ACOT9, a mitochondrial metabolism-related gene, promotes ROS-associated epithelial remodeling in laryngeal squamous cell carcinoma

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is characterized by mitochondrial metabolic reprogramming, but its prognostic significance and underlying molecular mechanisms remain insufficiently understood. This study aimed to develop a prognostic mitochondrial metabolism-related genes signature (MMGS) for LSCC and to investigate the biological role of ACOT9 in tumor progression. METHODS: Differentially expressed mitochondrial metabolism-related genes (MMGs) were identified from TCGA-LSCC data. A MMGS was established through machine-learning evaluation of 178 model combinations and validated in the TCGA training cohort, TCGA test cohort, and the independent GSE65858 dataset. To explore its biological relevance, immune microenvironment analysis, single-cell RNA sequencing analysis of GSE290927, scTenifoldKnk-based virtual knockout analysis, and a series of experimental assays, including immunohistochemistry, immunofluorescence, qRT-PCR, Western blotting, ROS detection, CCK-8, wound-healing, and apoptosis assays, were performed to characterize immune features, cellular heterogeneity, and the functional role of ACOT9. RESULTS: The MMGS demonstrated excellent prognostic performance, with a C-index greater than 0.7388, and independently predicted overall survival in LSCC patients (multivariate Cox, p < 0.05). High-risk patients exhibited increased M0 macrophage infiltration, impaired T-cell co-stimulation, downregulated co-stimulatory molecules, and enrichment of drug metabolism-related pathways. Single-cell analysis revealed elevated MMGS scores in LSCC epithelial and myeloid cells, while ACOT9 was markedly upregulated in malignant epithelial subtypes and positively correlated with stemness (R = 0.18, p < 0.01). Virtual knockout of ACOT9 in epithelial cells identified LAMC2 and KRT17 as the two most significantly perturbed genes, and enrichment analyses indicated a downstream epithelial stress-remodeling network involving keratinization, extracellular matrix organization, inflammatory/immune signaling, and lipid inflammatory pathways. Experimental validation confirmed ACOT9 overexpression, mitochondrial colocalization with TOM20, and elevated mitochondrial ROS levels in LSCC tissues and cells. Furthermore, mitochondrial ROS scavenging or ACOT9 knockdown significantly reduced cell viability and migration while promoting apoptosis. CONCLUSIONS: MMGS is a robust and reliable prognostic tool for LSCC. ACOT9 functions as a mitochondrial-associated oncogenic factor that may promote LSCC aggressiveness through a mitochondrial ROS-dependent epithelial remodeling program, highlighting its potential as a therapeutic target for risk-stratified clinical management.