Temozolomide Induces Glioblastoma Dedifferentiation into Highly Malignant Stem-like Cells Through Senescence-Associated Reprogramming via HIF1a/HIF2a Regulation [miRNA-seq]

A critical issue is that recurrent glioblastoma multiforme (GBM) after temozolomide (TMZ) exposure becomes more malignant, exhibiting higher invasion and stemness than the primary tumor. However, the detailed mechanism remains to be elucidated. While the majority of GBM cells succumb to TMZ treatment, some enter cell cycle arrest, adopt a senescence-associated secretory phenotype (SASP), and activate senescence-related signaling pathways. These cells later exit senescence, re-enter the cell cycle, and proliferate, forming aggregates with stemness characteristics, including high expression of stemness markers, colony formation, high invasion, migration, and chemotherapy resistance. Critically, these new aggregates promote the invasion, migration, and chemotherapy resistance of surrounding cells. Gene Set Enrichment Analysis (GSEA) and KEGG analysis of miRNA and mRNA sequences indicated that hallmark-hypoxia and HIF1-signaling pathways were activated. We verified that HIF1a and HIF2a levels changed before, during, and after TMZ treatment. Knocking out HIF1a and HIF2a in GBM cells and exposing them to TMZ resulted in fewer senescent cells and aggregates. This study clarifies how recurrent GBM becomes more malignant during and after TMZ treatment and highlights the regulatory roles of HIF1a and HIF2a, emphasizing that preventing senescence cell formation and inhibiting HIF1a and HIF2a expression are crucial for improving therapeutic outcomes. Overall design: After continuous induction with 50 µM TMZ, U-87MG and U-118MG cells formed resistant cell lines, which were then compared with sensitive cell lines using miRNA-seq.