Secretome-Based Identification and Characterization of Potential Biomarkers in Thyroid Cancer

Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital, Toronto, Ontario, Canada.
Journal of Proteome Research (Impact Factor: 4.25). 11/2010; 9(11):5757-69. DOI: 10.1021/pr100529t
Source: PubMed


In search of thyroid cancer biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of 46 high-confidence identifications, 6 proteins were considered for verification in thyroid cancer patients' tissue and blood. The localization of two proteins, nucleolin and prothymosin-α (PTMA), was confirmed in TPC-1 and CAL62 cells by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells in NOD/SCID/γ mice and human thyroid cancers (48 tissues). Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic compared to papillary and poorly differentiated carcinomas. Nuclear expression of nucleolin was observed in all subtypes of thyroid carcinomas, along with faint cytoplasmic expression in anaplastic cancers. Importantly, PTMA, nucleolin, clusterin, cysteine-rich angiogenic inducer 61, enolase 1, and biotinidase were detected in thyroid cancer patients' sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. In conclusion, we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify novel proteins that can be independently verified in cell lines, xenografts, tumor tissues, and blood samples of thyroid cancer patients. These observations support their potential utility as minimally invasive biomarkers for thyroid carcinomas and their application in management of these diseases upon future validation.

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    • "Previously, our study of the secretomes of two TC cell lines, a papillary-derived cell line (TPC-1) and an anaplasticderived cell line (CAL62), demonstrated that secretome proteins are detectable in sera and tissues of TC patients [24]. We identified 46 high-confidence protein IDs in the previous study and confirmed some of the identified proteins in TC patients' samples to assess their clinical significance. "
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    ABSTRACT: Using proteomics in tandem with bioinformatics, the secretomes of non-aggressive and aggressive thyroid carcinoma (TC) cell lines were analyzed to detect potential biomarkers for tumor aggressiveness. A panel of nine proteins, activated leukocyte cell adhesion molecule (ALCAM/ CD166), tyrosine-protein kinase receptor (AXL), amyloid beta A4 protein (APP), amyloid-like protein 2 (APLP2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), phosphoglycerate kinase 1 (PGK1), pyruvate kinase isozyme M2 (PKM2), phosphatase 2A inhibitor (SET), and protein kinase C inhibitor protein 1 (14-3-3 zeta) was chosen to confirm their expression in TC patients' sera and tissues. Increased pre-surgical circulating levels of ALCAM were associated with aggressive tumors (p = 0.04) and presence of lymph node metastasis (p = 0.018). Increased serum AXL levels were associated with extrathyroidal extension (p = 0.027). Furthermore, differential expression of APP, AXL, hnRNP K, PGK1, PKM2, and SET was observed in TC tissues compared to benign nodules. Decreased nuclear expression of AXL can detect malignancy with 90% specificity and 100% sensitivity (AUC = 0.995, p < 0.001). In conclusion, some of these proteins show potential for future development as serum and/or tissue-based biomarkers for TC and warrant further investigation in a large cohort of patients.
    Full-text · Article · Mar 2013 · Proteomics
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    • "Relative to healthy controls, the levels of serum vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) are elevated in late-stage PTC; however, as the levels of these markers do not differentiate between goiter and early stage PTC, they are not useful for improving diagnosis [8]. Levels of biotinidase, clusterin, cysteine-rich, angiogenic inducer, 61 (CYR61), enolase 1, nucleolin and prothymosin alpha (PTMA) have been compared between PTC patients and healthy controls but no comparison was made with patients with benign nodules [9]. In spite of these advances, there are currently no validated serum markers for the diagnosis of PTC or for risk stratification of thyroid masses. "
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    ABSTRACT: To identify serum biomarkers of papillary thyroid cancer. Prospective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors. A total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively. Serum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.
    Full-text · Article · Feb 2013 · Journal of otolaryngology - head & neck surgery = Le Journal d'oto-rhino-laryngologie et de chirurgie cervico-faciale
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    • "In search of new cancer biomarkers for this malignancy, we analyzed the secretome from thyroid cancer cell lines to identify cancer-relevant secreted proteins that can serve as potential biomarkers [7]. One of the candidate proteins identified in our study was biotinidase, an enzyme that catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. "
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    ABSTRACT: Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer (PTC, 34 benign thyroid tissues and 43 FNA samples and correlated with patients' prognosis. Overall biotinidase expression was decreased in PTC compared to benign nodules (p = 0.001). Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival (p = 0.019, Hazards ratio (HR) = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival (p = 0.014, HR = 5.4). Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors (p = 0.002, Odds ratio (OR) = 0.29) which was evident by its significant association with advanced T stage (p = 0.003, OR = 0.28), nodal metastasis (p<0.001, OR = 0.16), advanced TNM stage (p<0.001, OR = 0.21) and extrathyroidal extension (p = 0.001, OR = 0.23). However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas (p = 0.015, HR = 12.8). In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness.
    Full-text · Article · Jul 2012 · PLoS ONE
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