MEK-ERK Signaling Dictates DNA-Repair Gene MGMT Expression and Temozolomide Resistance of Stem-Like Glioblastoma Cells via the MDM2-p53 Axis

Abstract

Overcoming the resistance of glioblastoma cells against temozolomide, the first-line chemotherapeutic agent of choice for newly diagnosed glioblastoma, is a major therapeutic challenge in the management of this deadly brain tumor. The gene encoding O⁶-methylguanine DNA methyltransferase (MGMT), which removes the methyl group attached by temozolomide, is often silenced by promoter methylation in glioblastoma but is nevertheless expressed in a significant fraction of cases and is therefore regarded as one of the most clinically relevant mechanisms of resistance against temozolomide. However, to date, signaling pathways regulating MGMT in MGMT-expressing glioblastoma cells have been poorly delineated. Here in this study, we provide lines of evidence that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)–extracellular signal-regulated kinase (ERK)--murine double minute 2 (MDM2)-p53 pathway plays a critical role in the regulation of MGMT expression, using stem-like glioblastoma cells directly derived from patient tumor samples and maintained in the absence of serum, which not only possess stem-like properties but are also known to phenocopy the characteristics of the original tumors from which they are derived. We show that, in stem-like glioblastoma cells, MEK inhibition reduced MDM2 expression and that inhibition of either MEK or MDM2 resulted in p53 activation accompanied by p53-dependent downregulation of MGMT expression. MEK inhibition rendered otherwise resistant stem-like glioblastoma cells sensitive to temozolomide, and combination of MEK inhibitor and temozolomide treatments effectively deprived stem-like glioblastoma cells of their tumorigenic potential. Our findings suggest that targeting of the MEK-ERK-MDM2-p53 pathway in combination with temozolomide could be a novel and promising therapeutic strategy in the treatment of glioblastoma. STEM CELLS 2011;29:1942–1951.

INTRODUCTION

Glioblastoma multiforme, the most common primary neoplasm of the brain in adults, is one of the most aggressive types of human cancer with a dismal prognosis, the median survival being less than 2 years despite multimodality treatment. The standard of care for glioblastoma is composed of maximal surgical resection followed by radiotherapy with concomitant and adjuvant chemotherapy [1, 2]. Temozolomide is a monofunctional alkylating agent currently used as the first-line chemotherapeutic agent against newly diagnosed glioblastoma and is also a drug of choice for recurrent disease [3, 4]. Sensitivity of tumor cells to temozolomide is therefore key to successful management of this intractable disease; however, unfortunately glioblastoma cells often exhibit resistance against this alkylating agent. Among possible mechanisms, O⁶-methylguanine DNA methyltransferase (MGMT) expression has been well documented as the clinically most relevant mechanism of resistance against temozolomide-based glioblastoma therapies [5]. MGMT is a repair enzyme that rapidly removes the methyl group attached by temozolomide at the O⁶ position of the guanine residue and as such could theoretically counteract the antitumor effect of temozolomide. Indeed, accumulating evidence from correlative observations as well as from functional analyses, in either clinical settings or in vitro studies [5–9], now suggests that this is actually the case with glioblastoma, underscoring the absolute necessity for developing novel methods to inactivate MGMT in tumor cells to overcome temozolomide resistance. Understanding the molecular mechanism involved in the regulation of MGMT expression/function is vital for identification of therapeutic targets; however, signaling pathways controlling MGMT in glioblastoma cells remain poorly characterized.

In this study, we dissected the molecular pathway regulating MGMT expression using stem-like glioblastoma cells as a clinically relevant in vitro model of glioblastoma. We used stem-like cells directly derived from glioblastoma patients because (1) stem-like glioblastoma cells more faithfully phenocopy and represent the original tumors from which they are derived than their serum-cultured counterparts and conventional glioma cell lines [10, 11], and because (2) stem-like cancer cells are presumed, due to their inherent therapy resistance and high tumorigenic capacity, to be the source of post-treatment recurrence and hence the most critical target of therapy [12, 13]. Here, we provide lines of evidence that, in stem-like glioblastoma cells, the MEK-extracellular signal-regulated kinase (ERK) pathway dictates MGMT expression and temozolomide resistance via the murine double minute 2 (MDM2)-p53 axis. This study thus offers novel opportunities to develop molecular targeting drugs for MGMT inactivation in glioblastoma to be validated in future preclinical and clinical studies.

MATERIALS AND METHODS

Reagents and Antibodies

SL327 was purchased from Enzo Life Sciences (New York, NY, http://www.enzolifesciences.com), temozolomide was from LKT Laboratories (St. Paul, MN, http://www.lktlabs.com), Nutlin-3 was from Calbiochem (Darmstadt, Germany, http://www.emdchemicals.com), and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were from Peprotech (Princeton, NJ, http://www.peprotech.com). Anti-nestin (AB5922) and anti-p53 (OP-43) were from Chemicon (Billerica, MA, http://www.millipore.com). Anti-glial fibrillary acidic protein (AF2594), anti-βIII-tubulin (MAB1195), and anti-MDM2 (AF1244) were from R&D (Minneapolis, MN, http://www.rndsystems.com). Anti-ERK1/2 (#4695), anti-phospho-ERK1/2 (#9106), and anti-p21 (#2947) were from Cell Signaling Technology (Danvers, MA, http://www.cellsignal.com). Anti-MGMT (ab212628) was from Abcam (Cambridge, MA, http://www.abcam.com). Anti-MEK1 (sc-219) and anti-MEK2 (sc-525) were from Santa Cruz Biotechnology (CA, http://www.scbt.com). Anti-actin (A1978) was from Sigma (St. Louis, MO, http://www.sigmaaldrich.com). Horseradish peroxidase (HRP)-conjugated secondary antibodies for immunoblotting were from Jackson Immuno Research (Carlsbad, CA, http://www.invitrogen.com).

Human Glioblastoma Tissues and Isolation, Culture, and Characterization of Patient-Derived Stem-Like Glioblastoma Cells

Isolation and establishment of primary human stem-like glioblastoma cells (GS-Y01 and GS-Y02) were carried out essentially as previously described [14] in accordance with a protocol approved by the Institutional Review Boards of Yamagata University School of Medicine, and the stem-like cells were maintained in the monolayer culture condition [15, 16]. The capacity for self-renewal, multipotency of differentiation, and tumorigenicity were tested and verified as in Supporting Information Figure S1. To induce differentiation, cells were cultured in the differentiation culture condition (Dulbecco's modified Eagle's medium/F12 containing 10% fetal bovine serum). TGS01 and TGS04 are generous gifts from the Department of Neurosurgery, University of Tokyo. Characterization of TGS01 and TGS04 has been described elsewhere [17] and was also confirmed in this study (Supporting Information Fig. S1).

RNA Interference

Cells seeded in monolayer culture condition for stem-like cells on the previous day were transfected with the short-interfering RNAs (siRNAs) indicated below using Lipofectamin 2000 or Lipofectamin RNAi MAX (Invitrogen, Carlsbad, CA, http://www. invitrogen.com) in accordance with the manufacturer's protocols.

All the siRNAs used in this study were purchased from Invitrogen and are described below. 

Table  . 

Temozolomide Experiment and Cell Death Assay

Stem-like glioblastoma cells exposed to temozolomide for 4 hours were cultured on fresh, temozolomide-free culture medium for 72 hours after washout of temozolomide and then subjected to cell death assay. Dead cells were detected on the basis of their inability to exclude propidium iodide (PI). In brief, cells were incubated with 5 μg/ml PI and, for nuclear staining, with 10 μg/ml Hoechst 33342 for 10 minutes. The numbers of PI- and Hoechst-positive cells were scored under a fluorescence microscope (CKX41; Olympus, Tokyo, Japan; at least 500 cells per sample were examined), and the percentage of PI-positive cells (dead cells) against Hoechst-positive cells (total cells) was determined.

Immunoblot Analysis

For immunoblot analysis, cells were lysed in lysis buffer (10 mM Tris-HCl [pH 7.4], 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl, 1 mM EDTA, 1% protease inhibitor cocktail set III [Calbiochem]). After determination of protein concentrations using the BCA protein assay kit (Pierce, Rockford, IL, http://www.piercenet. com), cell lysates containing equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was probed with a primary antibody and then with an appropriate HRP-conjugated secondary antibody according to the protocol recommended by the manufacturer of each antibody. Signal was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, http://www.millipore. com) or ECL Western Blotting Detection Reagents (GE Healthcare, Amersham, UK, http://www.gelifesciences.com). Chemiluminescent images were captured using a CCD camera of Bio-Rad (Hercules, CA, http://www.bio-rad.com).

Animal Experiments

Stem-like glioblastoma cells (TGS01, 1 × 10⁴) pretreated for 3 days with or without SL327 (10 μM) were subsequently exposed to temozolomide (50 μM) or vehicle control (dimethyl sulfoxide) in the absence of SL327 for 4 hours. Then, after 3-day culture in temozolomide-free medium, the cells were injected stereotactically into the right corpus striatum (2.5 mm anterior and 2.5 mm lateral to the bregma, and 3.0 mm deep) of 5-week-old male BALB/c nu/nu mice (CLEA Japan, Inc.). All animal experiments were performed under a protocol approved by the Animal Research Committee of Yamagata University.

Statistical Analysis

The results of cell death analysis in Figures 1 and 2 are expressed as the means + standard deviations of three independent experiments and were analyzed using the unpaired Student's t test. In Figure 5, mouse survival was evaluated by the Kaplan-Meier method and analyzed using the log-rank test.

Figure 1. MGMT expression and temozolomide resistance of stem-like glioblastoma cells. (A): MGMT expression levels in the indicated stem-like glioblastoma cells were determined by immunoblot analysis. (B): The stem-like glioblastoma cells were exposed to TMZ at the indicated concentrations for 4 hours, and the percentage of dead cells was determined after 72 hours. The data represent the means + standard deviations of three independent experiments. (C, D, E): The stem-like glioblastoma cells were transfected with the indicated siRNAs against MGMT or with a nontargeting control (Cont.). The cells were then subjected to immunoblot analysis for MGMT expression (C) or to cell death assay using 50 μM temozolomide (D and E) 72 hours after transfection. In (D), representative images of cell death assay are shown, where the nuclei of propidium iodide-positive, dead cells are stained red while those of live cells are stained blue with Hoechst 33342 (scale bar = 200 μm). In (E), the graph shows the percentage of dead cells (means + standard deviations of three independent experiments). *, p < .01, **, p < .05, n.s., not significant. Abbreviations: MGMT, O⁶-methylguanine DNA methyltransferase; siRNA, short-interfering RNA; TMZ, temozolomide.

Figure 2. MEK/ERK pathway inhibition reduces MGMT expression and temozolomide resistance of stem-like glioblastoma cells. (A): The stem-like glioblastoma cells treated with or without 10 μM SL327 for 3 days were subjected to immunoblot analysis for the expression of MGMT and phospho-ERK. (B): The stem-like glioblastoma cells were transfected with the combination of siRNAs against MEK1 and MEK2 or with a nontargeting control (Cont.). After 3 days of transfection, the cells were analyzed by immunoblotting with the indicated antibodies. (C): The stem-like glioblastoma cells pretreated with or without 10 μM SL327 for 3 days were subsequently treated with or without temozolomide (TMZ, 50 μM) for 4 hours and then subjected to cell death assay 72 hours after the temozolomide treatment. The upper panels show representative images of the assay (red = dead cells, blue = live cells), and the graph shows the percentage of dead cells (means + standard deviations of three independent experiments). *, p < .01, **, p < .05, n.s., not significant. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; MGMT, O⁶-methylguanine DNA methyltransferase; siRNA, short-interfering RNA; TMZ, temozolomide.

RESULTS

MGMT Expression Level Is a Critical Determinant of Temozolomide Resistance of Stem-Like Glioblastoma Cells

To determine the role of MGMT in temozolomide resistance of stem-like glioblastoma cells, we first examined the expression level of the MGMT protein in four lines of stem-like glioblastoma cells used in this study, which were originally established in two independent institutions. The four lines expressed MGMT at varying levels, from barely detectable (TGS04) to very high (TGS01; Fig. 1A). When these cells were subjected to cell death assay after treatment with clinically relevant concentrations (∼50 μM) of temozolomide [18], we found that cell death was induced in close (inverse) correlation with their MGMT expression levels. For instance, whereas TGS04 expressing the lowest level of MGMT readily underwent cell death when treated with temozolomide at a lower concentration (25 μM), only minimal increase of cell death was observed with the highest expressor TGS01 even when treated at a higher concentration (50 μM) (Fig. 1B). These results are consistent with a recent report showing that nearly half of the brain tumor-initiating cell lines derived from glioblastoma expressed the MGMT protein and that those with MGMT expression, but not those without, showed resistance against temozolomide [7]. Such close association between MGMT expression and temozolomide sensitivity suggests a causal link between these two factors; however, this has not yet been established in stem-like glioblastoma cells, with only a single pilot study testing this point using two glioma-initiating cell lines so far [9]. Therefore, to definitively determine the role of MGMT expression in temozolomide resistance of stem-like glioblastoma cells, we knocked down MGMT expression in MGMT-expressing stem-like cells and examined its impact on their temozolomide sensitivity. We used three different sets of siRNA against MGMT, which inhibited MGMT with varying efficiencies (Fig. 1C). Significantly, cell death induced by temozolomide treatment directly correlated with the efficiency of knockdown, namely, the level of MGMT expression in all the three lines tested (Fig. 1D, 1E). Thus, the results indicate that the endogenous level of MGMT expression confers temozolomide resistance on stem-like glioblastoma cells in a quantitative, expression level-dependent manner.

MEK-ERK Signaling Is Required for the Maintenance of MGMT Expression and Temozolomide Resistance of Stem-Like Glioblastoma Cells

A previous study demonstrated that CD133-positive cancer stem cells derived from glioblastoma express much higher levels of MGMT mRNA than autologous CD133-negative tumor cells, suggesting that MGMT expression may be associated with stem-like state of glioblastoma cells [19]. We therefore reasoned that inhibition of MEK in stem-like glioblastoma cells, which we have recently shown to promote their differentiation [14], could also reduce MGMT expression along with other stem-like properties. We tested this idea first by treating the stem-like glioblastoma cells with chemical inhibitors of MEK, SL327 and U0126. Both inhibitors induced differentiation (Supporting Information Fig. S3) under conditions in which they inhibited ERK phosphorylation (Fig. 2A, Supporting Information Figs. S2 and S8), confirming that this is required for prevention of premature differentiation of stem-like glioblastoma cells, as we have shown using a different set of stem-like glioblastoma cells [14]. Under identical experimental conditions, both SL327 and U0126 suppressed MGMT expression, suggesting that the kinase activity of MEK is required for MGMT expression (Fig. 2A and Supporting Information Fig. S2). Knockdown experiments targeted against the MEK genes also demonstrated MEK requirement for MGMT expression, corroborating the results of the chemical inhibitor study (Fig. 2B). We then examined the impact of MEK inhibition-mediated downregulation of MGMT expression on temozolomide sensitivity of the stem-like glioblastoma cells. Intriguingly, when the cells were treated with SL327 for 3 days followed by washout of the inhibitor, MGMT expression remained suppressed for at least 3 days after the washout, despite apparent recovery of ERK phosphorylation (Supporting Information Fig. S4). We therefore treated stem-like glioblastoma cells first with SL327 for 3 days, and after washout of SL327, then treated them with temozolomide in the absence of the MEK inhibitor for 3 days to conduct cell death assay. The result clearly indicated that MEK inhibition preceding temozolomide treatment, while having a minimal effect on cellular viability by itself, remarkably promotes cell death induction by temozolomide in cells expressing MGMT (Fig. 2C, Supporting Information Fig. S5). In sharp contrast, MEK inhibition failed to promote temozolomide-induced death of TGS04 cells that express only a low level of MGMT and hence are originally sensitive to temozolomide (Figs. 1A, 1B, and 2C, Supporting Information Fig. S5), indicating that the cell death-promoting effect of MEK inhibition is dependent on MGMT expression. Together, these results suggest that MEK-ERK signaling contributes to temozolomide resistance of stem-like glioblastoma cells through maintenance of MGMT expression.

Upregulation of p53 is Responsible for MEK Inhibition-Mediated Suppression of MGMT Expression in Stem-Like Glioblastoma Cells

During our screening search for molecules differentially expressed in stem-like glioblastoma cells before and after differentiation, we noted remarkable upregulation of p53, together with its transcriptional target p21 (with the exception of TGS04 cells), in cells undergoing differentiation after SL327 treatment (Fig. 3A, Supporting Information Figs. S6A and S8A). Subsequent knockdown experiments targeted against the MEK genes confirmed that MEK inhibition is indeed responsible for the observed p53 activation (Fig. 3B, Supporting Information Figs. S6B and S8B). As previous studies indicated that p53 is capable of inhibiting MGMT expression [20–23], we investigated the role of this p53 upregulation in MEK inhibition-mediated suppression of MGMT. Under conditions where p53 expression was effectively knocked down by an siRNA against p53, suppression of MGMT expression either by SL327 (Fig. 3C, Supporting Information Fig. S6C) or by knockdown of the MEK genes (Fig. 3D, Supporting Information Fig. S6D) was abolished. Thus, suppression of MGMT expression of stem-like glioblastoma cells by MEK inhibition is dependent on p53.

Figure 3. p53 mediates MEK inhibition-induced MGMT downregulation in stem-like glioblastoma cells. (A): The expression levels of p53 and p21 in stem-like glioblastoma cells treated with or without 10 μM SL327 for 3 days were determined by immunoblot analysis. (B): The stem-like glioblastoma cells transfected with the combination of siRNAs against MEK1 and MEK2 or with a nontargeting control were subjected to immunoblot analysis for p53 and p21 expression 72 hours after transfection. (C): The stem-like glioblastoma cells transfected with a siRNA against p53 or with a nontargeting control were treated, 24 hours after transfection, with or without 10 μM SL327 for 3 days. The cells were then analyzed by immunoblotting with the indicated antibodies. (D): The stem-like glioblastoma cells were transfected first with an siRNA against p53 or with a nontargeting control and then, 24 hours later, transfected with the combination of siRNAs against MEK1 and MEK2 or with a nontargeting control. The cells were subjected to immunoblot analysis with the indicated antibodies 72 hours after the second transfection. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; MGMT, O⁶-methylguanine DNA methyltransferase; siRNA, short-interfering RNA.

MEK-ERK Signaling is Required for MDM2 Expression in Stem-Like Glioblastoma Cells, Which Prevents p53 Activation and Subsequent Suppression of MGMT Expression

To further delineate the molecular pathway involved in MEK inhibition-mediated suppression of MGMT expression, we next examined molecules that could modulate p53 expression in response to MEK inhibition. Among other candidates, we found in stem-like glioblastoma cells treated with SL327 decreased expression of MDM2 (Fig. 4A, Supporting Information Figs. S7A and S8A), a negative regulator of p53 expression and function [24], giving rise to the possibility that MEK-ERK signaling maintains MDM2 expression to prevent p53 activation and subsequent downregulation of MGMT expression in stem-like glioblastoma cells. To test this possibility, we first attempted functional inactivation of MDM2 using Nutlin-3, a chemical inhibitor of MDM2, which disrupts physical interaction between MDM2 and p53 [25]. Nutlin-3 treatment of stem-like glioblastoma cells increased the expression of the p53 protein accompanied by increased and decreased expressions of p21 and MGMT, respectively, and knockdown of p53 prevented the change of their expression levels (Fig. 4B, Supporting Information Fig. S7B). Similar results were obtained when MDM2 was inactivated by siRNA-mediated knockdown (Fig. 4C, Supporting Information Fig. S7C). In TGS04 cells, which originally express only a low level of MGMT, MDM2 inactivation led to increased p53 expression but again no change in p21 or MGMT expression level (Supporting Information Fig. S8C). Thus, inactivation of MDM2, be it either chemical or genetic, resulted in p53 activation and p53-dependent suppression of MGMT expression in stem-like glioblastoma cells with transcriptionally competent p53. Collectively, the results suggest that the MEK activity is required for MDM2 expression, which in turn is required for the maintenance of MGMT expression through negative regulation of p53.

Figure 4. MDM2 expression is MEK-dependent and contributes to the maintenance of MGMT expression through inhibition of p53 in stem-like glioblastoma cells. (A): The expression levels of MDM2 and phospho-ERK in stem-like glioblastoma cells treated with or without 10 μM SL327 for 3 days were determined by immunoblot analysis. (B): The stem-like glioblastoma cells transfected with an siRNA against p53 or with a nontargeting control were treated, 24 hours after transfection, with or without Nutlin-3 (10 μM) for 3 days. The cells were then analyzed by immunoblotting with the indicated antibodies. (C): The stem-like glioblastoma cells were transfected first with an siRNA against p53 or with a nontargeting control and then, 24 hours later, transfected with an siRNA against MDM2 or with a nontargeting control. The cells were subjected to immunoblot analysis with the indicated antibodies 72 hours after the second transfection. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; MDM2, murine double minute 2; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; MGMT, O⁶-methylguanine DNA methyltransferase.

Combination of Temozolomide Treatment with MEK Inhibition Promotes Elimination of Tumorigenic Population from Stem-Like Glioblastoma Cells

The results of this study thus far suggest that targeting of the molecule(s) in the MEK-ERK-MDM2-p53-MGMT pathway in combination with temozolomide treatment would be a rational therapeutic approach to glioblastoma. While stem-like glioblastoma cells maintained in the absence of serum generally closely phenocopy the original, bulk tumors from which they are derived, they are considered at the same time to specifically represent a highly tumorigenic subpopulation within the original tumors [10–12]. Our recent study showed that MEK inhibition promotes commitment of stem-like glioblastoma cells to differentiation and thereby reduces their tumorigenic potential; however, the suppressive effect of SL327 as a single-agent on stem-like glioblastoma cell tumorigenesis was limited [14]. We therefore asked in this study whether temozolomide treatment in combination with SL327, via the synergistic mechanism observed in vitro earlier in this study (Fig. 2C), further eliminates a subpopulation of stem-like glioblastoma cells that would have evaded commitment to differentiation and contributed to brain tumor formation if treated singly with SL327 (Fig. 5). In line with our earlier report using a different set of stem-like cells [14], nude mice transplanted intracranially with stem-like glioblastoma cells (TGS01) treated with SL327 survived significantly longer than control mice receiving vehicle-treated cells (median survival, 43 days for SL327 vs. 27 days for control). In contrast, temozolomide treatment (50 μM) alone extended survival of the mice significantly but modestly (median survival, 35 days), consistent with the in vitro observation that TGS01 cells are highly resistant against the cytotoxic effect of temozolomide unless pretreated with SL327. However, treatment of stem-like glioblastoma cells with temozolomide in combination with SL327 further extended mouse survival by more than 2 weeks (median survival, 60 days) compared with SL327 treatment alone. Thus, the results suggest that combination of temozolomide treatment with MEK inhibition is superior to MEK inhibition alone in terms of depleting the tumorigenic population of glioblastoma cells, presumably due to temozolomide-mediated killing of stem-like glioblastoma cells that remained undifferentiated but had reduced MGMT expression after MEK inhibition.

Figure 5. Effective elimination of tumor-initiating population of stem-like glioblastoma cells by combination of MEK inhibitor and temozolomide treatments. TGS01 stem-like glioblastoma cells pretreated with or without SL327 (10 μM) for 3 days were subsequently treated with or without TMZ (50 μM) for 4 hours and then, 72 hours after temozolomide treatment, implanted orthotopically into the brains of nude mice (five mice per group), as detailed in Materials and Methods section. Survival of mice was evaluated by Kaplan-Meier analysis. Abbreviations: DMSO, dimethyl sulfoxide; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; TMZ, temozolomide.

DISCUSSION

In this study, we have demonstrated that (1) a significant proportion (three out of four examined) of stem-like glioblastoma cells directly derived from glioblastoma tissues (patient-derived stem-like glioblastoma cells) express MGMT, (2) MGMT expression contributes to temozolomide resistance of stem-like glioblastoma cells in a quantitative manner, and (3) a signaling pathway, MEK-ERK-MDM2-p53-MGMT, is operative in stem-like glioblastoma cells and plays a key role in the regulation of their MGMT expression. Combined, these results imply that the molecules acting on this pathway could be promising targets for intervention to control MGMT expression and overcome temozolomide resistance of glioblastoma. Specifically, we have also demonstrated in this study that (4) MEK targeting in combination with temozolomide treatment effectively reduces tumorigenic potential of stem-like glioblastoma cells, suggesting that the combination could also contribute to prevention of tumor recurrence.

Although we have revealed that MEK-ERK signaling controls MGMT expression via the MDM2-p53 axis, the precise molecular links between ERK and MDM2 and between p53 and MGMT still remain elusive in this study. In this respect, it would be informative to point out that previous reports have established direct links between these molecules, demonstrating that the MEK-ERK pathway regulates MDM2 expression via direct control of the MDM2 promoter activity through Ets and transcription factor activator protein 1 as well as by control of MDM2 mRNA export to the cytoplasm [26, 27], and that p53 interferes with Sp1 activation of the MGMT promoter [23]. Nevertheless, the signaling pathway(s) controlling the expression of MGMT in stem-like glioblastoma cells may not be as simple as the linear model proposed in this study and may involve more complex regulation. For instance, MEK-ERK signaling may, in addition to MDM2, involve other p53 regulators to inhibit p53 activation. The regulation of the signaling pathway could be further complicated by interacting pathways activated by EGF and/or bFGF present in the stem cell culture medium. Although our results do not necessarily exclude such possibilities, the essence of our findings lies in the fact that, from a therapeutic perspective, targeting MEK or MDM2 was sufficient to reduce MGMT expression in stem-like glioblastoma cells. Notably, TGS04 was unique among the cells tested in this study in that p53 upregulation elicited by inhibition of MEK or MDM2 failed to induce p21 (Supporting Information Fig. S8A–S8C) and that MGMT expression remained low despite effective knockdown of p53 (Supporting Information Fig. S8D). In line with these findings, the results of further analyses suggested that the p53 protein in TGS04 is a transcriptionally inactive mutant (Supporting Information Fig. S9) and that MGMT expression is silenced via promoter methylation in TGS04 cells (Supporting Information Fig. S10). Although it still remains to be determined whether or not p53 mutation is associated with MGMT promoter methylation [28, 29], apparently, the regulatory mechanism of MGMT expression in glioblastomas harboring p53 mutation, which reportedly account for approximately one-third of glioblastoma cases [30], becomes an important subject of future investigation. In this regard, given a recent, small-scale study reporting that the p53 status had little to do with temozolomide sensitivity of glioblastoma-derived brain tumor stem cell lines [31], it would also be of significance to examine in a larger scale the sensitivity of stem-like glioblastoma cells with p53 mutation to temozolomide, alone and in combination with a MEK inhibitor.

To the best of our knowledge, this is not only the first report to demonstrate the involvement of the signaling pathway proposed here (MEK-ERK-MDM2-p53-MGMT) in the regulation of MGMT expression of glioblastoma cells in general but also the first study to dissect the molecular pathway regulating MGMT expression specifically in stem-like cells. The use of stem-like glioblastoma cells as an experimental model has great advantages from a clinical point of view. Aside from the fact that glioblastoma cells cultured in vitro under conditions designed to selectively propagate cells with stem-like properties (i.e., stem-like glioblastoma cells) may represent only a small fraction of tumor cells with high tumorigenic capacity, previous studies clearly demonstrated that these stem-like cells very closely, albeit not totally identically, mirror the genomic and transcriptional profiles of the original “bulk tumors” compared with serum-cultured cells and conventional cell lines [10, 11, 32]. We can therefore expect with a higher probability that the mechanism that we have disclosed in this study is indeed operative in patient glioblastomas. In addition, the findings obtained from this study are of direct use in targeting stem-like glioblastoma cells presumed to be the source of recurrence and should as such have great impact on the long-term outcome of glioblastoma patients [12, 13]. It is noteworthy that temozolomide reportedly preferentially depletes cancer stem cells in glioblastoma. However, it was also shown that glioblastoma cancer stem cells expressing a high level of MGMT are totally refractory to therapeutically relevant concentrations of temozolomide [33], again underscoring the necessity of developing methods to overcome temozolomide resistance of stem-like glioblastoma cells. In this regard, we have successfully demonstrated in this study that MEK inhibition reverses temozolomide resistance of stem-like glioblastoma cells and, when combined with temozolomide treatment, suppresses their tumorigenicity much more efficiently.

Another important aspect that needs to be taken into consideration in terms of clinical application is the specificity of the signaling pathway controlling MGMT expression. As exemplified by the case of O⁶-benzylguanine, a specific inhibitor of MGMT yet nonselective with respect to cell type, targeting molecules active in both normal and tumor tissues enhances temozolomide sensitivity of the tumor at the expense of increased temozolomide toxicity against normal tissues, inevitably limiting options for the route of drug delivery [34]. Viewed this way, the upstream components of the signaling pathway that we have disclosed in this study could reasonably be preferred targets for clinical application, given the previous reports demonstrating that MEK-ERK signaling is aberrantly activated in the majority of glioblastomas [35, 36] and that amplification of the MDM2 gene per se occurs in as much as 14% of glioblastomas [30]. Indeed, MEK inhibitor treatment of normal human fibroblasts, IMR-90, neither decreased MGMT expression nor increased temozolomide sensitivity of IMR-90 cells, which provides good support for this idea (Supporting Information Fig. S11). However, intriguingly, more detailed analyses of IMR-90 revealed that inhibition of MEK or MDM2 caused upregulation of p53 and p21 without alteration of the MGMT expression level (Supporting Information Fig. S11). Thus, IMR-90 may use a mechanism independent of the MEK-ERK-MDM2-p53-MGMT pathway to maintain MGMT expression, suggesting the existence of another level of selectivity in addition to the proposed ones mentioned above.

We obtained initial clues that led to this study when we noticed downregulation and upregulation of MGMT and p53, respectively, in stem-like glioblastoma cells undergoing MEK inhibition-induced differentiation. As MGMT downregulation was also observed in stem-like glioblastoma cells undergoing serum-induced differentiation (Supporting Information Fig. S12), we initially assumed that MGMT expression might be one of the stem-like properties of glioblastoma cells and that p53 might also be a master regulator of such stem-like properties including MGMT expression. However, our recent observation that p53 activation does accompany MEK inhibition-induced differentiation (this study), but not serum-induced differentiation of stem-like glioblastoma cells (Supporting Information Fig. S12), suggests that p53 activation may not always be essential for their differentiation and associated downregulation of MGMT expression. Although the exact role of MDM2 and p53 in the maintenance/differentiation of stem-like glioblastoma cells is still to be investigated, targeting molecules involved in the maintenance of both stem-like state and MGMT expression (e.g., MEK) may be more reasonable in that, by doing so, we can expect inhibition of tumorigenic potential of stem-like glioblastoma cells as well in addition to MGMT inactivation.

CONCLUSION

To date, a number of methods have been described to inhibit MGMT; however, their significance in clinical use for the purpose of overcoming temozolomide resistance is still limited or unknown [3, 37, 38]. Here, we have shown that the molecular pathway linking MEK to MGMT expression could offer novel and rational targets for MGMT inactivation, specifically MEK (by SL327) and MDM2 (by Nutlin-3). This study thus constitutes a significant step forward in the battle against glioblastoma, by adding new arms to our arsenal to combat this deadly disease.

Acknowledgements

We thank Drs. Tomoki Todo and Nobuhito Saito at the University of Tokyo for generously providing us with the TGS cells; Dr. Motoo Nagane at Kyorin University for valuable advice on xenograft experiments; Keita Shibuya and Dr. Tomoko Kagawa of our laboratory for his expertise in flow cytometry and for her continuous support/encouragements, respectively. This work was supported by Grants-in-Aid for Scientific Research, for Challenging Exploratory Research, and for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Grant-in-Aid from the Global COE Program of the Japan Society for the Promotion of Science, by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare of Japan, and by a grant from the Japan Brain Foundation.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no conflicts of interest.