TPI1 promotes malignant progression and ferroptosis resistance in head and neck squamous cell carcinoma.

Objective: Triosephosphate isomerase 1 (TPI1) is aberrantly overexpressed and exerts a critical oncogenic role in the development and progression of various human cancers by regulating multiple malignant biological phenotypes. However, the biological functions of TPI1 and the molecular mechanisms underlying its regulatory effect on ferroptosis sensitivity in head and neck squamous cell carcinoma (HNSCC) remain not fully elucidated. Methods: TPI1 expression was analyzed across 33 cancer types using The Cancer Genome Atlas pan-cancer dataset, and validated in 6 paired HNSCC tumor/adjacent normal tissues via Western blotting and immunohistochemistry. Clinical correlations were assessed using the Wilcoxon rank-sum test, and prognostic value was evaluated by Kaplan-Meier analysis and multivariate Cox regression. A prognostic nomogram integrating TPI1 and clinical covariates was constructed and validated by calibration curves. Single-cell RNA-sequencing (3 independent cohorts: GSE103322, GSE150321, GSE172577) and spatial transcriptomics (4 patient samples) were employed to map TPI1 cellular distribution in the tumor microenvironment. Gain- and loss-of-function assays were performed in FaDu (overexpression) and TU177 (knockdown) cell lines using lentiviral vectors. Cell proliferation was measured by CCK-8 and colony formation assays; migration and invasion were evaluated by wound healing and Transwell assays. Ferroptosis sensitivity was assessed by RSL3 dose-response curves, malondialdehyde (MDA) assay, BODIPY 581/591 C Results: TPI1 was significantly upregulated in HNSCC at both mRNA and protein levels, with an AUC of 0.914 for distinguishing tumor from normal tissues. High TPI1 expression correlated with male sex, high histologic grade, advanced pathologic stage, and T3-T4 stage, and independently predicted poor overall survival. The nomogram integrating TPI1, age, gender, stage, and grade showed robust prognostic performance with well-calibrated 1-, 3-, and 5-year survival predictions. Single-cell and spatial transcriptomics confirmed that TPI1 was predominantly and specifically expressed in malignant epithelial cells, with minimal expression in stromal and immune cells. Functionally, TPI1 overexpression significantly enhanced FaDu cell proliferation, migration, and invasion, while TPI1 knockdown markedly suppressed these phenotypes in TU177 cells. Mechanistically, TPI1 conferred ferroptosis resistance by attenuating RSL3-induced lipid peroxidation, reactive oxygen species accumulation, and intracellular Fe Conclusion: This study identifies TPI1 as a key tumor-promoting factor and independent prognostic biomarker in HNSCC. TPI1 promotes malignant progression by enhancing ferroptosis resistance, providing a promising therapeutic target for overcoming ferroptosis evasion in HNSCC.