IKBKE Promotes Radioresistance of Glioblastoma through AKT/FOXO3a Pathway

Abstract

Glioblastoma is an intracranial highly malignant primary tumor, and postoperative radiotherapy is a common treatment of glioblastoma. While radiotherapy resistance of glioblastoma is an important reason for treatment failure. IKBKE is overexpressed in gliomas, but its role in radiotherapy is unknown. This study confirmed that IKBKE can directly phosphorylate AKT protein to regulate FOXO3a, thus promoting the radioresistance of glioblastoma, and proposed a new therapeutic strategy to enhance the efficacy of radiotherapy. We used flow cytometry, tunel staining, plate cloning, a cell counting kit and WB to confirm the effects of IKBKE and FOXO3a on radioresistance of glioblastoma, and immunofluorescence and WB were used to detect the expression of γ-H2AX. Subcutaneous tumor formation in mice and immunohistochemical staining was performed. IP combined with mass spectrometry, immunofluorescence, endogenous and exogenous IP were used to confirm the interaction between IKBKE and AKT. Point mutation, IP and WB were used to confirm the phosphorylation site of AKT. IP and some small molecule inhibitors were used to confirm the relationship between IKBKE, AKT and PI3K. The effect of IKBKE on FOXO3a was confirmed by WB and qPCR. The protein relationship among IKBKE, FOXO3a and 14-3-3 was confirmed by CHX, MG132, ubiquitin test, immunofluorescence and IP. The above experiments were carried out to verify the effect of Amlexanox, an IKBKE inhibitor, on glioblastoma. And its pharmacokinetics in the brain was determined by LC-MS to provide a theoretical basis for further clinical use. It was found that IKBKE could increase the radioresistance of glioblastoma in vitro and in vivo. IKBKE could directly phosphorylate AKT, and its phosphorylation sites were Ser473 and Thr308. We also certified that IKBKE activated AKT independent of PI3K. IKBKE inhibited the expression of FOXO3a on protein level, promoted its ubiquitin degradation, enhanced its interaction with 14-3-3, and inhibited its transportation into the nucleus. FOXO3a can increase the radiosensitivity of glioblastoma. Amlexanox, an IKBKE inhibitor, can inhibit the radiosensitivity of glioblastoma and partially pass through the blood-brain barrier to enhance the radiosensitivity of intracranial tumors. IKBKE can activate AKT independent of PI3K by directly phosphorylating AKT Ser473 and Thr308, thus increasing the phosphorylation of FOXO3a. Phosphorylated FOXO3a promoted its ubiquitin degradation, and inhibited its transportation into the nucleus, causing radioresistance in glioblastoma. IKBKE inhibitor Amlexanox can pass through the blood-brain barrier and increase the radiosensitivity of intracranial tumor cells.