Metabolic Reprogramming and GM ‐ CSF Secretion in Areca Nut‐Activated Fibroblasts Drive Oral Precancer Progression

OBJECTIVE: Oral cancer, with a rising global incidence and poor prognosis, is associated with areca nut use in South and Southeast Asia. In this study, we addressed the effects of areca nut extract (ANE) on oral carcinogenesis by modulating fibroblast behavior in oral tissue. METHODS: Cell viability, migration, invasion, and flow cytometry assays were applied to study cell behaviors. Cytokine array, enzyme-linked immunosorbent assay, phospho-kinase array, Western blot analysis, and oxygen consumption and glycolysis assays were performed to evaluate cell functions. Immunohistochemical and immunofluorescent staining using human and hamster oral tissues was applied to validate in vitro findings. RESULTS: Increased alpha smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) were observed in fibroblasts from oral precancerous and cancer lesions. ANE increased mitochondrial metabolism in fibroblasts and induced myofibroblast transition. It enhanced epithelial-to-mesenchymal transition and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion in fibroblasts. Conditioned medium from ANE-treated fibroblasts and recombinant GM-CSF increased epidermal growth factor receptor (EGFR) phosphorylation and malignant transformation in dysplastic oral keratinocytes. In hamsters, ANE treatment increased GM-CSF expression in fibroblasts and EGFR phosphorylation in epithelial cells. CONCLUSION: ANE promotes epithelial-to-mesenchymal transition and GM-CSF secretion in fibroblasts, which activate EGFR signaling and malignant transformation of oral precancerous cells.