title: iTRAQ-multidimensional liquid chromatography and tandem mass spectrometry-based identification of potential biomarkers of oral epithelial dysplasia and novel networks between inflammation and premalignancy.
abstract: Chronic exposure of the oral mucosa to carcinogens in tobacco is linked to inflammation and development of oral premalignant lesions (OPLs) with high risk of progression to cancer; there is currently no clinical methodology to identify high-risk lesions. We hypothesized that identification of differentially expressed proteins in OPLs in relation to normal oral tissues using proteomic approach will reveal changes in multiple cellular pathways and aid in biomarker discovery. Isobaric mass tags (iTRAQ)-labeled oral dysplasias and normal tissues were compared against pooled normal control by online liquid chromatography and tandem mass spectrometry. Verification of biomarkers was carried out in an independent set of samples by immunohistochemistry, immunoblotting, and RT-PCR. We identified 459 nonredundant proteins in OPLs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of three best-performing biomarkers identified by iTRAQ analysis and verified by immunohistochemistrystratifin (SFN), YWHAZ, and hnRNPKachieved a sensitivity of 0.83, 0.91, specificity of 0.74, 0.95, and predictive value of 0.87 and 0.96, respectively, in discriminating dysplasias from normal tissues, thereby confirming their utility as potential OPL biomarkers. Pathway analysis revealed direct interactions between all the three biomarkers and their involvement in two major networks involved in inflammation, signaling, proliferation, regulation of gene expression, and cancer. In conclusion, our work on determining the OPL proteome unraveled novel networks linking inflammation and development of epithelial dysplasia and their key regulatory proteins may serve as novel chemopreventive/therapeutic targets for early intervention. Additionally, we identified and verified a panel of OPL biomarkers that hold promise for large-scale validation for ultimate clinical use.
authors.affiliation: Departments of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. ralhanr@yorku.ca
authors.forename: Ranju
authors.initials: R
authors.lastname: Ralhan
authors.forename: Leroi V
authors.initials: LV
authors.lastname: Desouza
authors.forename: Ajay
authors.initials: A
authors.lastname: Matta
authors.forename: Satyendra Chandra
authors.initials: SC
authors.lastname: Tripathi
authors.forename: Shaun
authors.initials: S
authors.lastname: Ghanny
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Dattagupta
authors.forename: Alok
authors.initials: A
authors.lastname: Thakar
authors.forename: Shyam S
authors.initials: SS
authors.lastname: Chauhan
authors.forename: K W Michael
authors.initials: KW
authors.lastname: Siu
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis; D002853:Chromatography, Liquid / Q000379:methods*; D004848:Epithelium / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D009061:Mouth Mucosa / Q000378:metabolism*; D009062:Mouth Neoplasms / Q000175:diagnosis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012372:ROC Curve; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D014408:Biomarkers, Tumor; D020543:Proteome