Arecoline suppresses cuproptosis and drives oral squamous cell carcinoma progression via a ROS-NRF2-MT2A axis

Abstract Betel nut chewing represents a primary risk factor for oral squamous cell carcinoma (OSCC), with arecoline as its principal bioactive alkaloid. Yet, the mechanisms underlying arecoline-driven malignant progression remain incompletely understood. Here, we show that arecoline activates a ROS-driven NRF2-MT2A axis to suppress cuproptosis in OSCC. Transcriptomic analyses revealed that betel nut–associated OSCC exhibited enhanced oxidative stress signatures, reduced cuproptosis scores, and elevated MT2A expression, which correlated with poorer overall and progression-free survival. Arecoline increased intracellular ROS, activated NRF2 signaling, and upregulated MT2A in OSCC cells, while suppressing cuproptosis and promoting copper toxicity resistance. Pharmacologic ROS scavenging or genetic silencing of NRF2 blunted arecoline‑induced MT2A expression, restored cuproptosis, and attenuated cell viability, clonogenicity, migration and invasion in vitro. CUT&Tag profiling demonstrated direct NRF2 occupancy on the MT2A promoter, and MT2A overexpression partially rescued the loss of pro‑survival and anti‑cuproptotic effects caused by NRF2 knockdown. In xenograft models, knockdown of NRF2 or MT2A significantly inhibited tumor growth, while enforced MT2A expression reversed the effect of NRF2 depletion. Moreover, copper chelation with TTM rescued the tumor suppressive effect of MT2A knockdown, further confirming the role of MT2A in protecting OSCC against cuproptosis in vivo. Collectively, these data define a ROS‑NRF2‑MT2A signaling axis that protects OSCC cells from cuproptosis and promotes malignant progression in the context of betel nut exposure, highlighting redox and copper‑dependent cell death pathways as potential therapeutic vulnerabilities in arecoline‑related OSCC.