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

Nature Medicine volume 13, pages1070–1077(2007)Cite this article

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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References

  1. 1

    Danial, N.N. & Korsmeyer, S.J. Cell death: critical control points. Cell 116, 205–219 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Fesik, S.W. Promoting apoptosis as a strategy for cancer drug discovery. Nat. Rev. Cancer 5, 876–885 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Dalton, W.S. & Friend, S.H. Cancer biomarkers—an invitation to the table. Science 312, 1165–1168 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Ashkenazi, A. & Dixit, V.M. Death receptors: signaling and modulation. Science 281, 1305–1308 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Ashkenazi, A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat. Rev. Cancer 2, 420–430 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Kischkel, F.C. et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579–5588 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Kischkel, F.C. et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611–620 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Igney, F. & Krammer, P. Death and anti-death: tumour resistance to apoptosis. Nat. Rev. Cancer 2, 277–288 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Ashkenazi, A. et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155–162 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Kelley, S. & Ashkenazi, A. Targeting death receptors in cancer with Apo2L/TRAIL. Curr. Opin. Pharmacol. 4, 333–339 (2004).

    CAS  Article  Google Scholar 

  11. 11

    LeBlanc, H. et al. Tumor-cell resistance to death receptor–induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax. Nat. Med. 8, 274–281 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Hang, H. & Bertozzi, C. The chemistry and biology of mucin-type O-linked glycosylation. Bioorg. Med. Chem. 13, 5021–5034 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Hanisch, F. O-glycosylation of the mucin type. Biol. Chem. 382, 143–149 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Ohtsubo, K. & Marth, J.D. Glycosylation in cellular mechanisms of health and disease. Cell 126, 855–867 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Ten Hagen, K.G., Fritz, T.A. & Tabak, L.A. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13, 1R–16R (2003).

    CAS  Article  Google Scholar 

  16. 16

    Wang, H. et al. Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, pp-GalNAc-T14. Biochem. Biophys. Res. Commun. 300, 738–744 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Delannoy, P. et al. Benzyl-N-acetyl-α-D-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation. Glycoconj. J. 13, 717–726 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Kuan, S.F., Byrd, J.C., Basbaum, C. & Kim, Y.S. Inhibition of mucin glycosylation by aryl-N-acetyl-α-galactosaminides in human colon cancer cells. J. Biol. Chem. 264, 19271–19277 (1989).

    CAS  PubMed  Google Scholar 

  19. 19

    Wei, M. et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727–730 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Boldin, M.P. et al. Self-association of the “death domains” of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects. J. Biol. Chem. 270, 387–391 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Hansen, J.E. et al. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj. J. 15, 115–130 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Hymowitz, S. et al. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol. Cell 4, 563–571 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Chan, F.K. et al. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 288, 2351–2354 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Clancy, L. et al. Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis. Proc. Natl. Acad. Sci. USA 102, 18099–18104 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Feig, C., Tchikov, V., Schutze, S. & Peter, M.E. Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling. EMBO J. 26, 221–231 (2007).

    CAS  Article  Google Scholar 

  26. 26

    Xu, Z. & Weiss, A. Negative regulation of CD45 by differential homodimerization of the alternatively spliced isoforms. Nat. Immunol. 3, 764–771 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Haines, N. & Irvine, K.D. Glycosylation regulates Notch signalling. Nat. Rev. Mol. Cell Biol. 4, 786–797 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Priatel, J.J. et al. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. Immunity 12, 273–283 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Brockhausen, I. Pathways of O-glycan biosynthesis in cancer cells. Biochim. Biophys. Acta 1473, 67–95 (1999).

    CAS  Article  Google Scholar 

  30. 30

    Dube, D. & Bertozzi, C. Glycans in cancer and inflammation—potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4, 477–488 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Fuster, M., Brown, J., Wang, L. & Esko, J. A disaccharide precursor of sialyl Lewis X inhibits metastatic potential of tumor cells. Cancer Res. 63, 2775–2781 (2003).

    CAS  PubMed  Google Scholar 

  32. 32

    Fuster, M. & Esko, J. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat. Rev. Cancer 5, 526–542 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Ohyama, C., Tsuboi, S. & Fukuda, M. Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells. EMBO J. 18, 1516–1525 (1999).

    CAS  Article  Google Scholar 

  34. 34

    Takada, A. et al. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res. 53, 354–361 (1993).

    CAS  PubMed  Google Scholar 

  35. 35

    Hoffman, E., Awad, T. & Palma, J. Expression profiling—best practices for data generation and interpretation in clinical trials. Nat. Rev. Genet. 5, 229–237 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Yauch, R. et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin. Cancer Res. 11, 8686–8698 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Sharp, D.A., Lawrence, D.A. & Ashkenazi, A. Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis. J. Biol. Chem. 280, 19401–19409 (2005).

    CAS  Article  Google Scholar 

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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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Author notes

  1. Klaus W Wagner

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

  2. Klaus W Wagner and Elizabeth A Punnoose: These authors contributed equally to this work.

Affiliations

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

    Kate Lancaster & Viswanatham Katta

  4. Department of Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, 94080, California, USA

    Sharon Fong Yee & Klara Totpal

  5. Department of Bioinformatics, Genentech, Inc., 1 DNA Way, South San Francisco, 94080, California, USA

    Guy Cavet

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

Corresponding author

Correspondence to Avi Ashkenazi.

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Competing interests

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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