Development and validation of a four-gene prognostic signature derived from disulfidptosis- and cuproptosis-related genes in oral squamous cell carcinoma

BACKGROUND: Disulfidptosis and cuproptosis are recently identified forms of regulated cell death whose component genes may harbor prognostic information in oral squamous cell carcinoma (OSCC). However, no validated multi-gene prognostic signature derived from these pathways has been established. METHODS: Differentially expressed genes from two GEO discovery cohorts (GSE30784 and GSE37991) were intersected with 28 disulfidptosis- and cuproptosis-related genes. A LASSO-Cox model was developed in a TCGA oral HNSC training cohort (n = 268) and validated in GSE41613 (n = 97), GSE65858 (n = 270), and GSE42743 (n = 55). An extended clinical-genomic model incorporating age, AJCC stage, histological grade, alcohol use, radiation, and chemotherapy was evaluated in a complete-case subcohort (n = 212). Single-cell RNA-seq data (GSE103322; 5,902 cells) were analyzed to characterize hub gene expression across cell types. Hub gene expression was validated by qRT-PCR and ELISA in OSCC cell lines. RESULTS: A four-gene signature (CDKN2A, FLNA, GLS, TLN1) was identified. The risk score was an independent prognostic factor (HR = 1.36 per SD, 95% CI: 1.08-1.72, P = 0.009) after adjustment for six clinical covariates. The extended combined model achieved a C-index of 0.669 and a 5-year AUC of 0.805, outperforming the extended clinical model (C-index 0.650; 5-year AUC 0.722) and gene-only model (C-index 0.563; 5-year AUC 0.684). The signature achieved significance in GSE41613 (P = 0.046, HR = 1.81) and GSE65858 (P = 0.0013, HR = 1.99), with a concordant trend in GSE42743 (P = 0.079, HR = 2.10). Single-cell analysis showed that CDKN2A was preferentially expressed in malignant cells, while FLNA and TLN1 were enriched in fibroblasts, consistent with ECM-mediated tumor-stromal crosstalk. CellChat revealed that high-activity malignant cells exhibited enhanced LAMININ, COLLAGEN, and THBS signaling. qRT-PCR and ELISA confirmed upregulation of all four hub genes in CAL-27 and SCC-9 cells (P < 0.01). CONCLUSIONS: A four-gene prognostic signature derived from disulfidptosis- and cuproptosis-related genes provides independent prognostic value in OSCC and improves stratification when combined with standard clinical variables.