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A C-terminal HSP90 inhibitor restores glucocorticoid sensitivity and relieves a mouse allograft model of Cushing disease

Abstract

One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH)1. Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism2,3,4. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases5,6. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

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Figure 1: Human corticotroph adenomas overexpress heat shock proteins; HSP90 inhibitors reduce AtT-20 cell proliferation.
Figure 2: Silibinin promotes the release of mature GR in AtT-20 cells through direct binding to the C-terminal domain of Hsp90.
Figure 3: Silibinin enhances GR activity in AtT-20 cells.
Figure 4: Effects of silibinin on primary cultures of human corticotroph adenomas and on a mouse allograft model of Cushing disease.

References

  1. Keller-Wood, M.E. & Dallman, M.F. Corticosteroid inhibition of ACTH secretion. Endocr. Rev. 5, 1–24 (1984).

    CAS  Article  PubMed  Google Scholar 

  2. Dahia, P.L.M. & Grossman, A.B. The molecular pathogenesis of corticotroph tumors. Endocr. Rev. 20, 136–155 (1999).

    CAS  Article  PubMed  Google Scholar 

  3. Newell–Price, J., Bertagna, X., Grossman, A.B. & Nieman, L.K. Cushing's syndrome. Lancet 367, 1605–1617 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Tritos, N.A., Biller, B.M. & Swearingen, B. Management of Cushing disease. Nat. Rev. Endocrinol. 7, 279–289 (2011).

    CAS  Article  PubMed  Google Scholar 

  5. Karl, M. et al. Cushing's disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc. Assoc. Am. Physicians 108, 296–307 (1996).

    CAS  PubMed  Google Scholar 

  6. Lamberts, S.W.J. Glucocorticoid receptors and Cushing's disease. Mol. Cell. Endocrinol. 197, 69–72 (2002).

    CAS  Article  PubMed  Google Scholar 

  7. Pratt, W.B. & Toft, D.O. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr. Rev. 18, 306–360 (1997).

    CAS  PubMed  Google Scholar 

  8. Picard, D. et al. Reduced levels of Hsp90 compromise steroid-receptor action in vivo. Nature 348, 166–168 (1990).

    CAS  Article  PubMed  Google Scholar 

  9. Kang, K.I. et al. The molecular chaperone Hsp90 can negatively regulate the activity of a glucocorticosteroid-dependent promoter. Proc. Natl. Acad. Sci. USA 96, 1439–1444 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Freeman, B.C. & Yamamoto, K.R. Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296, 2232–2235 (2002).

    CAS  Article  PubMed  Google Scholar 

  11. Marcu, M.G., Chadli, A., Bouhouche, I., Catelli, M. & Neckers, L.M. The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J. Biol. Chem. 275, 37181–37186 (2000).

    CAS  Article  PubMed  Google Scholar 

  12. Zhao, H., Brandt, G.E., Galam, L., Matts, R.L. & Blagg, B.S.J. Identification and initial SAR of silybin: An Hsp90 inhibitor. Bioorg. Med. Chem. Lett. 21, 2659–2664 (2011).

    CAS  Article  PubMed  Google Scholar 

  13. Trepel, J., Mollapour, M., Giaccone, G. & Neckers, L. Targeting the dynamic Hsp90 complex in cancer. Nat. Rev. Cancer 10, 537–549 (2010).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  14. García-Morales, P. et al. Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines. Oncogene 26, 7185–7193 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Whitesell, L. & Cook, P. Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells. Mol. Endocrinol. 10, 705–712 (1996).

    CAS  PubMed  Google Scholar 

  16. Segnitz, B. & Gehring, U. The function of steroid hormone receptors is inhibited by the Hsp90-specific compound geldanamycin. J. Biol. Chem. 272, 18694–18701 (1997).

    CAS  Article  PubMed  Google Scholar 

  17. Matts, R.L. et al. Elucidation of the Hsp90 C-terminal inhibitor binding site. ACS Chem. Biol. 6, 800–807 (2011).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  18. Young, J.C., Hoogenraad, N.J. & Hartl, F.U. Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell 112, 41–50 (2003).

    CAS  Article  PubMed  Google Scholar 

  19. Marcu, M.G., Schulte, T.W. & Neckers, L. Novobiocin and related coumarins and depletion of heat shock protein 90–dependent signaling proteins. J. Natl. Cancer Inst. 92, 242–248 (2000).

    CAS  Article  PubMed  Google Scholar 

  20. Yun, B.G., Huang, W., Leach, N., Hartson, S.D. & Matts, R.L. Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90–cochaperone–client interactions. Biochemistry 43, 8217–8229 (2004).

    CAS  Article  PubMed  Google Scholar 

  21. Dittmar, K.D., Demady, D.R., Stancato, L.F., Krishna, P. & Pratt, W.B. Folding of the glucocorticoid receptor by the heat shock protein (Hsp) 90-based chaperone machinery: the role of p23 is to stabilize receptor–Hsp90 heterocomplexes formed by Hsp90-p60-Hsp70. J. Biol. Chem. 272, 21213–21220 (1997).

    CAS  Article  PubMed  Google Scholar 

  22. Kirschke, E., Goswami, D., Southworth, D., Griffin, P. & Agard, D. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697 (2014).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  23. Genest, O. et al. Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast. Mol. Cell 49, 464–473 (2013).

    CAS  Article  PubMed  Google Scholar 

  24. Lorenz, O.R. et al. Modulation of the Hsp90 chaperone cycle by a stringent client protein. Mol. Cell 53, 941–953 (2014).

    CAS  Article  PubMed  Google Scholar 

  25. Páez-Pereda, M. et al. Retinoic acid prevents experimental Cushing syndrome. J. Clin. Invest. 108, 1123–1131 (2001).

    PubMed Central  PubMed  Article  Google Scholar 

  26. Kaul, S. et al. Mutations at positions 547–553 of rat glucocorticoid receptors reveal that Hsp90 binding requires the presence, but not defined composition, of a seven–amino acid sequence at the amino terminus of the ligand binding domain. J. Biol. Chem. 277, 36223–36232 (2002).

    CAS  Article  PubMed  Google Scholar 

  27. Philips, A. et al. Antagonism between Nur77 and glucocorticoid receptor for control of transcription. Mol. Cell. Biol. 17, 5952–5959 (1997).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  28. Liu, B., Hammer, G.D., Rubinstein, M., Mortrud, M. & Low, M.J. Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice. Mol. Cell. Biol. 12, 3978–3990 (1992).

    CAS  PubMed Central  PubMed  Google Scholar 

  29. Leung, C.K., Paterson, J., Imai, Y. & Shiu, R. Transplantation of ACTH-secreting pituitary tumor cells in athymic nude mice. Virchows Arch. A Pathol. Anat. Histol. 396, 303–312 (1982).

    CAS  Article  PubMed  Google Scholar 

  30. Bilodeau, S. et al. Role of Brg1 and HDAC2 in GR trans–repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev. 20, 2871–2886 (2006).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  31. Kino, T., De Martino, M.U., Charmandari, E., Mirani, M. & Chrousos, G.P. Tissue glucocorticoid resistance/hypersensitivity syndromes. J. Steroid Biochem. Mol. Biol. 85, 457–467 (2003).

    CAS  Article  PubMed  Google Scholar 

  32. Donnelly, A. & Blagg, B.S.J. Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket. Curr. Med. Chem. 15, 2702–2717 (2008).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  33. Fang, L., Ricketson, D., Getubig, L. & Darimont, B. Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain. Proc. Natl. Acad. Sci. USA 103, 18487–18492 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Vaughan, C.K. et al. Structure of an Hsp90–Cdc37–Cdk4 Complex. Mol. Cell 23, 697–707 (2006).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  35. Röhl, A., Rohrberg, J. & Buchner, J. The chaperone Hsp90: changing partners for demanding clients. Trends Biochem. Sci. 38, 253–262 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Kanelakis, K.C., Shewach, D.S. & Pratt, W.B. Nucleotide binding states of Hsp70 and Hsp90 during sequential steps in the process of glucocorticoid receptor–Hsp90 heterocomplex assembly. J. Biol. Chem. 277, 33698–33703 (2002).

    CAS  Article  PubMed  Google Scholar 

  37. Allan, R.K., Mok, D., Ward, B.K. & Ratajczak, T. Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization. J. Biol. Chem. 281, 7161–7171 (2006).

    CAS  Article  PubMed  Google Scholar 

  38. Söti, C., Rácz, A. & Csermely, P. A nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90: N-terminal nucleotide binding unmasks a C-terminal binding pocket. J. Biol. Chem. 277, 7066–7075 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Ramasamy, K. & Agarwal, R. Multitargeted therapy of cancer by silymarin. Cancer Lett. 269, 352–362 (2008).

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  40. Saller, R., Meier, R. & Brignoli, R. The use of silymarin in the treatment of liver diseases. Drugs 61, 2035–2063 (2001).

    CAS  Article  PubMed  Google Scholar 

  41. Morra, G. et al. Dynamics-based discovery of allosteric inhibitors: selection of new ligands for the C-terminal domain of Hsp90. J. Chem. Theory Comput. 6, 2978–2989 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was partly supported by a grant from the German Research Foundation (SFB1035 to M.S.), the Bayerisches Staatsministerium für Wirtschaft, Infrastruktur, Verkehr und Technologie (m4 Award to F.H. and M.P.-P.), Federal Ministry of Education and Research (BMBF; PersoMed to M.P.-P. and F.H.); and postdoctoral fellowships to L.F. from the European Commission (FP7-PEOPLE-20112011-IIF 301193, Hsp90NMR) and the European Molecular Biology Organization (EMBO ALTF 1255-2011). The expression plasmids for the HSP90-α CTD and GR were gifts from U. Hartl (Max Planck Institute of Biochemistry) and S. Simons Jr. (US National Institutes of Health), respectively. We thank J. Stalla and T. Kloss for technical assistance. We thank M. Theodoropoulou and K. Lucia for valuable comments on the manuscript.

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M.R. and M.P.-P. conceived of and designed the experiments. M.B. collected biopsies and diagnosed subjects. M.R., C.K., L.F., and M.P.-P. carried out experiments. M.R., M.S., F.H., G.K.S., and M.P.-P. analyzed the data. M.R., F.H., G.K.S., and M.P.-P. wrote the manuscript.

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Correspondence to Marcelo Paez-Pereda.

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Riebold, M., Kozany, C., Freiburger, L. et al. A C-terminal HSP90 inhibitor restores glucocorticoid sensitivity and relieves a mouse allograft model of Cushing disease. Nat Med 21, 276–280 (2015). https://doi.org/10.1038/nm.3776

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