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Background: Tobacco exposure (through smoking or chewing) is one of the predominant risk factors associated with the development of oral squamous cell carcinoma (OSCC). Despite the growing number of patients diagnosed with OSCC, there are few circulating biomarkers for identifying individuals at a higher risk of developing the disease. Successful identification of candidate molecular markers for risk assessment could aid in the early detection of oral lesions and potentially be used for community screening of high-risk populations., Objective: Identification of differentially expressed proteins in the serum of oral cancer patients which can serve as biomarkers for the diagnosis of the onset of oral cancer among tobacco users., Methods: We employed a tandem mass tag (TMT)-based quantitative proteomics approach to study alterations in the serum proteomes of OSCC patients based on their tobacco exposure habits (chewing and smoking) compared to healthy individuals with no history of using any form of tobacco or any symptoms of the disease., Results: Mass spectrometry-based analysis resulted in the identification of distinct signatures in the serum of OSCC patients who either chewed or smoked tobacco. Pathway analysis revealed opposing effects of dysregulated proteins enriched in the complement-coagulation signaling cascades with a high expression of the Serpin family of proteins observed in OSCC patients who chewed tobacco compared to healthy individuals whereas these proteins showed decreased levels in OSCC patients who smoked. ELISA-based validation further confirmed our findings revealing higher expression of SERPINA6 and SERPINF1 across serum of OSCC patients who chewed tobacco compared to healthy individuals., Conclusions: This study serves as a benchmark for the identification of serum-based protein markers that may aid in the identification of high-risk patients who either chew tobacco or smoke tobacco.

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.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Cigarette smoke and chewing tobacco are well known risk factors associated with ESCC. However, molecular mechanisms associated with development of ESCC among smokers and chewers are poorly understood. MicroRNAs play an important role in regulating physiological and disease processes including esophageal cancer., Objective and Methods: In this study, we developed an in vitro model by treating non-neoplastic Het- 1A esophageal cell line with cigarette smoke and chewing tobacco. We carried out miRNA sequencing on Illumina HiSeq 2500 platform and compared miRNA expression pattern across cigarette smoke and chewing tobacco treated Het-1A cells with untreated cells., Results: We identified and quantified 433 miRNAs in both smoke exposed and chewing tobacco treated cells, of which 13 miRNAs showed significantly altered expression in cigarette smoke exposed cells while 25 miRNAs showed significantly altered expression in chewing tobacco treated cells. In addition, we predicted novel miRNAs from these data-sets. We evaluated miRNAs that showed selective or context dependent expression pattern in cigarette smoke exposed or chewing tobacco treated cells., Conclusion: In this study, we have comprehensively mapped miRNA expression pattern in response to cigarette smoke and chewing tobacco in Het-1A cells. We identified miRNAs that show altered expression in these cell models., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins., Objective and Methods: In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line., Results: miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset., Conclusion: In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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 sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Cigarette smoking has been associated with multiple negative effects on human skin. Long-term physiological effects of cigarette smoke are through chronic and not acute exposure. Molecular alterations due to chronic exposure to cigarette smoke remain unclear. Primary human skin keratinocytes chronically exposed to cigarette smoke condensate (CSC) showed a decreased wound-healing capacity with an increased expression of NRF2 and MMP9. Using quantitative proteomics, we identified 4728 proteins, of which 105 proteins were overexpressed (≥2-fold) and 41 proteins were downregulated (≤2-fold) in primary skin keratinocytes chronically exposed to CSC. We observed an alteration in the expression of several proteins involved in maintenance of epithelial barrier integrity, including keratin 80 (5.3 fold, p value 2.5 × 10 -7 ), cystatin A (3.6-fold, p value 3.2 × 10 -3 ), and periplakin (2.4-fold, p value 1.2 × 10 -8 ). Increased expression of proteins associated with skin hydration, including caspase 14 (2.2-fold, p value 4.7 × 10 -2 ) and filaggrin (3.6-fold, p value 5.4 × 10 -7 ), was also observed. In addition, we report differential expression of several proteins, including adipogenesis regulatory factor (2.5-fold, p value 1.3 × 10 -3 ) and histone H1.0 (2.5-fold, p value 6.3 × 10 -3 ) that have not been reported earlier. Bioinformatics analyses demonstrated that proteins differentially expressed in response to CSC are largely related to oxidative stress, maintenance of skin integrity, and anti-inflammatory responses. Importantly, treatment with vitamin E, a widely used antioxidant, could partially rescue adverse effects of CSC exposure in primary skin keratinocytes. The utility of antioxidant-based new dermatological formulations in delaying or preventing skin aging and oxidative damages caused by chronic cigarette smoke exposure warrants further clinical investigations and multi-omics research.

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.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal cancer in India. Cigarette smoking and chewing tobacco are known risk factors associated with ESCC. However, genomic alterations associated with ESCC in India are not well-characterized. In this study, we carried out exome sequencing to characterize the mutational landscape of ESCC tumors from subjects with a varied history of tobacco usage. Whole exome sequence analysis of ESCC from an Indian cohort revealed several genes that were mutated or had copy number changes. ESCC from tobacco chewers had a higher frequency of C:G > A:T transversions and 2-fold enrichment for mutation signature 4 compared to smokers and non-users of tobacco. Genes, such as TP53, CSMD3, SYNE1, PIK3CA , and NOTCH1 were found to be frequently mutated in Indian cohort. Mutually exclusive mutation patterns were observed in PIK3CA – NOTCH1, DNAH5 – ZFHX4, MUC16 – FAT1 , and ZFHX4 – NOTCH1 gene pairs. Recurrent amplifications were observed in 3q22-3q29, 11q13.3-q13.4, 7q22.1-q31.1, and 8q24 regions. Approximately 53% of tumors had genomic alterations in PIK3CA making this pathway a promising candidate for targeted therapy. In conclusion, we observe enrichment of mutation signature 4 in ESCC tumors from patients with a history of tobacco chewing. This is likely due to direct exposure of esophagus to tobacco carcinogens when it is chewed and swallowed. Genomic alterations were frequently observed in PIK3CA-AKT pathway members independent of the history of tobacco usage. PIK3CA pathway can be potentially targeted in ESCC which currently has no effective targeted therapeutic options. [ABSTRACT FROM AUTHOR]

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits. [ABSTRACT FROM AUTHOR]

Cellular transformation owing to cigarette smoking is due to chronic exposure and not acute. However, systematic studies to understand the molecular alterations in lung cells due to cigarette smoke are lacking. To understand these molecular alterations induced by chronic cigarette smoke exposure, we carried out tandem mass tag (TMT) based temporal proteomic profiling of lung cells exposed to cigarette smoke for upto 12 months. We identified 2620 proteins in total, of which 671 proteins were differentially expressed (1.5-fold) after 12 months of exposure. Prolonged exposure of lung cells to smoke for 12 months revealed dysregulation of oxidative phosphorylation and overexpression of enzymes involved in TCA cycle. In addition, we also observed overexpression of enzymes involved in glutamine metabolism, fatty acid degradation and lactate synthesis. This could possibly explain the availability of alternative source of carbon to TCA cycle apart from glycolytic pyruvate. Our data indicates that chronic exposure to cigarette smoke induces mitochondrial metabolic reprogramming in cells to support growth and survival. [ABSTRACT FROM AUTHOR]