Areca nut chewing is associated with oral potentially malignant disorders (OPMDs), a spectrum of precancerous lesions with variable rates of malignant transformation (1.7–16.9%). This study investigates the molecular alterations underlying this process. Three immortalized oral keratinocyte lines were repeatedly exposed to arecoline, the primary alkaloid in areca nuts, for 5–8months. RNA-seq analysis and functional assays were conducted on vehicle control and arecoline-treated cell pairs. Two of the three cell lines transformed after arecoline exposure, exhibiting anchorage-independent growth in vitro and tumorigenicity in vivo. The upregulated pathways included reactive oxygen species metabolism, cell cycle progression, and regulation of stem cell. Increased expression of stemness-related genes was observed in clinical datasets including oral cancer and OPMDs. This change of gene expression suggests a potential association with epigenetic reprogramming, a recently identified hallmark of cancer. In summary, this study identifies potential molecular targets for intervention and highlights the role of epigenetic alterations in arecoline-induced malignant transformation.
