A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.

[1]  L. Silverman,et al.  Synthesis of α1-Antichymotrypsin and α1-Acid Glycoprotein by Human Breast Epithelial Cells , 1982 .

[2]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[3]  Kiyotoshi Inoue,et al.  High Serum Concentrations of Sialyl Lewisx Predict Multilevel N2 Disease in Non–Small-Cell Lung Cancer , 2006, Annals of Surgical Oncology.

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

[5]  T. Spector,et al.  Severe rheumatoid arthritis prohibits the pregnancy-induced decrease in alpha3-fucosylation of alpha1-acid glycoprotein. , 1998, Glycoconjugate journal.

[6]  S. Ōmura,et al.  Macrosphelide B suppressed metastasis through inhibition of adhesion of sLe(x)/E-selectin molecules. , 2002, Biochemical and biophysical research communications.

[7]  J. Haddow,et al.  Elevated serum acute phase protein levels as predictors of disseminated breast cancer , 1983, Cancer.

[8]  D. Kessel,et al.  Serum sialyl transferase levels in patients with metastatic breast cancer treated by chemotherapy. , 1982, British Journal of Cancer.

[9]  Pauline M Rudd,et al.  Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions. , 2006, Methods in molecular biology.

[10]  David J. Harvey,et al.  HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. , 2008, Analytical biochemistry.

[11]  G. Strecker,et al.  Determination of the primary structures of 16 asialo-carbohydrate units derived from human plasma alpha 1-acid glycoprotein by 360-MHZ 1H NMR spectroscopy and permethylation analysis. , 1978, Biochemistry.

[12]  C. Bertozzi,et al.  Glycans in cancer and inflammation — potential for therapeutics and diagnostics , 2005, Nature Reviews Drug Discovery.

[13]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Yehia Mechref,et al.  Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles. , 2008, Clinical chemistry.

[15]  Melinda Fitzgerald,et al.  Immunol. Cell Biol. , 1995 .

[16]  E. B. D. Brinkman-van der Linden,et al.  Inflammation-induced expression of sialyl Lewisx is not restricted to α1-acid glycoprotein but also occurs to a lesser extent on α1-antichymotrypsin and haptoglobin , 1998, Glycoconjugate Journal.

[17]  B. Zetter,et al.  Cancer biomarkers: knowing the present and predicting the future. , 2005, Future oncology.

[18]  D. Harvey.  Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides. , 1993, Rapid communications in mass spectrometry : RCM.

[19]  C. Ohyama,et al.  Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Kannagi,et al.  Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases. , 1997, Japanese journal of clinical oncology.

[21]  S. Thompson,et al.  Elevated levels of abnormally-fucosylated haptoglobins in cancer sera. , 1987, British Journal of Cancer.

[22]  T. Spector,et al.  Severe rheumatoid arthritis prohibits the pregnancy-induced decrease in α3-fucosylation of α1-acid glycoprotein , 1998, Glycoconjugate Journal.

[23]  R. Kannagi,et al.  Carbohydrate‐mediated cell adhesion in cancer metastasis and angiogenesis , 2004, Cancer science.

[24]  R. Dwek,et al.  Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA. , 2006, Glycobiology.

[25]  M. Fukuda.  Possible roles of tumor-associated carbohydrate antigens. , 1996, Cancer research.

[26]  R. Dwek,et al.  Changes of serum glycans during sepsis and acute pancreatitis. , 2007, Glycobiology.

[27]  W. van Dijk,et al.  Inflammation-induced expression of sialyl Lewis X-containing glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera , 1993, The Journal of experimental medicine.

[28]  F. Altmann,et al.  Comprehensive glyco‐proteomic analysis of human α1‐antitrypsin and its charge isoforms , 2006, Proteomics.

[29]  S. Hakomori,et al.  Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. , 2001, Advances in experimental medicine and biology.

[30]  Kazutaka Nakashima,et al.  Combined measurement of serum sialyl Lewis X with serum CA15-3 in breast cancer patients. , 2006, Japanese journal of clinical oncology.

[31]  D. Harvey.  Fragmentation of negative ions from carbohydrates: Part 3. Fragmentation of hybrid and complex N-linked glycans , 2005, Journal of the American Society for Mass Spectrometry.

[32]  S. Hakomori,et al.  Biosynthesis of the sialyl-Lex determinant carried by type 2 chain glycosphingolipids (IV3NeuAcIII3FucnLc4, VI3NeuAcV3FucnLc6, and VI3NeuAcIII3V3Fuc2nLc6) in human lung carcinoma PC9 cells. , 1986, The Journal of biological chemistry.

[33]  L. Silverman,et al.  Synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. , 1982, Cancer research.

[34]  T. Nagayasu,et al.  Expression of ABH/Lewis-related antigens as prognostic factors in patients with breast cancer , 2002, Journal of Cancer Research and Clinical Oncology.

[35]  R. Dwek,et al.  Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry. , 2008, Analytical biochemistry.

[36]  T. Hall,et al.  Enzyme activity in invasive tumors of human breast and colon. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[37]  R. Dwek,et al.  Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure , 2004, FEBS letters.

[38]  Shinzaburo Noguchi,et al.  mRNA expression level of estrogen‐inducible gene, α1‐antichymotrypsin, is a predictor of early tumor recurrence in patients with invasive breast cancers , 2004, Cancer science.

[39]  R. Kannagi,et al.  Gene expression of fucosyl- and sialyl-transferases which synthesize sialyl Lewisx, the carbohydrate ligands for E-selectin, in human breast cancer. , 1998, International journal of oncology.

[40]  Kojiro Matsumoto,et al.  Binding of sialyl Lewis X antigen to lectin-like receptors on NK cells induces cytotoxicity and tyrosine phosphorylation of a 17-kDa protein. , 2006, Biochimica et biophysica acta.

[41]  M. Duffy,et al.  Serum tumor markers in breast cancer: are they of clinical value? , 2006, Clinical chemistry.

[42]  B. Cantwell,et al.  Abnormally-fucosylated haptoglobin: a cancer marker for tumour burden but not gross liver metastasis. , 1991, British Journal of Cancer.

[43]  G. Strecker,et al.  Determination of the primary structures of 16 asialo-carbohydrate units derived from human plasma α1-acid glycoprotein by 360-MHz 1H NMR spectroscopy and permethylation analysis , 1978 .

[44]  T. Paavonen,et al.  Endothelial and epithelial expression of sialyl Lewisx and sialyl Lewisa in lesions of breast carcinoma , 1997, International journal of cancer.

[45]  CHARGE-SHIFT PROBES OF MEMBRANE POTENTIAL. SYNTHESIS , 1984 .

[46]  T. Goi,et al.  Expression of nm23-H1 Gene and Sialyl Lewis X Antigen in Breast Cancer , 1998, Oncology.

[47]  K. Okuno,et al.  Role of sialyl Lewis X in liver metastasis in view of liver-associated immunity. , 2003, Hepato-gastroenterology.

[48]  B. Domon,et al.  A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates , 1988, Glycoconjugate Journal.

[49]  A. Kobata,et al.  Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours , 2005, Immunology and cell biology.

[50]  Ying Zhang,et al.  Regulation of metastasis‐suppressive gene Nm23‐H1 on glycosyl‐transferases involved in the synthesis of sialyl Lewis antigens , 2005, Journal of cellular biochemistry.

[51]  J. Magnani.  The discovery, biology, and drug development of sialyl Lea and sialyl Lex. , 2004, Archives of biochemistry and biophysics.

[52]  D. Hochstrasser,et al.  Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients , 1997, Electrophoresis.