Chronic shisha exposure alters phosphoproteome of oral keratinocytes

Shisha smoking has been epidemiologically linked to development of oral lesions as well as various cancers. However, few molecular studies investigate the pathobiology of shisha induced cancer. In this study we investigate the effects of chronic shisha exposure in an in vitro model using normal, immortalized, non-transformed oral keratinocytes. Quantitative proteomic and phosphoproteomic analyses were performed on OKF6/TERT1 cells exposed to shisha for a period of 8 months. Gene Ontology and pathway analysis of dysregulated proteins and phosphoproteins were carried out to identify significantly enriched biological processes and pathways in shisha exposed cells compared to parental cells. Chronic shisha exposure resulted in increased cell scattering phenomenon in OKF6/TERT1 cells. Quantitative data analysis revealed differential phosphorylation of 164 phosphopeptides (fold change ≥1.5, p≤0.05) corresponding to 136 proteins. Kinases such as PRKCD were seen to be hyperphosphorylated. Pathway analysis revealed an enrichment of hyperphosphorylated and/or overexpressed proteins involved in the mTORC1 and EIF4F complexes. These complexes are associated with initiation of protein translation and are known to the affected in cancers. Network analysis also highlighted a downregulation of proteins associated with Type I interferon signaling in shisha exposed cells. High throughput phosphoproteomic analysis revealed global perturbations to the molecular milieu of oral keratinocytes upon shisha exposure. Further studies are needed to validate putative targets in oral cancer patients with shisha smoking history.