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

Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40–60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines (FaDu, UTSCC8 and UTSCC42a) in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated data set was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and over-expressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of five markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western blotting, respectively. IHC-based validation conducted in two independent cohorts comprising of 40 and 39 HNSCC biopsies revealed that high tumor expression of PLAU, IGFBP7, MMP14 and THBS1 were associated with inferior disease-free survival, and increased risk of disease progression or relapse. Furthermore, as demonstrated using ELISAs, circulating levels of PLAU and IGFBP7 were significantly higher in the plasma of HNSCC patients compared with healthy individuals. Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40–60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines (FaDu, UTSCC8 and UTSCC42a) in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated data set was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and over-expressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of five markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western blotting, respectively. IHC-based validation conducted in two independent cohorts comprising of 40 and 39 HNSCC biopsies revealed that high tumor expression of PLAU, IGFBP7, MMP14 and THBS1 were associated with inferior disease-free survival, and increased risk of disease progression or relapse. Furthermore, as demonstrated using ELISAs, circulating levels of PLAU and IGFBP7 were significantly higher in the plasma of HNSCC patients compared with healthy individuals. Head and Neck Squamous Cell Carcinoma (HNSCC) 1The abbreviations used are:HNSCChead and neck squamous cell carcinomaSCCsquamous cell carcinomaCMconditioned mediaIHCimmunohistochemistryWCLwhole cell lysateMudPITmultidimensional protein identification technologyHNChead and neck cancerNOEnormal oral epithelialqRT-PCRquantitative real-time PCRDFSdisease-free survivalPLAUurokinaseIGFBP7insulin-like growth factor binding protein 7FDRfalse discovery rateSpCspectral countingFCfold changeHPAhuman protein atlasOSoverall survivalKMKaplan–MeierHRhazard ratioCIconfidence intervalMMP14matrix metalloproteinase 14THBS1thrombospondin-1TGFBItransforming growth factor, beta-induced, 68kDa.1The abbreviations used are:HNSCChead and neck squamous cell carcinomaSCCsquamous cell carcinomaCMconditioned mediaIHCimmunohistochemistryWCLwhole cell lysateMudPITmultidimensional protein identification technologyHNChead and neck cancerNOEnormal oral epithelialqRT-PCRquantitative real-time PCRDFSdisease-free survivalPLAUurokinaseIGFBP7insulin-like growth factor binding protein 7FDRfalse discovery rateSpCspectral countingFCfold changeHPAhuman protein atlasOSoverall survivalKMKaplan–MeierHRhazard ratioCIconfidence intervalMMP14matrix metalloproteinase 14THBS1thrombospondin-1TGFBItransforming growth factor, beta-induced, 68kDa. is the sixth most common cancer worldwide, with ∼600,000 new cases diagnosed every year (1Leemans C.R. Braakhuis B.J. Brakenhoff R.H. The molecular biology of head and neck cancer.Nat. Rev. Cancer. 2011; 11: 9-22Crossref PubMed Scopus (1887) Google Scholar). HNSCCs include squamous cell carcinomas (SCCs) of the oral cavity, pharynx (nasopharynx, oropharynx, and hypopharynx), and larynx. Despite improvements in therapeutic approaches, the overall 5-year survival rate of HNSCC patients has not improved over the past three decades (1Leemans C.R. Braakhuis B.J. Brakenhoff R.H. The molecular biology of head and neck cancer.Nat. Rev. Cancer. 2011; 11: 9-22Crossref PubMed Scopus (1887) Google Scholar, 2Matta A. Ralhan R. DeSouza L.V. Siu K.W. Mass spectrometry-based clinical proteomics: head-and-neck cancer biomarkers and drug-targets discovery.Mass Spectrom. Rev. 2010; 29: 945-961Crossref PubMed Scopus (36) Google Scholar). head and neck squamous cell carcinoma squamous cell carcinoma conditioned media immunohistochemistry whole cell lysate multidimensional protein identification technology head and neck cancer normal oral epithelial quantitative real-time PCR disease-free survival urokinase insulin-like growth factor binding protein 7 false discovery rate spectral counting fold change human protein atlas overall survival Kaplan–Meier hazard ratio confidence interval matrix metalloproteinase 14 thrombospondin-1 transforming growth factor, beta-induced, 68kDa. head and neck squamous cell carcinoma squamous cell carcinoma conditioned media immunohistochemistry whole cell lysate multidimensional protein identification technology head and neck cancer normal oral epithelial quantitative real-time PCR disease-free survival urokinase insulin-like growth factor binding protein 7 false discovery rate spectral counting fold change human protein atlas overall survival Kaplan–Meier hazard ratio confidence interval matrix metalloproteinase 14 thrombospondin-1 transforming growth factor, beta-induced, 68kDa. Clinical management of HNSCC is faced by several challenges, including early detection of the primary tumor, and site-specific control of the disease (3Schaaij-Visser T.B. Brakenhoff R.H. Leemans C.R. Heck A.J. Slijper M. Protein biomarker discovery for head and neck cancer.J. Proteomics. 2010; 73: 1790-1803Crossref PubMed Scopus (64) Google Scholar, 4de Jong M.C. Pramana J. Knegjens J.L. Balm A.J. van den Brekel M.W. Hauptmann M. Begg A.C. Rasch C.R. HPV and high-risk gene expression profiles predict response to chemoradiotherapy in head and neck cancer, independent of clinical factors.Radiother. Oncol. 2010; 95: 365-370Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Early diagnosis is often hampered by the asymptomatic nature of HNSCC development and the inadequacy of current diagnostic methods to identify HNSCCs with sufficient specificity and sensitivity. As a result, over 60% of HNSCC patients present with advanced stages III or IV, harboring lymph node metastases (3Schaaij-Visser T.B. Brakenhoff R.H. Leemans C.R. Heck A.J. Slijper M. Protein biomarker discovery for head and neck cancer.J. Proteomics. 2010; 73: 1790-1803Crossref PubMed Scopus (64) Google Scholar). The outcome of HNSCC patients is reduced by the development of local recurrences, which can develop in ∼10–30% of cases, even when surgical margins are pathologically tumor-free (5Tabor M.P. Brakenhoff R.H. Ruijter-Schippers H.J. Kummer Leemans C.R. Braakhuis B.J. as of head and neck a PubMed Scopus Google Scholar). to patients with high risk of tumor in to for in HNSCC biomarkers have including and of human or (1Leemans C.R. Braakhuis B.J. Brakenhoff R.H. The molecular biology of head and neck cancer.Nat. Rev. Cancer. 2011; 11: 9-22Crossref PubMed Scopus (1887) Google Scholar, J. A. J. J. and survival in carcinoma of the head and J. PubMed Scopus Google Scholar, J. R. R. R. and survival of patients with J. 2010; PubMed Scopus Google Scholar, M. A. J. J. J. Siu M. of compared with in for human squamous Oncol. PubMed Scopus Google Scholar). has on HNSCC in survival for advanced remains J. J. J. for advanced head and neck 5-year survival data from a and and Oncol. 2010; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). are biomarkers which have management of in the identification of secreted biomarkers for in or plasma have with of the and which the discovery of biomarkers in M. and for quantitative proteomics using Proteomics. 73: PubMed Scopus Google Scholar). cell a to or plasma for the discovery of potential and have used as a for discovery of secreted biomarkers for and (3Schaaij-Visser T.B. Brakenhoff R.H. Leemans C.R. Heck A.J. Slijper M. Protein biomarker discovery for head and neck cancer.J. Proteomics. 2010; 73: 1790-1803Crossref PubMed Scopus (64) Google Scholar, of conditioned media from three cancer cell a for biomarkers and therapeutic Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar, biomarkers for cancer identified from the of cancer cell lines and the human protein Proteomics. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, of conditioned media from the and cancer cell discovery and validation of cancer PubMed Scopus Google Scholar, C.R. A. of conditioned media from cancer cell lines markers of epithelial cancer.J. PubMed Scopus Google Scholar, M. A. proteomics for discovery of cancer Proteomics. 2010; 73: PubMed Scopus Google Scholar, C.R. of five cancer biomarkers by proteomics of conditioned media of cancer cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). The cancer cell is of proteins secreted or shed by tumor cells in and in the conditioned media secreted or shed by cancer cells have a of and have the potential to in in a clinical for disease diagnosis or of conditioned media from three cancer cell a for biomarkers and therapeutic Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). In vitro cells to in normal growth media by in media for of The is for proteomic and biomarker candidates are and using as or immunohistochemistry associated with biomarker discovery using is the of secreted proteins for can present as a of cell Hence, cell to cell Furthermore, proteomic profiling of whole cell can used as a to and for secreted with the that the relative protein of a secreted protein higher in the compared with the of metalloproteinase in Oncol. PubMed Scopus Google Scholar, M.P. M. M.W. of cancer cells of protein and of PubMed Scopus Google Scholar, proteomics to the cell and of cancer cells and of protein PubMed Scopus Google Scholar, M.C. the of as a for Early 11: PubMed Scopus Google Scholar). In the current study, on multidimensional protein identification technology analyses of and whole cell lysate of three head and neck cancer (HNC) cell and with gene expression as a for discovery of putative secreted HNSCC markers M.P. of the by multidimensional protein identification PubMed Scopus Google Scholar, on a PubMed Scopus Google Scholar). cell lines were from two of the head and the and the larynx and on protein and expression candidates with increased in the the and increased expression as compared with the normal oral epithelial were selected and for validation using publicly available candidates were selected for in vitro validation at the protein Western the of cancer cell lines and control and at the using quantitative real-time PCR In using demonstrated that of candidates have higher protein expression in compared with and that were associated with disease-free survival and increased risk of disease two of the PLAU and IGFBP7 growth factor binding protein were significantly in the plasma of HNSCC patients compared with healthy which in to with increased risk of in the cancer squamous cell cancer cell UTSCC8 and squamous cell cancer cell lines, from R. and normal cells were M. 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A. R. J. J. of associated with and potential of cancer by PubMed Scopus Google Scholar). The the expression profiles of HNSCC and from the patients and squamous cell and validation of in head and neck PubMed Scopus Google Scholar). The the expression profiles of squamous cell carcinomas and normal A. A. R. J. J. of associated with and potential of cancer by PubMed Scopus Google Scholar). candidates to at one of of which were at in HNSCCs for validation. The gene expression profiles for of several candidates in HNSCCs as compared with proteins were secreted the or using and proteins in the cell using and PubMed Scopus Google Scholar, of proteins and PubMed Scopus Google Scholar). of the 90 candidates were to secreted by one of and for validation by to candidates that to the extracellular a was performed to identify biomarker candidates that have in cancer in candidates with available for and ELISAs were for validation. The candidates were selected and in vitro using qRT-PCR and Western the of HNSCC and cells PLAU, IGFBP7, MMP14 metalloproteinase THBS1 and growth factor, were verified as at the and protein levels in HNSCC cells and as compared with the protein was verified THBS1 was to in the HNSCC cell lines at the THBS1 protein was not in the of cells of a at the protein was by with high In the relative and protein expression levels of markers by qRT-PCR and Western a to the levels identified gene expression profiling and spectral respectively. As the to the expression of markers in a tumor of HNSCC using in tumor and that five proteins were in to proteins were in primary human HNSCC that five proteins were in primary HNSCC biopsies expression to to the and not the cancer the potential of five proteins was that for five proteins were on a set by a validation of selected patients and were for clinical including at and and a the not of of the or for the in the of expression levels of PLAU, IGFBP7, and THBS1 were significantly higher in HNSCCs in the set to was in the validation was to that a of expression was in the independent validation set for for for and for on human HNSCC was used to the levels of biomarker candidates in to HNSCCs of and the of PLAU, IGFBP7, MMP14 and THBS1 was of and using the The expression data were for the and validation cohorts in to the potential of The patients were two on the for high with high expression of of proteins a significantly compared with with expression for PLAU, IGFBP7, and Furthermore, with high IGFBP7 and THBS1 expression were associated with for IGFBP7 and the hazard for disease progression or were significantly higher for high expression of proteins IGFBP7 MMP14 or THBS1 were associated with increased risk of disease were for increased risk of for IGFBP7 and THBS1 that biomarkers were identified from the of proteins in the plasma of HNSCC MMP14 THBS1 were secreted cancer patients and healthy PLAU, and IGFBP7 were significantly over-expressed in compared with healthy of and In the of IGFBP7 was higher in compared with levels in healthy in of the of IGFBP7 a potential diagnostic for the patients for plasma IGFBP7 a was a a higher plasma of IGFBP7 in not patients were high and plasma PLAU or IGFBP7 expression on the patients with higher PLAU or IGFBP7 levels a increased risk of for PLAU and for a proteomics approach, with three HNSCC cell lines of and PLAU, IGFBP7, and THBS1 were identified as potential tumor markers for clinical outcome. Furthermore, for the PLAU and IGFBP7 were to in the plasma of with a higher risk of from disease for with higher circulating levels at of the of approaches in and biomarkers for approaches for identification and of secreted cancer biomarkers for in vitro tumor have by with in including the of mass biomarker and validation methods M. A. proteomics for discovery of cancer Proteomics. 2010; 73: PubMed Scopus Google Scholar). the potential diagnostic or of protein candidates have and are one of the primary of biomarker to to and of available have circulating biomarkers with to promising clinical A. M. cell a biomarker for J. Cancer. PubMed Scopus Google Scholar, J. M. J. 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THBS1 is a secreted in tumor progression of extracellular matrix and in remains to THBS1 has to tumor the urokinase tumor cell the urokinase in head and neck cancer Full Text Full Text PDF PubMed Scopus Google Scholar). proteins to have as are of a extracellular matrix Furthermore, as by the the tumor of markers not are tumor progression extracellular matrix cell growth and are at the cell or the extracellular of tumor as that proteins in were identified in Ralhan the profiling of a the of the human cell in addition to oral cell lines, which has in protein of a proteomics R. L.V. A. M. Siu K.W. of proteins secreted by head and neck cancer cell lines using for discovery of 2011; 11: PubMed Scopus Google Scholar). our by validation in and plasma proteins were identified the by Ralhan and the current study, including PLAU, IGFBP7, and the was to our in protein expression in in two independent of primary HNSCC of the tumor and of the of and of the secreted markers in plasma of HNSCC patients and controls. As a result, in a survival that high tumor PLAU, IGFBP7, and THBS1 expression with inferior and increased risk of and disease Furthermore, plasma levels of PLAU and IGFBP7 used to with and data that PLAU and IGFBP7 as diagnostic or biomarkers for squamous cell carcinomas of the larynx and the plasma PLAU and IGFBP7 levels in HNSCC patients of increased risk of validation in a of HNSCC plasma in the was plasma IGFBP7 and PLAU levels with disease progression or of the in In proteins were in plasma to PLAU and IGFBP7 demonstrated a significantly higher expression in the HNSCC expression was not for MMP14 and are secreted by cell the from the tumor M. J. can as a in patients with PubMed Scopus Google Scholar, in head and neck PubMed Scopus Google Scholar). to study, in that the clinical to our was a with a in the of patients with HNSCC in the the the validation set stages and The primary is on the plasma from healthy HNSCC patients were not or with the significantly the and of higher of with and are potential to the current validation using cohorts biomarkers can advanced to clinical management M. A.J. J. A. A. C.R. M. A.C. M. J. and revealed by J. PubMed Scopus Google Scholar). the of plasma and the discovery of putative markers from in vitro cell are for In the cancer cell a of proteins for biomarker discovery using a In the current study, the identification of several biomarker candidates for validation to the diagnostic and of markers in and to specificity and in to clinical a study, to PLAU and IGFBP7 as biomarkers to disease or as early of disease relapse. with

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