Nucleolin-Sle A Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells

Background: Gastric cancer (GC) is a major health burden worldwide, with half of patients developing metastases within 5 years after treatment, urging novel biomarkers for diagnosis and efficient therapeutic targeting. Sialyl-Lewis A (SLeA), a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. It is rarely expressed in healthy tissues and blood cells, while it is present in highly metastatic cell lines and metastases. SLeA is also involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. Methods and Results: To improve cancer specificity, we have explored the SLeA-glycoproteome of six GC cell models, with emphasis on glycoproteins showing affinity for E-selectin. A novel bioinformatics-assisted algorithm identified nucleolin (NCL), a nuclear protein, as a potential targetable biomarker potentially involved in metastasis. Several immunoassays, including Western blot and in situ proximity ligation reinforced the existence of cell surface NCL-SLeA glycoforms in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues. Conclusions: NCL-SLeA is a biomarker of poor prognosis in GC holding potential for precise cancer targeting. This is the first report describing SLeA in preferentially nuclear protein, setting a new paradigm for cancer biomarkers discovery and targeted therapies. The authors wish to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant SFRH/BD/103571/2014 (EF), SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA), SFRH/BD/127327/2016 (CG); and assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT funds for CI-IPOP research unit (PEst-OE/SAU/UI0776/201), CI-IPOP funds (CI-IPOP-29-2014; CI-IPOP-58-2015; CI-IPOP-Proj.70-bolsa2019-GPTE), FCT/MCTES funds for the LAQV research unit (UIDB/50006/2020) and PhD Programs in Biomedical Sciences and Pathology and Molecular Genetics of ICBAS-University of Porto. The authors EF, DF and MRS also acknowledge the financial support of the Research Group of Digestive Cancers (GICD), the ?Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)? framework (NORTE-01-0145-FEDER-000027), IPO-Score (DSAIPA/DS/0042/2018) and ?Towards a single therapy with a synergistic combination against triple negative breast cancer and neuroblastoma by nucleolin mediated multicellular targeting? (EU joint call ENMed/0006/2015). This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by FCT/MCTES funds in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274).