Metabolic Reprogramming and GM-CSF Secretion in Areca Nut-Activated Fibroblast Drives Oral Precancer Progression

Oral cancer, marked by a rising global incidence and dismal prognosis, is associated with areca nut use, particularly in South and Southeast Asia. Yet, its influence on the oral tissue microenvironment is not well elucidated. This research aimed to elucidate the effects of areca nut extract (ANE) on oral carcinogenesis by modulating fibroblast behavior within the microenvironment. In this study, cell viability assay, transwell migration and invasion assays, and flow cytometry were employed to study cell behaviors. Cytokine array, ELISA, phospho-kinase array, Western blot analysis, extracellular O2 consumption, and glycolysis assays were conducted to assess cellular functionalities. In vivo experiments, complemented by immunohistochemical and immunofluorescent staining of oral lesions, were performed to corroborate in vitro observations. Our results showed that upregulation of α-SMA and FAP was observed in fibroblasts within oral pre-cancer and cancer lesions. ANE enhanced mitochondrial metabolism in fibroblasts and induced their transition into myofibroblasts. Additionally, ANE triggered epithelial-to-mesenchymal transition (EMT) and stimulated GM-CSF secretion in fibroblasts, thereby advancing the progression of oral pre-cancer. The conditioned medium from ANE-treated human gingival fibroblasts, along with recombinant GM-CSF, elevated EGFR phosphorylation and facilitated oncogenic transformation in DOK dysplastic oral keratinocytes. Moreover, in a hamster model, daily application of ANE on buccal pouches for 37 weeks replicated the in vitro findings for ANE-induced GM-CSF expression in fibroblasts and EGFR phosphorylation in mucosa epithelial cells. In conclusion, this investigation provides in vitro and in vivo evidence suggesting that ANE promotes EMT and GM-CSF secretion in fibroblasts, which activates EGFR and the malignant transformation of oral pre-cancer cells.