NCKAP1 Is Essential for SLC7A11-Dependent Disulfide Stress-Induced Cell Death Under Glucose Starvation in an Oral Squamous Cell Model

Background: Oral squamous cell carcinoma (OSCC) has a poor prognosis and requires new therapeutic strategies. Disulfidptosis is a recently defined form of cell death that occurs in SLC7A11-high cells under glucose starvation and has been linked to collapse of the actin cytoskeleton. NCKAP1 has been linked to this process in other tumors, but its role in OSCC remains unknown. The aim of this study was to determine whether NCKAP1 mediates glucose-starvation-induced, disulfide stress-driven cell death in an OSCC cell model and to examine whether its contribution depends on SLC7A11-mediated cystine import.Methods: Expression of NCKAP1 and SLC7A11 was assessed in OSCC cell lines and normal oral keratinocytes by qRT-PCR and Western blotting. A glucose-starvation model was established in SCC-9 cells, and the resulting cell death was characterized using cell viability assays, lactate dehydrogenase (LDH) release assays, flow cytometry, a panel of death-pathway inhibitors, and reducing agents. NCKAP1 was manipulated using siRNA, plasmid overexpression, and stable shRNA knockdown. Features of disulfide stress were evaluated by measuring free thiols and NADPH/NADP+ levels, as well as by analyzing the glutathione pool, including total glutathione, oxidized glutathione (GSSG), reduced glutathione (GSH) and the GSH/GSSG ratio. Proliferation and migration were assessed using colony formation, wound healing, and transwell assays.Results: NCKAP1 and SLC7A11 were higher in the two OSCC cell lines examined than in normal keratinocytes, each cultured in its respective routine medium (p < 0.001). In SCC-9 cells, glucose starvation triggered a rapid, non-apoptotic death that was not inhibited by inhibitors of apoptosis, necroptosis, autophagy, or ferroptosis, but was largely prevented by co-treatment with dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) (p < 0.001). This response was accompanied by decreased NADPH/NADP+ ratios, depletion of the glutathione pool with GSSG accumulation, an approximately 89% reduction in the GSH/GSSG ratio, and loss of free thiols (all p < 0.001). NCKAP1 knockdown preserved cell viability and partially preserved intracellular free thiols, whereas NCKAP1 overexpression aggravated thiol depletion (p < 0.001). NCKAP1 overexpression failed to reduce cell viability when SLC7A11 was silenced. Manipulation of NCKAP1 did not materially alter total glutathione or GSSG, but produced modest changes in reduced GSH, the GSH/GSSG ratio and, after knockdown, the NADPH/NADP+ ratio, recovering less than 10% of the starvation-induced changes. These data argue against a major role of NCKAP1 in the upstream redox collapse without excluding a minor metabolic contribution, and are consistent with a functional dependency in which the contribution of NCKAP1 requires SLC7A11-mediated cystine import. Under normal culture conditions, NCKAP1 knockdown also reduced colony formation, wound closure, and migration (all p < 0.001).Conclusion: NCKAP1 is required for glucose-starvation-induced, disulfide stress-driven cell death and contributes to SCC-9 proliferation and migration, identifying the Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE) regulatory complex component NCKAP1 as a factor involved in this death modality.