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Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Abstract

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non–small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine–galactose–sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.

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Figure 1: Specific O-glycosylation enzyme expression correlates with sensitivity to Apo2L/TRAIL.
Figure 2: Inhibition of O-glycosylation enzymes reduces sensitivity to Apo2L/TRAIL.
Figure 3: Overexpression of O-glycosylation enzymes promotes sensitivity to Apo2L/TRAIL.
Figure 4: Modulation of O-glycosylation enzymes affects caspase-8 activation.
Figure 5: Identification of potential O-glycosylation sites in the ectodomain of DR4 and DR5.
Figure 6: Inhibition of O-glycosylation impairs Apo2L/TRAIL-induced receptor clustering.

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Acknowledgements

We thank S. Marsters and M. Nagel for plasmids and purification of recombinant DR5 respectively; S. Ross and M. Go for execution of xenograft studies; and W. Forrest for statistical analysis.

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K.W.W., D.L. and M.V.G. performed the cell line characterization. K.W.W. and G.C. performed the microarray analysis. E.A.P., T.J., D.A.L., R.M.P. and K.T. performed the functional and mechanistic studies. E.A.P., L.H., K.L. and V.K. performed the glycosylation and mass spectrometric analyses. S.F.Y. conducted the in vivo experiments. S.G.H. carried out the structural modeling. K.W.W., E.A.P., L.A. and A.A. guided the project and contributed to the experimental design and to data interpretation. K.W.W., E.A.P. and A.A. wrote the manuscript.

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Correspondence to Avi Ashkenazi.

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All authors except K.W.W. and D.L. are presently employed by Genentech and may own Genentech shares.

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Supplementary Figures 1–7, Supplementary Tables 1–2, Supplementary Methods (PDF 3522 kb)

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Wagner, K., Punnoose, E., Januario, T. et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med 13, 1070–1077 (2007). https://doi.org/10.1038/nm1627

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