A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer

• Published in: Biochimica et biophysica acta. General subjects 2018-09, Vol.1862 (9), p.2069-2080
• Main Authors: Carrascal, M.A., Silva, M., Ferreira, J.A., Azevedo, R., Ferreira, D., Silva, A.M.N., Ligeiro, D., Santos, L.L., Sackstein, R., Videira, P.A.
• Format: Article
• Language: English
• Subjects: Breast cancer; CD13; CD44; E-selectin ligand; HCELL; sLeX
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2018.05.013

The glycan moieties sialyl-Lewis-X and/or -A (sLeX/A) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. We observed that the CF1_T cell line expressed sLeX, but not sLeA and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLeX-CD44 and sLeX-CD13 was confirmed in clinical breast cancer tissue samples. Both CD44 and CD13 glycoforms display sLeX in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target. [Display omitted] •Glycoforms of CD13 and CD44 are E-selectin ligands in the CF1_T breast cancer cell line.•CD13 and CD44 glycoproteins are decorated with sLeX glycans in breast cancer tissues.•Most E-selectin ligands in CF1_T breast cancer cell line are N-glycosylated glycoproteins.