Autophagy inhibition synergizes with calcium mobilization to achieve efficient therapy of malignant gliomas

Abstract

Autophagy plays a critical role in tumorigenesis, but how autophagy contributes to cancer cells’ responses to chemotherapeutics remains controversial. To investigate the roles of autophagy in malignant gliomas, we used CRISPR/CAS9 to knock out the ATG5 gene, which is essential for autophagosome formation, in tumor cells derived from patients with glioblastoma. While ATG5 disruption inhibited autophagy, it did not change the phenotypes of glioma cells and did not alter their sensitivity to temozolomide, an agent used for glioblastoma patient therapy. Screening of an anticancer drug library identified compounds that showed greater efficacy to ATG5‐knockout glioma cells compared to control. While several selected compounds, including nigericin and salinomycin, remarkably induced autophagy, potent autophagy inducers by mTOR inhibition did not exhibit the ATG5‐dependent cytoprotective effects. Nigericin in combination with ATG5 deficiency synergistically suppressed spheroid formation by glioma cells in a manner mitigated by Ca2+ chelation or CaMKK inhibition, indicating that, in combination with autophagy inhibition, calcium‐mobilizing compounds contribute to efficient anticancer therapeutics. ATG5‐knockout cells treated with nigericin showed increased mitochondria‐derived reactive oxygen species and apoptosis compared to controls, indicating that autophagy protects glioma cells from mitochondrial reactive oxygen species‐mediated damage. Finally, using a patient‐derived xenograft model, we demonstrated that chloroquine, a pharmacological autophagy inhibitor, dramatically enhanced the efficacy of compounds selected in this study. Our findings propose a novel therapeutic strategy in which calcium‐mobilizing compounds are combined with autophagy inhibitors to treat patients with glioblastoma.

Cyclosporine A sensitizes lung cancer cells to crizotinib through inhibition of the Ca2+/calcineurin/Erk pathway

“…Ca 2+ is not only involved in the regulation of Erk1/2 signaling but is also involved in the regulation of AKT and STAT3 signaling [62,63]. Ca 2+ also plays an important role in regulating autophagy [64,65] and epithelial-mesenchymal transition (EMT) [63], both of which are related to drug resistance to TKIs [28,66]. As a result, blocking Ca 2+ signaling may be a promising strategy that enhances the clinical efficacy of TKIs.…”

Applications of CRISPR-Cas9 Technology to Genome Editing in Glioblastoma Multiforme

“…A study has shown inhibition of autophagy induction by CRISPR-Cas9-mediated ATG5 gene knockout in TGS01 and TGS04 cells in conjunction with a calcium mobilization agent (nigericin) that works together to increase mitochondrial reactive oxygen species for cell death [ 55 ]. Another study showed that CRISPR-Cas9 mediated knockout of TSC2 (Tuberous Sclerosis 2) gene, an autophagy promoting molecule, in GBM LN18 cells rendering them to be more susceptible to cell death in response to photodynamic therapy (PDT) [ 56 ].…”

Ionophores: Potential Use as Anticancer Drugs and Chemosensitizers

“…Autophagy inhibition potentiates NIG treatment. In a recent study Vu et al demonstrated a reduction in spheroid formation by ATG5 deficient glioma cells on treatment with NIG [ 138 ].…”