Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy

Abstract

Oncogenic activation of the mTOR signaling pathway occurs frequently in tumor cells and contributes to the devastating features of cancer, including breast cancer. mTOR inhibitors rapalogs are promising anticancer agents in clinical trials; however, rapalogs resistance remains an unresolved clinical challenge. Therefore, understanding the mechanisms by which cells become resistant to rapalogs may guide the development of successful mTOR-targeted cancer therapy. In this study, we found that eEF-2K, which is overexpressed in cancer cells and is required for survival of stressed cells, was involved in the negative-feedback activation of Akt and cytoprotective autophagy induction in breast cancer cells in response to mTOR inhibitors. Therefore, disruption of eEF-2K simultaneously abrogates the two critical resistance signaling pathways, sensitizing breast cancer cells to rapalogs. Importantly, we identified mitoxantrone, an admitted anticancer drug for a wide range of tumors, as a potential inhibitor of eEF-2K via a structure-based virtual screening strategy. We further demonstrated that mitoxantrone binds to eEF-2K and inhibits its activity, and the combination treatment of mitoxantrone and mTOR inhibitor resulted in significant synergistic cytotoxicity in breast cancer. In conclusion, we report that eEF-2K contributes to the activation of resistance signaling pathways of mTOR inhibitor, suggesting a novel strategy to enhance mTOR-targeted cancer therapy through combining mitoxantrone, an eEF-2K inhibitor.

Fig. 1. eEF-2K-eEF2-AMPK signaling pathway is involved in the Akt activation induced by mTOR inhibitor.

MDA-MB-231 or MCF-7 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF-2K, followed by treatment with 100 nM rapamycin (A) or everolimus (B) for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. C MDA-MB-231 or MCF-7 cells were pretreated with 0.25 μM NH125 for 1 h, followed by treatment with 100 nM everolimus for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. D, E MDA-MB-231 or MCF-7 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF2. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. F The content of ATP in MCF-7 cells (shCtrl and sheEF2) was analyzed by the ATPlite Luminescence Assay Kit. The results are displayed as the means ± SD of triplicate measurements from one of three identical experiments; **P < 0.01, t test. G MCF-7 and MDA-MB-231 cells were transfected with nontargeting shRNA or shRNA targeting AMPK. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. H MCF-7 and MDA-MB-231 cells (shCtrl and sheEF2) were further transfected with HA-AMPK. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. All experiments were repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2. Inhibition of eEF-2K abrogates rapalogs-induced autophagy.

MDA-MB-231 or MCF-7 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF-2K, followed by treatment with 100 nM rapamycin (A) or everolimus (B) for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. C MDA-MB-231 or MCF-7 cells were pretreated with 0.25 μM NH125 for 1 h, followed by treatment with 100 nM rapamycin for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. All experiments were repeated three times.

Fig. 3. Inhibition of eEF-2K enhanced the anticancer effect of mTOR inhibitors in vitro.

A MCF-7 or MDA-MB-231 cells (shCtrl and sheEF-2K) were treated with 100 nM rapamycin for indicated durations. Cell proliferation was analyzed by cell counting. **P < 0.01, ***P < 0.001, t test. B MCF-7 or MDA-MB-231 cells (shCtrl and sheEF-2K) were seeded for colony-formation assay. The results are displayed as means ± SD (n = 3). **P < 0.01, ***P < 0.001, t test. C MCF-7 cells which were transfected with nontargeting siRNAs or siRNAs targeting eEF-2K or MDA-MB-231 cells (shCtrl and sheEF-2K) followed by treatment with 100 nM everolimus for 24 h. Cell proliferation was detected by Edu incorporation assay. The results are displayed as means ± SD (n = 3). **P < 0.01, ***P < 0.001, t test. Scale bar: 100 μm. D MDA-MB-231 cells (shCtrl and sheEF-2K) were treated with 100 nM everolimus for 48 h. Cell cycle was assessed by PI staining and flow cytometry. E MDA-MB-231 cells (shCtrl and sheEF-2K) were treated with 100 nM everolimus for 48 h. Cell cycle-related proteins were analyzed by western blot. Tubulin was used as a loading control. F MDA-MB-231 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF-2K or siRNAs targeting Beclin 1, followed by treatment with different concentrations of rapamycin or everolimus for 72 h. Cell viability was analyzed by CCK8 reagent. The results are displayed as the means ± SD of triplicate measurements from one of three identical experiments; *P < 0.05, **P < 0.01, ***P < 0.001, t test.

Fig. 4. Inhibiting eEF-2K sensitizes breast cancer to everolimus in vivo.

A Growth curve, B dissected tumors, C tumor weight, and D mouse body weight for the xenograft experiments with MDA-MB-231 cells (shCtrl and sheEF-2K) inoculated subcutaneously into flanks of nude mice treated with 5 mg/kg everlomus. Visible tumors were measured every 2 days. E Effect of everolimus treatment on in vivo proliferation was evaluated by IHC staining of ki67 (a marker of proliferative cells). Scale bars: 200 μm.

Fig. 5. Discovering new eEF-2K inhibitor from FDA-approved drugs.

A The sequence alignment result between the template protein and eEF-2K. B The Ramachandran diagram is used for validating the eEF-2K homology model. C The 3D eEF-2K structure from the homology modeling together with its corresponding binding site.

Fig. 6. Mitoxantrone blocked the activation of Akt and autophagy induced by mTOR inhibitor by targeting eEF-2K.

A MDA-MB-231 cells in growth media or starved were treated with indicated concentrations of mitoxantrone for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control. B SPR was performed using purified recombinant eEF-2K with increasing concentrations of mitoxantrone. C Schematic illustration of the interaction between eEF-2K and mitoxantrone. D MDA-MB-231 or MCF-7 cells were pretreated with 1 μM mitoxantrone for 1 h, followed by treatment with 100 nM rapamycin or everolimus (E) for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. β-actin was used as a loading control.

Fig. 7. Combination treatment with mTOR inhibitor and mitoxantrone synergistically inhibits the growth of breast cancer cells in vitro and in vivo.

A MCF-7 cells were treated with 1 μM mitoxantrone or NH125 in the presence or absence of 100 nM everolimus. Colony formation was assessed by crystal violet staining quantification. The results are displayed as means ± SD (n = 3). **P < 0.01, t test. B MCF-7 or MDA-MB-231 cells were pretreated with 1 μM mitoxantrone or NH125 for 1 h, followed by treatment with rapamycin for 72 h. Cell viability was analyzed by CCK8 reagent. The results are displayed as the means ± SD of triplicate measurements from one of three identical experiments; *P < 0.05, **P < 0.01, ***P < 0.001, t test. C Dissected tumors, D tumor weight, E growth curve, and F mouse body weight for the xenograft experiments with MDA-MB-231 cells inoculated subcutaneously into flanks of nude mice treated with 5 mg/kg everolimus and 0.5 mg/kg mitoxantrone. Visible tumors were measured every 2 days. G Effect of everolimus and mitoxantrone treatment on in vivo proliferation was evaluated by IHC staining of ki67 (a marker of proliferative cells). Scale bars: 200 μm. H Liver and kidney toxicities were observed after the course of therapy by detecting blood ALT, AST, nitrogen urea, and creatinine.

Fig. 8. Schematic diagram for eEF-2K-dependent regulation of rapalogs-induced Akt and autophagy activation.

The model shows that eEF-2K regulates AMPK activity by controlling the process of peptide elongation, thereby regulating Akt phosphorylation. Mitoxantrone abrogates eEF-2K-mediated Akt activation and autophagy, enhanced mTOR inhibitors anti-breast cancer effect in vivo.