TTK Defines a High-Risk Oral Squamous Cell Carcinoma Subtype Through Dual mTORC1/NF-κB Activation.

OBJECTIVE: Oral squamous cell carcinoma remains difficult to treat because of its marked tumor heterogeneity, highlighting the need to identify molecular subtypes with distinct therapeutic vulnerabilities. This study aimed to comprehensively characterize OSCC molecular subtypes and identify potential therapeutic targets. MATERIALS AND METHODS: We conducted an integrated analysis combining single-cell and bulk RNA sequencing from 709 OSCC cases. Protein interactions were examined using mass spectrometry and immunoprecipitation assays. The functional roles of key regulators were evaluated through in vitro cell proliferation and invasion assays, RNA sequencing of knockdown cell lines, and in vivo xenograft models. Statistical analyses included differential gene expression analysis, pathway enrichment, and IC50 determination for drug sensitivity. RESULTS: We identified a distinct molecular OSCC subtype marked by concurrent activation of the mTORC1 and NF-κB pathways, with TTK emerging as a central regulator of this co-activation. Patients in this subtype exhibited pronounced genomic instability, reflected by increased tumor mutational burden, higher TP53 mutation frequency, copy number amplifications across multiple genomic regions. Mechanistically, mass spectrometry and co-immunoprecipitation assays showed that TTK directly interacts with the TAK1-TAB protein complex, thereby activating the NF-κB pathway. RNA sequencing of TTK knockdown cell lines demonstrated significant downregulation of both mTOR and NF-κB signaling upon TTK suppression. Functional assays confirmed that TTK inhibition strongly reduced OSCC cell proliferation and invasion and markedly enhanced cisplatin sensitivity in vitro and in vivo. CONCLUSION: Our findings establish TTK as a pivotal mediator defining a high-risk OSCC molecular subtype characterized by simultaneous activation of the mTORC1 and NF-κB pathways and severe genomic instability. The discovery of a direct interaction between TTK and the TAK1-TAB complex provides novel mechanistic insight into NF-κB activation, while its inhibition significantly improves cisplatin sensitivity. These results warrant further clinical evaluation of TTK inhibitors as a promising therapeutic strategy to improve outcomes in aggressive OSCC.