Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.

Smoking is the leading cause of preventable death worldwide. Though cigarette smoke is an established cause of head and neck cancer (including oral cancer), molecular alterations associated with chronic cigarette smoke exposure are poorly studied. To understand the signaling alterations induced by chronic exposure to cigarette smoke, we developed a cell line model by exposing normal oral keratinocytes to cigarette smoke for a period of 12 months. Chronic exposure to cigarette smoke resulted in increased cellular proliferation and invasive ability of oral keratinocytes. Proteomic and phosphoproteomic analyses showed dysregulation of several proteins involved in cellular movement and cytoskeletal reorganization in smoke exposed cells. We observed overexpression and hyperphosphorylation of protein kinase N2 (PKN2) in smoke exposed cells as well as in a panel of head and neck cancer cell lines established from smokers. Silencing of PKN2 resulted in decreased colony formation, invasion and migration in both smoke exposed cells and head and neck cancer cell lines. Our results indicate that PKN2 plays an important role in oncogenic transformation of oral keratinocytes in response to cigarette smoke. The current study provides evidence that PKN2 can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients with a history of smoking.