title: Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry.
abstract: Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3sigma); YWHAZ (14-3-3zeta); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin alpha (PTHA); L-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.
authors.affiliation: Department of Chemistry, York University, Toronto, Ontario M2J 1P3, Canada. 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: Siddartha
authors.initials: S
authors.lastname: Datta Gupta
authors.forename: Sudhir
authors.initials: S
authors.lastname: Bahadur
authors.forename: K W Michael
authors.initials: KW
authors.lastname: Siu
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004848:Epithelium / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry; D005095:Exoribonucleases; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; C089242:S100A7 protein, human; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human