The mTORC1/mTORC2 inhibitor AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells

Abstract

Background: The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).

Methods: mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation.

Results: Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments.

Conclusions: These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.

Keywords: AZD2014; Radiation; glioblastoma; mTOR; orthotopic xenograft; tumor stem cell.

Fig. 1.

Effect of AZD2014 on mTORC1 and mTORC2 activities in CD133+ GBMJ1 cells. (A) Cells in monolayer culture were exposed to the indicated concentration of AZD2014 for 1 hour and collected for immunoblot analysis. (B) Cells were exposed to AZD2014 (2 µM) for the specified time and collected for analysis. β-actin was used as a loading control; blots are representative of 2 independent experiments.

Fig. 2.

Influence of radiation on mTORC1 and mTORC2 activities. GBMJ1 CD133+ cells were irradiated (2 Gy) and collected at the specified times for immunoblot analysis. β-actin was used as a loading control; blots are representative of 2 independent experiments.

Fig. 3.

Effects of AZD2014 on GSC radiosensitivity. (A) GBMJ1 CD133+ (B) NSC23 CD133+, (C) GBAM1 CD133+, (D) 0927 CD15+. Cells were seeded into poly-L-lysine coated tissue culture plates and allowed to attach overnight with AZD2014 (2 µM) then added 1 hour before irradiation. Twenty-four hours after irradiation, media was removed, and fresh drug-free media was added. Colony-forming efficiency was determined 21 days later, and survival curves were generated after normalizing for cytotoxicity induced from drug alone. Values represent the mean ± SE of 3 independent experiments.

Fig. 4.

The influence of timing of AZD2014 treatment on GSC radiosensitivity. GBMJ1 CD133+ cells were seeded and allowed to attach overnight. AZD2014 (2 µM) was added to cultures 24 hours before irradiation (24 h Pre-IR), 2 hours before (2 h Pre-IR), 1 hour before (1 h Pre-IR), or 1 hour after (1 h Post-IR) irradiation. Twenty-four hours after irradiation, media was removed, and fresh drug-free media was added. Colony-forming efficiency was determined 21 days later, and survival curves were generated after normalizing for cytotoxicity induced from drug alone. Values represent the mean ± SE of 3 independent experiments.

Fig. 5.

Influence of AZD2014 on the G2/M checkpoint and H2AX foci levels in irradiated GBMJ1 and GBAM1 cells. (A) G₂/M checkpoint activation was determined by mitotic index (% cells in mitosis). Left panel: GBMJ1; right panel: GBAM1. AZD2014 (2 µM) was added 1 hour before irradiation (IR) (2 Gy), which was followed by immediate addition of nocodazole (50 ng/mL). Cells were collected at specified time points for cell cycle distribution analysis and determination of phospho-H3 expression. Values represent the mean ± SE of 3 independent experiments. (B) Radiation-induced γH2AX foci formation and dispersal. Left panel: GBMJ1; right panel: GBAM1. AZD2014 (2 µM) was added 1 hour prior to irradiation (2 Gy) with cells collected at specified times. The number γH2AX foci were determined in at least 50 nuclei per treatment condition. Values represent the mean ± SE of 3 independent experiments, *P < .05.

Fig. 6.

Effects of AZD2014 on mTOR activity in orthotopic xenografts initiated from CD133+ GBMJ1 cells. At the onset of morbidity (mean, 52 days), mice bearing orthotopic xenografts were exposed to vehicle or AZD2014 (50 mg/kg, oral gavage) and collected 2 hours later for immunohistochemical evaluation: total 4EBP1 (green), p4E-BP1 t37/46 (green), AKT (green), pAKT s473 (green), nestin to identify human tumor cells (red), and nuclei (blue), 40x magnification.

Fig. 7.

Influence of AZD2014 on the radioresponse of orthotopic xenografts initiated from CD133+ GBMJ1 cells. At 12 days after orthotopic implant, mice were randomized and treatment initiated as described. Mice were followed until the onset of morbidity. Kaplan–Meier survival curves were generated with log-rank analysis for comparison.