Autophagy Flux Blockade Alters Cholesterol Metabolism and Sustains Temozolomide Resistance in Glioblastoma

Temozolomide (TMZ) resistance remains a major barrier to effective glioblastoma therapy and is increasingly linked to metabolic adaptation and cellular stress responses. In this study, we investigated the relationship between autophagy dysregulation and cholesterol metabolism during glioma progression and TMZ resistance. Tissue microarray analysis of astrocytoma and glioblastoma specimens demonstrated progressive accumulation of LC3β puncta together with increased FDPS expression, suggesting coordinated alterations in autophagy and cholesterol metabolism in high-grade tumors. To explore this relationship mechanistically, TMZ-resistant U251 glioblastoma cells were generated and compared with TMZ-sensitive counterparts. Resistant cells exhibited epithelial-to-mesenchymal transition–like morphology, reduced proliferation, mitotic quiescence, mitochondrial remodeling, and diminished respiratory reserve capacity, consistent with a therapy-tolerant metabolic phenotype. Ultrastructural and biochemical analyses revealed persistent autophagosome accumulation and impaired autophagy flux in resistant cells. This defect was accompanied by suppression of de novo cholesterol biosynthesis, reduced expression of SREBP2 and LDLR, and extensive remodeling of cholesterol ester species identified by lipidomic profiling. Although inhibition of the mevalonate pathway with simvastatin altered cholesterol ester composition, it failed to restore TMZ sensitivity or induce apoptosis in resistant cells. Together, these findings indicate that persistent autophagy flux impairment and altered cholesterol metabolism characterize TMZ-resistant glioblastoma.