Abstract
Glioblastoma Multiforme (GBM), a highly aggressive primary brain tumor, often develops resistance to standard treatments such as ionizing radiation (IR), which leads to a median survival of about 15 months. GBM contains glioblastoma stem cells (GSCs), which are crucial in driving therapeutic resistance and recurrence. In our study, we conducted comprehensive analyses to evaluate the molecular changes associated with radioresistance and explored the role of the Spy1-CLIP3 axis as a key regulator of GSC maintenance. Our methods included quantitative PCR and immunoblotting to measure CLIP3 mRNA and protein levels, revealing a significant reduction in expression among radioresistant GBM cells. We found that exposure to IR leads to the upregulation of Spy1 and downregulation of CLIP3, contributing to the development of radioresistance in surviving GBM cells. We used metabolic flux analysis techniques to observe changes in glycolytic activity, which increased in correlation with CLIP3 downregulation. IR induces CLIP3 downregulation, increasing glycolytic activity by facilitating the trafficking of GLUT3, the primary glucose transporter in GSCs. Additionally, colony-forming ability was evaluated using colony-forming assay after treatment of glimepiride with or without IR. Glimepiride didn’t reduce colony-forming ability in non-irradiated cells, but when combined with IR, it significantly reduced colony-forming ability, possibly by disrupting maintenance of GSCs. Importantly, our data reveal that glimepiride, a CLIP3 activator targets GSCs metabolism and enhances radiosensitization while maintaining low toxicity. The radioresistant GBM cells that survive radiotherapy exhibit increased stemness and glycolytic activity mediated by the Spy1-CLIP3 axis. Overall, our findings suggest that clinical trials using glimepiride for GBM patients may potentially improve survival rates, particularly for those experiencing recurrence after radiotherapy. This research highlights the importance of targeting cellular metabolism and stem cell properties in the context of GBM radioresistance and suggests potential avenues for improving the effectiveness of existing treatments.