Identification of proteins secreted by head and neck cancer cell lines using LC‐MS/MS: Strategy for discovery of candidate serological biomarkers

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.

Global secretome analysis identifies novel mediators of bone metastasis

“…To filter out likely intracellular contaminants and retain proteins most likely to be secreted by live cells, we used the SignalP [26,27] and Gene Ontology (GO) [28,29] databases to retain only proteins either experimentally observed to be extracellular or otherwise containing predicted cleavable signal peptides targeting them to the secretory pathway. This analysis yielded a total of 298 unique, secretory-predicted proteins across the eight cell lines, which is in line with previous quantities and percentages of secreted proteins found in comparable secretome analyses [8,[30][31][32][33][34][35][36][37][38][39][40].…”

“…The first breast cancer metastasis secretome study profiled the secretomes of the MCF10A metastasis progression series, finding proteins such as alpha-1-antichymotrypsin and galectin-3-binding protein to be upregulated in conditioned media from the metastatic variants [48]. More recently, many secretome analyses of cell lines representing several different cancer types have been reported [8,[30][31][32][33][34][35][36][37][38][39][40]. Most secretome studies have used methodology similar to ours in terms of initial protein harvesting conditions and then have typically used either concentrating columns or total protein precipitation methods for secreted protein isolation.…”

Potentially Novel Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma Identified Using an Integrated Cell Line-based Discovery Strategy

“…These are key biological processes occurring at the cell surface or the extracellular vicinity of tumor cells; thus, it serves as no surprise that proteins involved in these diverse processes were identified in this study. Ralhan et al reported the only secretome profiling of a similar HNC subsite, examining the CM of the human laryngeal SCC cell line (SCC38), in addition to oral SCC cell lines, which has resulted in fewer protein identifications, likely because of a different proteomics analysis strategy (53). We further extended our results by systematic validation in wellannotated patient tissue and plasma samples.…”

Quantitative Proteomic Analysis in Metastatic Renal Cell Carcinoma Reveals a Unique Set of Proteins with Potential Prognostic Significance

“…Each set was then separated via off-line SCX chromatography using an HP1050 HPLC instrument (Agilent, Palo Alto, CA) with a 2.1-mm internal diameter ϫ 100-mm length PolyLC Polysulfoethyl A column packed with 5-m beads with 300-Å pores (The Nest Group, Southborough, MA) as described elsewhere (27). Separation was performed using a linear binary gradient over 1 h (see details in supplemental Table S3) of Buffer A and Buffer B; Buffer B was composed of Buffer A and 350 mM potassium chloride.…”