IFIT3 silencing improves the sensitivity of gliomas to temozolomide by inactivating the PI3K/AKT signaling pathway.

Background

Gliomas are highly aggressive primary brain tumors with a dismal prognosis. Temozolomide (TMZ) serves as the first-line chemotherapeutic agent for glioma patients. However, the clinical efficacy of TMZ is severely limited by the inevitable development of acquired chemoresistance, which ultimately leads to tumor recurrence and treatment failure. Unraveling the molecular mechanisms underlying TMZ resistance is therefore critical for improving glioma prognosis. This study aimed to identify key genes driving TMZ resistance and explore their underlying mechanisms to provide novel therapeutic targets for overcoming this clinical challenge.

Methods

Differentially expressed genes (DEGs) between TMZ-resistant (LN229TR, U251TR, and U87TR) and TMZ-sensitive glioma cells were screened using GEO datasets. The intersecting DEGs were subjected to protein-protein interaction (PPI) network construction via the STRING database and visualized using Cytoscape software. Hub genes were identified by integrating the results from the Maximal Clique Centrality (MCC) and Density of Maximum Neighborhood Component (DMNC) algorithms. The expression patterns of candidate hub genes were validated in glioma cells, clinical tissues, and the Gene Expression Profiling Interactive Analysis (GEPIA) database. Functional assays, including cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine, colony formation, transwell, flow cytometry, and sphere formation assays, were performed to evaluate cell viability, proliferation, migration, apoptosis, and stem-like properties in vitro. Furthermore, a subcutaneous xenograft tumor model in mice was established to assess the in vivo therapeutic effects.

Results

A total of 320 intersecting DEGs were extracted from the three cell line groups, and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) along with 2'-5'-oligoadenylate synthetase like (OASL) were ultimately identified as the core hub genes. IFIT3 was selected for further investigation due to its significant upregulation in both low-grade gliomas and glioblastoma compared to normal brain tissues. Consistently, IFIT3 expression was remarkably elevated in TMZ-resistant glioma tissues and cell lines (P<0.05), which exhibited significantly higher half-maximal inhibitory concentration (IC50) values of TMZ than their sensitive counterparts (P<0.05). Functionally, IFIT3 silencing significantly decreased the IC50 of TMZ (P<0.05), suppressed cell proliferation (P<0.05), migration (P<0.05), and stem-like traits (P<0.05), and induced apoptosis (P<0.05) in resistant glioma cells. Conversely, ectopic IFIT3 expression exerted opposite effects on cell proliferation, migration, and stem-like traits and notably increased the ratios of phosphorylated phosphoinositide 3-kinase (PI3K) to total PI3K and phosphorylated AKT to total AKT (P<0.05); however, these effects induced by IFIT3 overexpression were effectively reversed by the PI3K inhibitor LY294002 (P<0.05). In vivo experiments demonstrated that knocking down IFIT3 expression remarkably reduced tumor volume and weight upon TMZ treatment, accompanied by decreased expression levels of IFIT3, nuclear proliferation marker (Ki-67), and phosphorylated AKT in tumor tissues (P<0.05).

Conclusion

IFIT3 overexpression conferred TMZ resistance and promoted multiple malignant phenotypes in gliomas by activating the PI3K/AKT signaling pathway. Clinically, targeting IFIT3 could effectively re-sensitize resistant gliomas to TMZ, thereby offering a novel and actionable strategy to overcome chemoresistance and improve clinical outcomes for glioma patients.