Glycoproteomic identification of potential glycoprotein biomarkers in ovarian cancer proximal fluids

Abstract Background: Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in ovarian cancer. Several sialyltransferase genes have been shown to be up-regulated at both the mRNA and protein levels in a number of cancers, including that of the ovary. ST6GAL1 (β-galactosamide α2,6-sialyltranferase 1) gene expression has previously been shown to be upregulated in ovarian cancers of all major subtypes. Methods: We have identified the sialome (i.e., sialic acid containing glycoproteins) of biological fluids from ovarian cancer patients and ovarian cancer cell lines utilizing tandem mass spectrometry as a potential pool of novel biomarker candidates. The sialoglycopeptides from four ovarian cancer cell lines, pooled ascites (n=13) and ovarian cyst (n=14) fluids from ovarian cancer patients were enriched utilizing affinity to agarose-immobilized Elderberry lectin (Sambucus nigra agglutinin) and magnetic hydrazide beads folowing periodate-mediated oxidation of sialic acids. Benign ovarian cyst (n=10) and peritoneal effusion (n=20) fluids were analyzed in the same fashion to serve as controls. PNGase F deglycosylated peptides were identified using electrospray ionization-LTQ Orbitrap tandem mass spectrometry. Results: In all of the samples analyzed in the glycoproteomic portion of the study, we have identified 579 glycosylation sites on 333 proteins. Of these, 13 were exclusively identified in biological fluids from ovarian cancer patients, and another eight were common to these fluids and the ovarian cancer cell line supernatants. Conclusions: The proteins identified in the present study could form the basis for future studies examining and quantifying their sialylation status as biomarkers of ovarian cancer.

[1]  S. Bellis,et al.  Sialylation of the Fas Death Receptor by ST6Gal-I Provides Protection against Fas-mediated Apoptosis in Colon Carcinoma Cells* , 2011, The Journal of Biological Chemistry.

[2]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[3]  E. Diamandis,et al.  Mining the Ovarian Cancer Ascites Proteome for Potential Ovarian Cancer Biomarkers*S , 2009, Molecular & Cellular Proteomics.

[4]  D. Bullard,et al.  ST6Gal-I Regulates Macrophage Apoptosis via α2-6 Sialylation of the TNFR1 Death Receptor* , 2011, The Journal of Biological Chemistry.

[5]  Pauline M Rudd,et al.  Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG. , 2007, Glycobiology.

[6]  C. Santiskulvong,et al.  Cisplatin and PI3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix‐metalloproteinase expression , 2010, Cytoskeleton.

[7]  E. Diamandis,et al.  Separation of kallikrein 6 glycoprotein subpopulations in biological fluids by anion‐exchange chromatography coupled to ELISA and identification by mass spectrometry , 2012, Proteomics.

[8]  M. Baumann,et al.  Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis. , 1996, The Biochemical journal.

[9]  Pauline M. Rudd,et al.  Glycosylation Changes on Serum Glycoproteins in Ovarian Cancer May Contribute to Disease Pathogenesis , 2009, Disease markers.

[10]  I. Shih,et al.  Oncoproteomic Analysis Reveals Co-Upregulation of RELA and STAT5 in Carboplatin Resistant Ovarian Carcinoma , 2010, PloS one.

[11]  A. Yamanaka,et al.  Sialic acid in fibrinogen: effects of sialic acid on fibrinogen-fibrin conversion by thrombin and properties of asialofibrin clot. , 1993, Biological & pharmaceutical bulletin.

[12]  Jiri Petrak,et al.  Decreased concentrations of retinol-binding protein 4 in sera of epithelial ovarian cancer patients: a potential biomarker identified by proteomics. , 2011, Oncology reports.

[13]  Károly Héberger,et al.  Mass spectrometric and linear discriminant analysis of N-glycans of human serum alpha-1-acid glycoprotein in cancer patients and healthy individuals. , 2008, Journal of proteomics.

[14]  L. Moreno,et al.  Relationship between immunoinflammatory proteins containing sialic acid and low-density lipoprotein serum concentrations. , 1996, Clinica chimica acta; international journal of clinical chemistry.

[15]  B. Milutinovic,et al.  Assessment of Sialic Acid Diversity in Cancer- and Non-Cancer Related CA125 Antigen Using Sialic Acid-Binding Ig-Like Lectins (Siglecs) , 2012, Disease markers.

[16]  Wu Cy,et al.  Different enzyme activities of sialyltransferases in gynecological cancer cell lines. , 2002 .

[17]  H. Parkinson,et al.  A global map of human gene expression , 2010, Nature Biotechnology.

[18]  Ajit Varki,et al.  alpha 2-6-Linked sialic acids on N-glycans modulate carcinoma differentiation in vivo. , 2008, Cancer research.

[19]  E. Diamandis,et al.  Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System* , 2009, Molecular & Cellular Proteomics.

[20]  L. Drahos,et al.  Investigation of genetic variants of α-1 acid glycoprotein by ultra-performance liquid chromatography–mass spectrometry , 2009, Analytical and bioanalytical chemistry.

[21]  K A Baggerly,et al.  New tumor markers: CA125 and beyond , 2005, International Journal of Gynecologic Cancer.

[22]  D. Ankerst,et al.  Afamin and Apolipoprotein A-IV: Novel Protein Markers for Ovarian Cancer , 2009, Cancer Epidemiology Biomarkers & Prevention.

[23]  K. Partheen,et al.  Four potential biomarkers as prognostic factors in stage III serous ovarian adenocarcinomas , 2008, International journal of cancer.

[24]  G. Lauc,et al.  Enzyme linked lectin assay (ELLA) for direct analysis of transferrin sialylation in serum samples. , 2007, Clinical biochemistry.

[25]  Dylan J. Sorensen,et al.  A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma. , 2011, Analytical biochemistry.

[26]  Michèl Schummer,et al.  The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. , 2003, Cancer research.

[27]  I. Bairati,et al.  Immunohistochemical analysis of possible chemoresistance markers identified by micro-arrays on serous ovarian carcinomas , 2008, Modern Pathology.

[28]  Susan J Fisher,et al.  Sweetening the pot: adding glycosylation to the biomarker discovery equation. , 2010, Clinical chemistry.

[29]  M. Hodson,et al.  Altered sialyl- and fucosyl-linkage on mucins in cystic fibrosis patients promotes formation of the sialyl-Lewis X determinant on salivary MUC-5B and MUC-7 , 2001, Pflügers Archiv.

[30]  P. Rudd,et al.  Glycosylation of liver acute‐phase proteins in pancreatic cancer and chronic pancreatitis , 2010, Proteomics. Clinical applications.

[31]  M. Schindl,et al.  Cathepsin D in ovarian cancer: prognostic value and correlation with p53 expression and microvessel density. , 2004, Gynecologic oncology.

[32]  S. Bellis,et al.  ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function , 2008, Journal of ovarian research.

[33]  B. Têtu,et al.  Gene expression profiling of paired ovarian tumors obtained prior to and following adjuvant chemotherapy: molecular signatures of chemoresistant tumors. , 2006, International journal of oncology.

[34]  G. Hall,et al.  Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer , 2007, Clinical Cancer Research.

[35]  J. W. Kim,et al.  Identification of genes with differential expression in chemoresistant epithelial ovarian cancer using high-density oligonucleotide microarrays. , 2006, Oncology research.

[36]  I. Christensen,et al.  Cleaved Forms of the Urokinase Plasminogen Activator Receptor in Plasma Have Diagnostic Potential and Predict Postoperative Survival in Patients with Ovarian Cancer , 2008, Clinical Cancer Research.

[37]  B. Milutinovic,et al.  Glycoforms of CA125 antigen as a possible cancer marker. , 2008, Cancer biomarkers : section A of Disease markers.

[38]  I. Shih,et al.  Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions , 2010, Journal of Cellular and Molecular Medicine.

[39]  E. Fung,et al.  A novel proteomic biomarker panel as a diagnostic tool for patients with ovarian cancer. , 2011, Gynecologic oncology.

[40]  K. Oliva,et al.  Identification of ovarian cancer‐associated proteins in symptomatic women: A novel method for semi‐quantitative plasma proteomics , 2012, Proteomics. Clinical applications.

[41]  D R Schwartz,et al.  Coordinately up-regulated genes in ovarian cancer. , 2001, Cancer research.

[42]  L. Kiemeney,et al.  Polymorphisms in Stromal Genes and Susceptibility to Serous Epithelial Ovarian Cancer: A Report from the Ovarian Cancer Association Consortium , 2011, PloS one.

[43]  A. Berchuck,et al.  Co-expression of angiogenic markers and associations with prognosis in advanced epithelial ovarian cancer: a Gynecologic Oncology Group study. , 2007, Gynecologic oncology.

[44]  N. Karlsson,et al.  O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose. , 2012, Glycobiology.

[45]  Peng-Hui Wang,et al.  Altered mRNA expressions of sialyltransferases in ovarian cancers. , 2005, Gynecologic oncology.

[46]  Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display. , 2011, Journal of biotechnology.

[47]  R. Bast,et al.  Status of tumor markers in ovarian cancer screening. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[48]  E. Appella,et al.  The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. , 1990, The Journal of biological chemistry.

[49]  M. Schummer,et al.  Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the established marker CA125. , 2012, Gynecologic oncology.

[50]  Richard D. Cummings,et al.  The repertoire of glycan determinants in the human glycome. , 2009, Molecular bioSystems.

[51]  D. Xie,et al.  Overexpression of Clusterin in Ovarian Cancer is Correlated With Impaired Survival , 2009, International Journal of Gynecologic Cancer.

[52]  A. Gadducci,et al.  Preoperative serum intercellular adhesion molecule-1 (ICAM-1) and E-selectin (endothelial cell leukocyte adhesion molecule, ELAM-1) in patients with epithelial ovarian cancer. , 1995, Anticancer research.

[53]  O. Hashim,et al.  Patients with ovarian carcinoma excrete different altered levels of urine CD59, kininogen-1 and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin , 2010, Proteome Science.

[54]  L. Hefler,et al.  The prognostic value of plasma fibrinogen levels in patients with endometrial cancer: a multi-centre trial , 2010, British Journal of Cancer.

[55]  A. Mes-Masson,et al.  Protease inhibitor SERPINA1 expression in epithelial ovarian cancer , 2010, Clinical & Experimental Metastasis.

[56]  M. Sasaki,et al.  Isolation and characterization of sulfated glycoprotein from human pancreatic juice. , 1988, Biochimica et biophysica acta.