Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death

Abstract

BackgroundThe ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated.ResultsUsing the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays.ConclusionResults herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance.

ST6GAL1: A key player in cancer (Review)

“…An upstream regulator of DNA repair pathways is EGFR, known to regulate DNA repair, DNA replication and maintenance of genome stability when found in the nucleus (105). A study of ST6GAL1 sialylation in ovarian cancer, suggested that increased ST6GAL1 sialylation confers resistance to chemotherapeutic intervention (106). Of interest, they postulated that this chemoresistance was through direct sialylation of EGFR by ST6GAL1, resulting in heightened activation of EGFR.…”

ST6GAL1-mediated heterogeneity in sialic acid levels potentiates invasion of breast cancer epithelia

“…Several published reports note hypersialylation of cancer cells, including that of the neoplasms of breast (22) cancer. Hypersialylation is associated with, and suggested to be causal to, increased aggressiveness, stemness, resistance to chemotherapeutic agents, ability to survive in stressful conditions like hypoxia and impaired nutrient supply (24,25,27,38). However, when we performed flow cytometry to isolate the two subpopulations showing distinct α2,6 sialic acid levels, the one showing lower levels (which we denoted as medium 2,6-Sial cells) showed greater invasion than high 2,6-Sial cells in both transwell assay and in 3D cultures.…”

“…Hypersialylation is one of the most frequently observed changes in glycosylation in many cancer types (22,23). Selective enrichment of terminal α2,6linked sialic acids (referred here-onward as α2,6-Sial), due to overexpression of ST6GAL1, in cancer cell glycocalyces can elicit a wide range of biological outcomes like protection from hypoxia, resistance to chemotherapy, pro-survival and conferral of cancer stem cell phenotype (24)(25)(26)(27).…”

Don’t sugarcoat it: How glycocalyx composition influences cancer progression

“…Sialylation by ST6Gal-1 is thought to maintain a more stem-like state in cancer cells (Schultz et al, 2016) and has been implicated in bladder (Antony et al, 2014), colon (Zhang et al, 2017), breast (Lu et al, 2014), and ovarian cancers (Christie et al, 2008). Multiple works have demonstrated how ST6Gal-1 both confers resistance to chemotherapy (Britain et al, 2018; Schultz et al, 2013) and protects cancer cells from apoptosis by sialylation of Fas (Swindall and Bellis, 2011) and tumor necrosis factor receptor (Holdbrooks et al, 2018). Interestingly, ST6Gal-1 is also the only glycosyltransferase to date in which its soluble form can “extrinsically” glycosylate outside the cell (Manhardt et al, 2017).…”