Molecular alterations elicited by chronic exposure to cigarette smoke and chewing tobacco in oral keratinocytes

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore investigated molecular alterations in oral keratinocytes (OKF6/TERT1) chronically exposed to smoke and chewing tobacco. OKF6/TERT1 cells acquired an oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed both quantitative proteomics and whole exome sequencing (WES) approaches to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes. Distinct molecular alterations were observed in smoke and chewing tobacco exposed cells compared to parental cells. MAPK1 was identified as a key regulator of proteins dysregulated in smoke exposed cells whereas ERBB2 and RABL6 were key regulators in chewing tobacco exposed cells. Exome sequencing revealed a much higher rate of C>A transversions in smoke exposed cells in conjunction with previous studies. In contrast, C>G transversions were observed to be higher in chewing tobacco exposed cells. We identified diverse mutations in smoke exposed cells compared to chewing tobacco treated cells thereby highlighting the distinct effects of each exposure on the genome. This study can serve as a reference for fundamental damage on oral cells as a consequence of exposure to different forms of tobacco. Future studies are needed to validate some of the genetic and proteomic alterations unique to each form of tobacco exposure. This could lead to validation of potential markers that may aid in stratifying patients based on tobacco usage history.