Abstract
Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by TNF induces either cell survival or cell death. However, the mechanisms mediating these distinct outcomes remain poorly understood. In this study, we report that the ST6Gal-I sialyltransferase, an enzyme up-regulated in numerous cancers, sialylates TNFR1 and thereby protects tumor cells from TNF-induced apoptosis. Using pancreatic and ovarian cancer cells with ST6Gal-I knockdown or overexpression, we determined that α2-6 sialylation of TNFR1 had no effect on early TNF-induced signaling events, including the rapid activation of NF-κB, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt (occurring within 15 min). However, upon extended TNF treatment (6-24 h), cells with high ST6Gal-I levels exhibited resistance to TNF-induced apoptosis, as indicated by morphological evidence of cell death and decreased activation of caspases 8 and 3. Correspondingly, at these later time points, high ST6Gal-I expressers displayed sustained activation of the survival molecules Akt and NF-κB. Additionally, extended TNF treatment resulted in the selective enrichment of clonal variants with high ST6Gal-I expression, further substantiating a role for ST6Gal-I in cell survival. Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization. The α2-6 sialylation of TNFR1 was found to inhibit TNF-induced TNFR1 internalization. Thus, by restraining TNFR1 at the cell surface via sialylation, ST6Gal-I acts as a functional switch to divert signaling toward survival. These collective findings point to a novel glycosylation-dependent mechanism that regulates the cellular response to TNF and may promote cancer cell survival within TNF-rich tumor microenvironments.
Highlights
Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by Tumor necrosis factor (TNF) induces either cell survival or cell death
Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization
The level of ␣2-6 sialylation on TNFR1 was subsequently examined. ␣2-6 –sialylated proteins were precipitated by SNA–agarose beads, and precipitates were immunoblotted for TNFR1
Summary
The addition of ␣2-6 sialic acid to TNFR1 was shown to block TNF-induced apoptosis in human monocytic cells as well as monocytes from ST6Gal-I– overexpressing mice [13] In this prior investigation, neither the mechanism by which sialylation regulates TNFR1 nor the specific signaling cascades downstream of TNFR1 sialylation were examined, limiting a fundamental understanding of the role of glycosylation in regulating TNFR1 function. Using ovarian and pancreatic cancer cell models with ST6Gal-I overexpression or knockdown, we find that ST6Gal-I–mediated TNFR1 sialylation blocks the apoptotic arm of TNFR1 signaling while leaving NF-B- and Akt-mediated survival signaling intact. These findings highlight the importance of receptor glycosylation in the regulation of a key cell survival pathway critical to numerous physiologic and pathophysiologic processes
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