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

Nature Medicine volume 13, pages 10701077 (2007) | Download Citation

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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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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.

Author information

Author notes

    • Klaus W Wagner

    Present address: Indiana University School of Medicine, 1001 West 10th Street, Indianapolis, Indiana 46202, USA.

    • Klaus W Wagner
    •  & Elizabeth A Punnoose

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Diagnostics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Klaus W Wagner
    • , Elizabeth A Punnoose
    • , Thomas Januario
    • , Dori Lee
    • , Melissa von Goetz
    • , Ling Huw
    •  & Lukas Amler
  2. Department of Molecular Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Elizabeth A Punnoose
    • , David A Lawrence
    • , Robert M Pitti
    •  & Avi Ashkenazi
  3. Department of Analytical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Kate Lancaster
    •  & Viswanatham Katta
  4. Department of Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Sharon Fong Yee
    •  & Klara Totpal
  5. Department of Bioinformatics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Guy Cavet
  6. Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    • Sarah G Hymowitz

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Contributions

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.

Competing interests

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

Corresponding author

Correspondence to Avi Ashkenazi.

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    Supplementary Figures 1–7, Supplementary Tables 1–2, Supplementary Methods

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DOI

https://doi.org/10.1038/nm1627

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