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Theranostics 2019; 9(24):7431-7446. doi:10.7150/thno.33858 This issue Cite

Research Paper

Carcinoembryonic antigen carrying SLe^X as a new biomarker of more aggressive gastric carcinomas

Catarina Gomes^1,2✉, Andreia Almeida^3,4, Ana Barreira^1,2, Juliana Calheiros^1,2, Filipe Pinto^1,2, Rafaela Abrantes^1,2, Ana Costa^1,2, António Polonia^1,2, Diana Campos^1,2, Hugo Osório^1,2,5, Hugo Sousa⁵, João Pinto-de-Sousa^2,5, Daniel Kolarich^3,4, Celso A. Reis^1,2,5,6✉

1. Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal
2. Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal
3. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany
4. Institute for Glycomics, Griffith University, Southport, QLD, Australia
5. Faculty of Medicine, University of Porto, Porto, Portugal
6. Institute of Biomedical Sciences of Abel Salazar, ICBAS, Porto, Portugal

✉ Corresponding authors: Celso A. Reis, Glycobiology in Cancer, IPATIMUP, i3S. E-mail: celsorpt; Catarina Gomes, Glycobiology in Cancer, IPATIMUP, i3S. E-mail: cgomespt

Abstract

Malignant transformation of gastric cells is accompanied by the deregulated expression of glycosyltransferases leading to the biosynthesis of tumor-associated glycans such as the sialyl-Lewis X antigen (SLe^x). SLe^x presence on cell surface glycoconjugates increases the invasive capacity of gastric cancer cells and is associated with tumor metastasis. ST3Gal IV enzyme is involved in the synthesis of SLe^x antigen and overexpressed in gastric carcinomas. Herein, we identified the glycoproteins carrying SLe^x in gastric cancer cells overexpressing ST3Gal IV enzyme and evaluated their biomarker potential for gastric carcinoma.

Methods: SLe^x modified glycoproteins were identified applying western blot and mass spectrometry. Immunoprecipitation, proximity ligation assay (PLA), E-selectin binding assay and CRISPR/cas9 knockout experiments were performed to characterize the presence of SLe^x on the identified glycoprotein. Protein N-glycans of the SLe^x protein carrier were in deep analyzed by porous-graphitized-carbon liquid-chromatography and tandem mass spectrometry glycomics. In silico expression analysis of α2-3 sialyltransferase ST3Gal IV and SLe^x protein carrier was performed and the conjoint expression of the SLe^x modified glycoproteins evaluated by immunohistochemistry and PLA in a series of gastric carcinomas.

Results: Carcinoembryonic antigen (CEA; CEACAM5) was identified and validated by different methodologies as a major carrier of SLe^x. N-glycomics of CEA revealed that complex N-glycans are capped with α2-3 linked sialic acid (Neu5Acα2-3Galβ1-4GlcNAc). Data set analysis of ST3Gal IV and CEA showed that ST3Gal IV expression was associated with patient´s poor survival, whereas CEA did not show any prognostic value. The co-expression of both CEA and SLe^X was observed in 86,3% of gastric carcinoma cases and 74,5% of the total cases displayed the conjoint CEA+SLe^x in situ PLA expression. This expression was associated with clinicopathological features of the tumors, including infiltrative pattern of tumor growth, presence of venous invasion and patient's poor survival. CEA immunoprecipitation from gastric carcinoma tissues also confirmed the presence of SLe^x.

Conclusion: CEA is the major glycoprotein carrying SLe^x in gastric carcinoma and the conjoint detection of CEA-SLe^x is associated with aggressive tumor features highlighting its PLA detection as a biomarker of gastric cancer patient prognosis for theranostic applications.

Keywords: Gastric Cancer, Carcinoembryonic antigen, Glycosylation, ST3Gal IV, Sialyl-Lewis X

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