Prognostic Significance of Head-and-Neck Cancer Biomarkers Previously Discovered and Identified Using iTRAQ-Labeling and Multidimensional Liquid Chromatography−Tandem Mass Spectrometry

Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Journal of Proteome Research (Impact Factor: 5). 06/2008; 7(5):2078-87. DOI: 10.1021/pr7007797
Source: PubMed

ABSTRACT Diagnostic oncoproteomics is an emerging field; at present, studies on evaluation of prognostic utility of potential biomarkers identified using proteomic techniques are limited. Analysis with isobaric mass tags (iTRAQ) by multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) to identify proteins that are differentially expressed in human head-and-neck/oral squamous cell carcinomas (HNOSCCs) versus noncancerous head-and-neck tissues has led to the discovery, identification, and verification of consistently increased expression of a panel of proteins, including stratifin (14-3-3sigma) and YWHAZ (14-3-3zeta), that may serve as potential cancer biomarkers. Herein, we describe the prognostic utility of these two candidate biomarkers for head-and-neck/oral squamous cell carcinoma (HNOSCC). To determine the clinical significance of stratifin and YWHAZ in head-and-neck tumorigenesis, the expressions of these two proteins were analyzed in HNOSCCs (51 cases) and nonmalignant tissues (39 cases) using immunohistochemistry. Significant increase in stratifin expression was observed in the HNOSCCs as compared to the nonmalignant mucosa [p=0.003, Odd's Ratio (OR)=3.8, 95% CI=1.6-9.2]. Kaplan-Meier survival analysis reveals correlation of stratifin overexpression with reduced disease-free survival of HNOSCC patients (p=0.06). The most intriguing finding is the significant decrease in median disease-free survival (13 months) in HNOSCC patients showing overexpression of both stratifin and YWHAZ proteins, as compared to patients that did not show overexpression of these proteins (median disease-free survival=38 months, p=0.019), underscoring their utility as adverse prognosticators for HNOSCCs. Co-immunoprecipitation assays show the formation of stratifin-YWHAZ heterodimers in HNOSCC cells and tissue samples, and interactions with NFkappaB, beta-catenin, and Bcl-2 proteins. These results suggest the involvement of these proteins in the development of head-and-neck cancer and their association with adverse disease outcome.

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    • "14-3-3 proteins have been found to interact with target proteins involved in the regulation of multiple cellular processes, such as cell cycle control, protein trafficking, antiapoptosis , metabolism, signal transduction, inflammation, and cell adhesion/motility (Wilker and Yaffe, 2004; Morrison, 2009). YWHAZ has been identified as a clinically relevant prognostic marker for breast cancer (Lu et al, 2009; Neal et al, 2009), lung cancer (Fan et al, 2007), and head and neck cancer (Matta et al, 2008) and may allow for the identification of patients whose "
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    ABSTRACT: Background: Several studies have demonstrated that YWHAZ (14-3-3ζ), included in the 14-3-3 family of proteins, has been implicated in the initiation and progression of cancers. We tested whether YWHAZ acted as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Methods: We analysed 7 GC cell lines and 141 primary tumours, which were curatively resected in our hospital between 2001 and 2003. Results: Overexpression of the YWHAZ protein was frequently detected in GC cell lines (six out of seven lines, 85.7%) and primary tumour samples of GC (72 out of 141 cases, 51%), and significantly correlated with larger tumour size, venous and lymphatic invasion, deeper tumour depth, and higher pathological stage and recurrence rate. Patients with YWHAZ-overexpressing tumours had worse overall survival rates than those with non-expressing tumours in both intensity and proportion expression-dependent manner. YWHAZ positivity was independently associated with a worse outcome in multivariate analysis (P=0.0491, hazard ratio 2.3 (1.003–5.304)). Knockdown of YWHAZ expression using several specific siRNAs inhibited the proliferation, migration, and invasion of YWHAZ-overexpressing GC cells. Higher expression of the YWHAZ protein was significantly associated with the lower expression of miR-375 in primary GC tissues (P=0.0047). Conclusion: These findings suggest that YWHAZ has a pivotal role in tumour cell proliferation through its overexpression, and highlight its usefulness as a prognostic factor and potential therapeutic target in GC.
    British Journal of Cancer 02/2013; 108(6). DOI:10.1038/bjc.2013.65 · 4.82 Impact Factor
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    • "The protein samples were then heated at 601C for 1 h in the presence of 5 mM DTT, cooled to room temperature, and then alkylated by incubation with 10 mM iodoacetamide for 1 h in dark. Sequencing grade trypsin (Promega, Madison, WI) at 1:20 w/w in 50 mM ammonium bicarbonate was then added, and the samples were incubated at 371C overnight [46]. The digested samples were then dried under vacuum and redissolved in 10 mL of 0.1% formic acid. "
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    ABSTRACT: In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.
    Proteomics 06/2011; 11(12):2363-76. DOI:10.1002/pmic.201000186 · 3.97 Impact Factor
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