BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor and exhibits marked resistance to standard therapies, including temozolomide (TMZ). Circadian rhythm disruption is increasingly recognized in GBM; however, the molecular factors linking circadian dysregulation to metabolic adaptability and therapy resistance remain poorly defined. This study investigates the role of transmembrane protein 5 (TMEM5; ribitol xylosyltransferase 1, RXYLT1) as a circadian-associated regulator in GBM.
METHODS: Integrative in silico analyses of TCGA-GBM and CircaDB datasets were performed to evaluate TMEM5 expression, circadian rhythmicity, and clinical relevance. TMEM5 function was examined using shRNA-mediated knockdown in primary GBM cells, followed by circadian gene expression profiling across Zeitgeber times, TMZ sensitivity assays, RNA sequencing, and pathway enrichment analyses. Functional assays included tumorsphere formation, migration and invasion assays, co-immunoprecipitation to assess TMEM5-CLOCK association, and Seahorse extracellular flux analysis to evaluate metabolic capacity. Candidate pharmacological modulators were identified using CellMinerDCB and evaluated alone or in combination with TMZ in vitro and in vivo.
RESULTS: TMEM5 expression was significantly elevated in GBM tissues, correlated with CLOCK and CRY1 expression, and associated with poor patient survival. TMEM5 knockdown disrupted Zeitgeber time-dependent oscillation of multiple core clock genes, including CLOCK, CRY1, PER1, BMAL1, CRY2, and PER2, and enhanced TMZ sensitivity. Transcriptomic profiling revealed coordinated alterations in circadian regulation, glycolytic metabolism, glycosylation, angiogenesis, and immune-related pathways. TMEM5 depletion suppressed GBM stem-like properties, migration, and invasion, and reduced both glycolytic and mitochondrial respiratory capacity. Pharmacological agents associated with reduced TMEM5 expressions, including dabrafenib and Nutlin-3, exhibited favourable interaction profiles with TMZ.
CONCLUSION: TMEM5 is associated with circadian-gene expression patterns, temozolomide response, and bioenergetic readouts in GBM models. TMEM5 knockdown was accompanied by altered time-dependent expression profiles of multiple clock genes, reduced OCR/ECAR parameters under the tested conditions, and increased TMZ sensitivity. These findings support TMEM5 as a circadian-associated factor in GBM and provide a rationale for future studies to define whether TMEM5 directly influences circadian regulation and metabolic pathway remodeling in vivo.
