Abstract
The TNF-TNFR1 signaling pathway plays a pivotal role in regulating the balance between cell survival and cell death. Upon binding to TNF, plasma membrane-localized TNFR1 initiates survival signaling, whereas TNFR1 internalization promotes caspase-mediated apoptosis. We previously reported that the α2-6 sialylation of TNFR1 by the tumor-associated sialyltransferase ST6GAL1 diverts signaling toward survival by inhibiting TNFR1 internalization. In the current investigation, we interrogated the mechanisms underlying sialylation-dependent regulation of TNFR1 and uncovered a novel role for α2-6 sialylation, but not α2-3 sialylation, in mediating apoptosis-resistance. Our studies utilized HEK293 cells with deletion of sialyltransferases that modify N-glycans with either α2-3-linked sialic acids (ST3GAL3/4/6) or α2-6-linked sialic acids (ST6GAL1/2). Additionally, ST6GAL1 was re-expressed in cells with ST6GAL1/2 deletion to restore α2-6 sialylation. Using total internal reflection fluorescence (TIRF) microscopy and BS3 cross-linking, we determined that, under basal conditions, cells expressing TNFR1 devoid of α2-6 sialylation displayed enhanced TNFR1 oligomerization, an event that poises cells for activation by TNF. Moreover, upon stimulation with TNF, greater internalization of TNFR1 was observed via time-lapse TIRF and flow cytometry, and this correlated with increased caspase-dependent apoptosis. These effects were reversed by ST6GAL1 re-expression. Conversely, eliminating α2-3 sialylation did not significantly alter TNFR1 clustering, internalization or apoptosis. We also evaluated the Fas receptor, given its structural similarity to TNFR1. As with TNFR1, α2-6 sialylation had a selective effect in protecting cells against Fas-mediated apoptosis. These results collectively suggest that ST6GAL1 may serve a unique function in shielding cancer cells from apoptotic stimuli within the tumor microenvironment.