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
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 (SLex). SLex 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 SLex antigen and overexpressed in gastric carcinomas. Herein, we identified the glycoproteins carrying SLex in gastric cancer cells overexpressing ST3Gal IV enzyme and evaluated their biomarker potential for gastric carcinoma.
Methods: SLex 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 SLex on the identified glycoprotein. Protein N-glycans of the SLex 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 SLex protein carrier was performed and the conjoint expression of the SLex 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 SLex. 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 SLeX was observed in 86,3% of gastric carcinoma cases and 74,5% of the total cases displayed the conjoint CEA+SLex 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 SLex.
Conclusion: CEA is the major glycoprotein carrying SLex in gastric carcinoma and the conjoint detection of CEA-SLex 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