Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Inhibition of proteasome deubiquitinating activity as a new cancer therapy

Abstract

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles1,2. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target3. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle–associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: b-AP15 inhibits the UPS.
Figure 2: b-AP15 inhibits deubiquitination by the 19S regulatory particle (RP).
Figure 3: b-AP15 inhibits the 19S regulatory particle (RP) deubiquitinases UCHL5 and USP14.
Figure 4: b-AP15 inhibits tumor growth in vivo.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Rechsteiner, M., Hoffman, L. & Dubiel, W. The multicatalytic and 26 S proteases. J. Biol. Chem. 268, 6065–6068 (1993).

    CAS  PubMed  Google Scholar 

  2. Chu-Ping, M., Vu, J.H., Proske, R.J., Slaughter, C.A. & DeMartino, G.N. Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20 S proteasome. J. Biol. Chem. 269, 3539–3547 (1994).

    CAS  PubMed  Google Scholar 

  3. Adams, J. & Kauffman, M. Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest. 22, 304–311 (2004).

    Article  CAS  Google Scholar 

  4. Berndtsson, M. et al. Induction of the lysosomal apoptosis pathway by inhibitors of the ubiquitin-proteasome system. Int. J. Cancer 124, 1463–1469 (2009).

    Article  CAS  Google Scholar 

  5. Erdal, H. et al. Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis. Proc. Natl. Acad. Sci. USA 102, 192–197 (2005).

    Article  CAS  Google Scholar 

  6. Lamb, J. et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006).

    Article  CAS  Google Scholar 

  7. Adams, J. et al. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg. Med. Chem. Lett. 8, 333–338 (1998).

    Article  CAS  Google Scholar 

  8. Shibata, T. et al. An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome. Biochemistry 42, 13960–13968 (2003).

    Article  CAS  Google Scholar 

  9. Yang, H., Chen, D., Cui, Q.C., Yuan, X. & Dou, Q.P. Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res. 66, 4758–4765 (2006).

    Article  CAS  Google Scholar 

  10. Yang, H., Shi, G. & Dou, Q.P. The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from “Indian winter cherry”. Mol. Pharmacol. 71, 426–437 (2007).

    Article  CAS  Google Scholar 

  11. Menéndez-Benito, V., Verhoef, L.G., Masucci, M.G. & Dantuma, N.P. Endoplasmic reticulum stress compromises the ubiquitin-proteasome system. Hum. Mol. Genet. 14, 2787–2799 (2005).

    Article  Google Scholar 

  12. Mimnaugh, E.G., Chen, H.Y., Davie, J.R., Celis, J.E. & Neckers, L. Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response. Biochemistry 36, 14418–14429 (1997).

    Article  CAS  Google Scholar 

  13. Sheaff, R.J. et al. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol. Cell 5, 403–410 (2000).

    Article  CAS  Google Scholar 

  14. Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682–685 (1995).

    Article  CAS  Google Scholar 

  15. Maki, C.G., Huibregtse, J.M. & Howley, P.M. In vivo ubiquitination and proteasome-mediated degradation of p53(1). Cancer Res. 56, 2649–2654 (1996).

    CAS  PubMed  Google Scholar 

  16. Rosenberg-Hasson, Y., Bercovich, Z., Ciechanover, A. & Kahana, C. Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent. Eur. J. Biochem. 185, 469–474 (1989).

    Article  CAS  Google Scholar 

  17. Shieh, S.Y., Ikeda, M., Taya, Y. & Prives, C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334 (1997).

    Article  CAS  Google Scholar 

  18. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).

    Article  CAS  Google Scholar 

  19. Ling, X., Calinski, D., Chanan-Khan, A.A., Zhou, M. & Li, F. Cancer cell sensitivity to bortezomib is associated with survivin expression and p53 status but not cancer cell types. J. Exp. Clin. Cancer Res. 29, 8 (2010).

    Article  Google Scholar 

  20. Paoluzzi, L. et al. The BH3-only mimetic ABT-737 synergizes the antineoplastic activity of proteasome inhibitors in lymphoid malignancies. Blood 112, 2906–2916 (2008).

    Article  CAS  Google Scholar 

  21. Mullally, J.E. & Fitzpatrick, F.A. Pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p53-independent cell death. Mol. Pharmacol. 62, 351–358 (2002).

    Article  CAS  Google Scholar 

  22. Guterman, A. & Glickman, M.H. Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome. J. Biol. Chem. 279, 1729–1738 (2004).

    Article  CAS  Google Scholar 

  23. Borodovsky, A. et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001).

    Article  CAS  Google Scholar 

  24. Lam, Y.A., DeMartino, G.N., Pickart, C.M. & Cohen, R.E. Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26 S proteasomes. J. Biol. Chem. 272, 28438–28446 (1997).

    Article  CAS  Google Scholar 

  25. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002).

    Article  CAS  Google Scholar 

  26. Yao, T. & Cohen, R.E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002).

    Article  CAS  Google Scholar 

  27. Kramer, G. et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res. 64, 1751–1756 (2004).

    Article  CAS  Google Scholar 

  28. Olofsson, M.H. et al. Specific demonstration of drug-induced tumour cell apoptosis in human xenografts models using a plasma biomarker. Cancer Biomark. 5, 117–125 (2009).

    Article  CAS  Google Scholar 

  29. Reyes-Turcu, F.E., Ventii, K.H. & Wilkinson, K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

    Article  CAS  Google Scholar 

  30. Lee, B.H. et al. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 (2010).

    Article  CAS  Google Scholar 

  31. Koulich, E., Li, X. & DeMartino, G.N. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol. Biol. Cell 19, 1072–1082 (2008).

    Article  CAS  Google Scholar 

  32. Dimmock, J.R. et al. A conformational and structure-activity relationship study of cytotoxic 3,5-bis(arylidene)-4-piperidones and related N-acryloyl analogues. J. Med. Chem. 44, 586–593 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Glickman (Department of Biology, Technion-Israel Institute of Technology) for providing the Ub-GFP constructs, N. Dantuma (Department of Cell and Molecular Biology, Karolinska Institutet) for providing the MelJuSo Ub-YFP reporter cell line, B. Vogelstein (Sidney Kimmel Comprehensive Cancer Center, John Hopkins University) for providing the HCT-116 cell lines with targeted disruptions, L. Perup Segerström for technical advice, Uppsala Array Platform and L. Gatti for drug formulation. We thank Cancerfonden, Radiumhemmets forskningsfonder, Vetenskapsrådet, Strategiska forskningsstiftelsen, Vinnova, European Union CHEMORES, Frame Program 6 (LSHC-CT-2007-037665), Lions Cancerforskningsfond and Swedish Children Cancer Society for support.

Author information

Authors and Affiliations

Authors

Contributions

All authors were involved in designing experiments and interpreting data. P.D. and S.B. carried out most of the experiments and contributed equally to this work. P.D. and S.L. wrote the manuscript. M.H.O. performed the immunohistochemisty. M.F. and R.L. performed the CMAP analysis and in vitro cytotoxicity analysis. K.L. performed deubiquitinase labeling. M.D.C. and P.P. performed the in vivo study on colon carcinoma xenografts. B.S. and M.H. performed the in vivo study and staining on the AML model.

Corresponding author

Correspondence to Stig Linder.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 3893 kb)

Supplementary Data

CMAP data of b-AP15 treated cells (XLS 964 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

D'Arcy, P., Brnjic, S., Olofsson, M. et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med 17, 1636–1640 (2011). https://doi.org/10.1038/nm.2536

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2536

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing