Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFRmediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.sialic acid | glycoproteomics | glycan sequencing | click chemistry | mass spectrometry

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKIsensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKIresistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKIresistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.E pidermal growth factor receptor (EGFR), one of the most studied receptor tyrosine kinases, is a drug target for cancer therapy, because its kinase activity correlates with tumorigenicity (1). Under normal conditions, EGFR forms dimers upon ligand binding and induces kinase activation (2-6). The conformational change of EGFR from tethered to extended form induced by ligand binding involves the exposure of the interface, followed by dimerization, activation, and autophosphorylation (7). The phosphorylation code of EGFR determines the propensity of the downstream signaling network to regulate cell proliferation, survival, migration, and angiogenesis (8, 9).In a significant fraction of patients with nonsmall cell lung cancer (NSCLC), especially patients in Asia and those with the adenocarcinoma subtype, mutations in the kinase domain of EGFR cause constitutive activation and have been identified as an important factor in EGFR dysregulation (10, 11). Particularly, mutation from leucine to arginine at position 858 (L858R) and, less significantly, deletion of exon 19 that eliminates four amino acids (LREA) account for ∼90% of the mutations involved in the constitutive activation of EGFR. These mutations are commonly found in patients with increased sensitivity to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib (12-14). However, most patients with such mutations show resistance within months after TKI therapy, and >50% of them develop a second EGFR mutation, T790M, which confers TKI resi...