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BACKGROUND: Shisha smoking has been associated with multiple diseases including oral cancer. However, a mechanistic study to investigate alteration of secreted proteins in oral cells due to shisha smoking is lacking. OBJECTIVES: Elucidation of differentially secreted proteins by immortalized human normal oral keratinocytes (OKF6/TERT1) upon chronic exposure to shisha. METHODS: OKF6/TERT1 was chronically treated with 0.5% shisha extract for 8 months. Conditioned media from shisha treated (OKF6/TERT1-Shisha) and untreated (OKF6/TERT1-Parental) cells were subjected to TMT-based quantitative proteomic analysis. Bioinformatics analysis of differentially secreted proteins was carried out using SignalP, SecretomeP and TMHMM. Immunoblot validation of selected proteins was carried out to confirm the proteomics results. RESULTS: Proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha secretome resulted in the identification of 1,598 proteins, of which 218 proteins were found to be differentially secreted (⩾ 1.5-fold; p-value ⩽ 0.05) in shisha treated cells. Bioinformatics analysis using prediction tools showed secretory potential of differentially secreted proteins identified in OKF6/TERT1-Shisha. Western blotting validated the expression of AKR1C2, HSPH1 and MMP9 in OKF6/TERT1-Shisha secretome in agreement with proteomic data. CONCLUSION: This study serves as a useful resource to understand the effect of chronic shisha smoking on the milieu of secreted proteins of oral cells. In vivo studies are warranted to supplement our in vitro data to elucidate the role of these proteins as early diagnostic biomarkers for oral carcinogenesis among shisha smokers.

Background: Tobacco is smoked in different form including cigarettes and water pipes. One popular form of water pipe smoking especially in Middle Eastern countries is shisha smoking. Shisha has been associated with various diseases including oral cancer. However, genomic alterations and gene expression changes associated with chronic shisha exposure have not been previously investigated. Objectives: Whole‐exome sequencing and gene expression profiling of immortalized human oral keratinocytes (OKF6/TERT1) cells chronically treated with 0.5% shisha extract for a period of 8 months was undertaken to characterize molecular alterations associated with shisha exposure. Methods: Genomic DNA and RNA were extracted and preprocessed as per manufacturer's instruction and subjected to whole‐exome and transcriptome sequencing using Illumina HiSeq2500 platform. Exome was analyzed using GATK pipeline whereas RNA‐Seq data was analyzed using HiSat2 and HTSeq along with DESeq to elucidate differentially expressed genes. Results: Whole‐exome sequence analysis led to identification of 521 somatic missense variants corresponding to 389 genes RNA‐Seq data revealed 247 differentially expressed genes (≥2‐fold, P‐value

Aberrant promoter methylation of several known or pu-tative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been con-si der ed as a po ten ti al mol ec u la r ma rke r fo r ca nc er. We examined t he methylation status of n ine g enes ( APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1 , and TM S1 ), by quantitative methylation specific P CR . Synchronous preinvasive lesions (atypical ductal hyper-plasia and/or ductal carcinoma in situ ) and invasive duc-tal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor a nd 20 normal brea st distant from t umor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1 ,and GSTP1 . H ow ever, hierar chic al m ix ed model and Gener alized Estimating Equations model a nalyses show ed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.0 4, Mann -Wh itn ey test wi th permu tat io n corr ec -tion). The analysis of APC, CDH1 , and CTNNB1 methyl-ation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confi-dence i nterval, 60-71%) a nd a s pecificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involveme nt of APC, CDH1 ,and CTNNB1 promoter methylation in the early stages of breast cancer progres-si on and su gge st th at th ey ma y re p re se n t a use ful to o l for t he detection of t umor cells in clinical specimens.

Current morphology-based cervical cancer screening is associated with significant false-positive and false-negative results. Tumor suppressor gene hypermethylation is frequently present in cervical cancer. It is unknown whether a cervical scraping reflects the methylation status of the underlying epithelium, and it is therefore unclear whether quantitative hypermethylation specific PCR (QMSP) on cervical scrapings could be used as a future screening method augmenting the current approach. Cervical scrapings and paired fresh frozen cervical tissue samples were obtained from 53 cervical cancer patients and 45 controls. All scrapings were morphologically scored and analyzed with QMSP for the genes APC, DAPK, MGMT, and GSTP1. To adjust for DNA input, hypermethylation ratios were calculated against DNA levels of a reference gene. Hypermethylation ratios of paired fresh frozen tissue samples and scrapings of cervical cancer patients and controls were strongly related (Spearman correlation coefficient, 0.80 for APC, 0.98 for DAPK, and 0.83 for MGMT; P