SPINT2 inhibits NEDD4L-mediated ACSL4 ubiquitination to promote ferroptosis and suppress gallbladder cancer progression.

Gallbladder cancer (GBC) is a highly aggressive malignancy characterized by poor prognosis and limited therapeutic options, largely due to late-stage diagnosis and a lack of defined molecular targets. This is the first study to elucidate the molecular mechanism by which SPINT2 governs GBC progression through regulation of ferroptosis, linking it to ACSL4 stability and tumor suppression. Although SPINT2 has been implicated as a tumor suppressor in multiple cancer types, its biological function and mechanistic role in GBC have remained elusive. In this study, we identify SPINT2 as a key regulator of ferroptosis in GBC. Functional assays demonstrated that SPINT2 suppresses tumor cell proliferation in vitro and tumorigenicity in vivo. Metabolomic profiling revealed that SPINT2 deficiency alters lipid metabolism and reduces susceptibility to ferroptosis. Mechanistically, SPINT2 interacts with ACSL4 and prevents its ubiquitination by the E3 ligase NEDD4L, thereby stabilizing ACSL4 protein and promoting ferroptotic cell death. Clinically, low SPINT2 expression was significantly associated with poor differentiation, advanced tumor stage, and worse overall survival. Collectively, these findings position SPINT2 as a pivotal modulator of the ferroptotic pathway in GBC and highlight its translational promise as both a prognostic biomarker and a therapeutic entry point for ferroptosis-based interventions in GBC patients.