Hypoxia Enhanced Glioblastoma Resistance to Erastin-Induced Ferroptosis by Up-Regulating GPX4 via PI3K/AKT/HIF-1α Axis

Background Glioblastoma is the deadliest type of primary brain tumor with a high rate of recurrence and treatment resistance. Hypoxia contributed much to radiotherapy resistance and chemoresistance of cancer. Ferroptosis is a nonapoptotic, oxidative cell death and identified as a potential anticancer mechanism in recent years. Erastin acts as a ferroptosis activator and shows a potential role in tumor treatment but the relationship between hypoxia and erastin resistance in glioblastoma has not been explained. This study aimed to investigate the role and underlying mechanism of hypoxia in erastin-induced ferroptosis in glioblastoma. Methods Cell proliferation and viability were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, TUNEL assays, and clone formation assay. Lipid peroxides level was analyzed by Malondialdehyde (MDA) assay and flow cytometry using C11-BODIPY dye. The correlation between HIF-1α and GPX4 expression was detected in data from the TCGA database and was determined by ChIP-qPCR assay and luciferase reporter assay. Subcutaneous xenograft and orthotopic xenograft models were established to test our findings in vivo. Results Hypoxia for at least 16 hours significantly suppressed erastin-induced ferroptosis by up-regulating glutathione peroxidase 4 (GPX4) expression in U87 and U251 cells. Hypoxia promotes GPX4 expression via enhancing the PI3K/AKT/HIF-1α pathway. Mechanistically, HIF-1α directly bound to the GPX4 gene promoter region and promoted GPX4 transcription. AKT inhibitor MK-2206 and HIF-1α inhibitor PX-478 could significantly reverse the effect. Besides, under normoxia, PX-478 could induce a higher lipid peroxidation level by decreasing GPX4 expression in U87 and U251 cells but cannot induced cell death directly, and it could significantly enhance the tumor cell killing effect of erastin. In vivo, combination of PX-478 and erastin had a coordinated intensification effect on anticancer activity uncovered by subcutaneous xenograft and orthotopic xenograft mouse models. Conclusions Hypoxia enhanced glioblastoma resistance to erastin-induced ferroptosis by activating PI3K/AKT/HIF-1α pathway and promoting GPX4 expression in a transcriptional regulation way. Combination therapy of PX-478 and erastin may be a potential strategy against glioblastoma.