in cells treated with both KU0063794 and TMZ, there was an increase in MGMT protein expression. This occurred even though global protein synthesis rates were reduced by around 80% under these conditions. These data suggest that although dual mTOR inhibition was indeed impairing the formation of the translation initiation complex, reducing global protein synthesis rates and causing a disaggregation of polysomes from mRNA, protein levels were still increasing. To decipher if this unexpected maintenance of MGMT protein levels was reliant on an alternative translation mechanism, we assessed the translation status of MGMT mRNA. This demonstrated that during conditions of reduced global translation rates, and therefore reduced mRNA association with polysomes, MGMT mRNA remained associated with heavy polysomes, although GAPDH mRNA shifted to sub polysomal fractions. This is strong evidence that MGMT mRNA might be translated using a cap-independent mechanism, or preferentially recruited to ribosomes under conditions of low levels of eIF4F , but in the absence of increased levels of eIF2α phosphorylation (Figure 3). This is further supported by the presence of a number of GC-rich short stem loops in the annotated 3’-UTR of the MGMT mRNA, which may also have a role in this process. Additional extensive biochemical analysis would be needed to determine if this is the case. Alternative mechanisms controlling translation have been identified under hypoxic stress conditions, which rely on protein complexes involving the alternative eIF4E isoform eIF4E2, specifically recruiting target mRNAs to polysomes during periods of reduced cap dependant translation . These findings could compliment the preferential translation of MGMT under stress conditions, but further investigations would need to be carried out to confirm this.
In addition to the mechanisms regulating translational control of MGMT mRNA, the stability of the MGMT protein was also investigated, as this is of paramount importance when considering its manipulation for effective chemotherapeutic treatment. The inhibition of protein synthesis elongation with emetine in the presence of TMZ caused a dramatic decrease in MGMT protein level, reflecting the rapid degradation of the protein following DNA repair activity (Figure 5). However, the combination of KU0063794 and TMZ compared with TMZ alone resulted in a lengthened half-life of MGMT protein to around 40 hours. This is an important observation, as an increase in MGMT stability has the potential to greatly impede treatment of GBM with DNA methylating agents.
Previous work has used the mTORC1 inhibitor rapamycin to inhibit the mTOR/PI3K pathway in an attempt to inhibit the growth of GBM [30–32]. It was therefore a logical progression to investigate specific mTOR signalling inhibition in GBM cells and their possible use in conjunction with TMZ. Even though research in this area is surprisingly limited, recent studies have indicated combination treatments involving compounds that negatively regulate the mTOR signalling pathway may impact MGMT regulation , further compounding the need for research to increase the efficacy of TMZ. Indeed a number of clinical trials combining mTOR inhibitors with radiation and TMZ have been initiated and are recruiting patients (e.g. everolimus (RTOG 0913) and BKM120).
The data presented here reveal that in vitro, rather than inhibiting translation of MGMT, mTOR inhibition promotes steady state levels of the MGMT protein and counteracts the depleting effects of TMZ. We therefore conclude that further in vivo analysis of mTOR inhibition is necessary to determine if this effect is also found in patients exhibiting GBM. If so, mTOR inhibition would not be a useful adjunct to TMZ therapy in a clinical setting and could exacerbate tumour growth.
The work was supported by a PhD studentship ref# BB/F01645X/1 from the Biotechnology and Biological Sciences Research Council (BBSRC).
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