Integrated Transcriptomic and Functional Analyses Identify SERPINE1 as a Ferroptosis-Associated Mediator of Glioblastoma Radioresistance

Background: Radiotherapy failure remains a major challenge in glioblastoma (GBM), yet the stress-adaptive programs that enable tumor cells to survive irradiation-induced lethal injury remain incompletely defined. This study investigated whether serpin family E member 1 (SERPINE1) contributes to GBM radioresistance by modulating ferroptosis-associated lipid peroxidation, a process linked to iron-dependent oxidative injury. Methods: Radiotherapy-treated GBM cohorts from TCGA (n = 93; 56 radiosensitive and 37 radioresistant cases), the Chinese Glioma Genome Atlas (CGGA)-325 (n = 100), and CGGA-693 (n = 193), as well as Clinical Proteomic Tumor Analysis Consortium (CPTAC) protein data and the Gene Expression Omnibus (GEO) single-cell RNA sequencing (scRNA-seq) dataset GSE131928, were analyzed using differential gene expression, weighted gene co-expression network analysis (WGCNA), machine-learning feature selection, FerrDb annotation, survival analysis, pathway enrichment, scRNA-seq, and tumor microenvironment (TME) signature profiling. Radiosensitive disease was defined as a complete or partial response after radiotherapy, whereas radioresistant disease was defined as stable or progressive disease. Statistical analyses included Wilcoxon rank-sum tests, Spearman correlations, log-rank tests, multivariable Cox regression for overall survival (OS), and time-dependent receiver operating characteristic (ROC) analysis. Functional validation was performed in two GBM cell lines (U87 and U251) using SERPINE1 knockdown or overexpression, X-ray irradiation, ferrostatin-1 (Fer-1), and RAS-selective lethal 3 (RSL3). Results: Integrative screening identified SERPINE1 as a ferroptosis-associated mediator of radioresistance and poor survival. SERPINE1 expression was elevated in primary GBM tumors (n = 199) compared with normal tissues (n = 18). SERPINE1-high tumors showed enrichment of ferroptosis driver, suppressor, and marker gene signatures, consistent with a ferroptosis-stressed but ferroptosis-defensive state. Single-cell analysis localized SERPINE1 mainly to mesenchymal-like malignant cells, where high expression co-occurred with hypoxia, extracellular matrix remodeling, radioresistance, and ferroptosis-defense programs. In U87 cells, SERPINE1 knockdown enhanced radiosensitivity and increased irradiation-induced lipid reactive oxygen species (ROS), malondialdehyde (MDA), and Fe2+ accumulation, whereas SERPINE1 overexpression in U251 cells attenuated ferroptosis-associated lipid peroxidation and iron accumulation and improved post-irradiation cell survival. Fer-1 partially rescued the radiosensitizing effect of SERPINE1 depletion, whereas RSL3 weakened SERPINE1-mediated radioprotection. Conclusions: SERPINE1 appears to link mesenchymal adaptation, redox stress regulation, and ferroptosis-associated lipid peroxidation defense in GBM radioresistance. These findings support further evaluation of SERPINE1-guided ferroptosis-based radiosensitization strategies. Public datasets were obtained from TCGA, CGGA, CPTAC, and GEO (GSE131928).