Mol Cell Proteomics. 2015 Jun;14(6):1616-29. doi: 10.1074/mcp.M114.046862. Epub 2015 Mar 26.

Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery.

Campos D¹, Freitas D², Gomes J², Magalhães A², Steentoft C³, Gomes C², Vester-Christensen MB³, Ferreira JA⁴, Afonso LP⁵, Santos LL⁶, Pinto de Sousa J⁷, Mandel U³, Clausen H³, Vakhrushev SY⁸, Reis CA⁹.

Author information

1: From the ‡Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal;
2: §IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal;
3: From the ‡Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
4: ¶Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Rua Dr. António Bernardino de Almeida 4200-072 Porto, Portugal; ‖QOPNA, Department of Chemistry of the University of Aveiro, Campus Universitário de Santiago 3810-193 Aveiro, Portugal;
5: **Department of Pathology, Portuguese Institute of Oncology, Rua Dr. António Bernardino de Almeida 4200-072 Porto, Portugal;
6: ¶Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Rua Dr. António Bernardino de Almeida 4200-072 Porto, Portugal;
7: ‡‡Faculty of Medicine of the University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal;
8: From the ‡Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; seva@sund.ku.dk celsor@ipatimup.pt.
9: §IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal; ‡‡Faculty of Medicine of the University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal; §§Institute of Biomedical Sciences Abel Salazar, ICBAS, Rua de Jorge Viterbo Ferreira n.228, 4050-313 Porto, Portugal seva@sund.ku.dk celsor@ipatimup.pt.

Abstract

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.