Inhibition of proteasome deubiquitinating activity as a new cancer therapy

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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.

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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.

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References

  1. 1

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

  2. 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).

  3. 3

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

  4. 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).

  5. 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).

  6. 6

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

  7. 7

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

  8. 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).

  9. 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).

  10. 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).

  11. 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).

  12. 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).

  13. 13

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

  14. 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).

  15. 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).

  16. 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).

  17. 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).

  18. 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).

  19. 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).

  20. 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).

  21. 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).

  22. 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).

  23. 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).

  24. 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).

  25. 25

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

  26. 26

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

  27. 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).

  28. 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).

  29. 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).

  30. 30

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

  31. 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).

  32. 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).

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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

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.

Correspondence to Stig Linder.

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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)

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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) doi:10.1038/nm.2536

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