Habitual Betel Nut Chewing Primes Oral Tumourigenesis Through Global DNA Hypomethylation and Oncogenic Pathway Activation

ABSTRACT Introduction and Aims Oral squamous cell carcinoma (OSCC) is a major malignancy of the oral-maxillofacial region. In addition to tobacco and alcohol use, betel nut chewing is a prominent aetiological factor for OSCC, particularly in East, South, and Southeast Asia; however, its underlying mechanisms remain poorly understood. This study aimed to investigate the role of habitual betel nut chewing in OSCC tumourigenesis and to provide possible predictive biomarkers. Methods Analysis of a clinical cohort comprising 1108 patients was performed. Global DNA methylation levels of OSCC and normal adjacent tissues (NATs) were analysed. The results were validated in vitro by treating cells with arecoline. Multiomics integration of DNA methylation and transcriptomic data was performed to identify key genes and signalling pathways. The findings were validated using data from The Cancer Genome Atlas, clinical samples, and an OSCC hamster model. Results Habitual betel nut chewing was specifically associated with an increased incidence of second primary tumours in the oral cavity. While OSCC tissues showed global DNA hypomethylation compared with NATs, NATs from habitual chewers exhibited significant hypomethylation. Multiomics integration revealed that habitual betel nut chewing altered NATs, making them more similar to OSCC tissues. Five key hub genes, ANLN, DEPC1B, ECT2, PLK1 , and SPC25 , were potential drivers of OSCC tumourigenesis. The upregulation of these genes was confirmed in clinical samples and mostly in a betel nut–induced OSCC hamster model, which also recapitulated increased OSCC formation under persistent betel nut exposure. Conclusion Our findings demonstrate that habitual betel nut chewing promotes OSCC development and may do so by inducing global DNA hypomethylation and activating oncogenic pathways in nonmalignant oral mucosa. Clinical Relevance Assessing DNA methylation status and key gene expression could serve as biomarkers and targets for early OSCC detection and prevention.