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.

  • Article
  • Published:

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Nature Chemical Biology volume 7, pages 818–826 (2011)Cite this article

Subjects

Abstract

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: PU-H71 interacts with a restricted fraction of Hsp90 that is more abundant in cancer cells.
Figure 2: PU-H71 is selective for and isolates Hsp90 in complex with oncoproteins and cochaperones.
Figure 3: PU-H71 identifies the aberrant signalosome in CML cells.
Figure 4: PU-H71-identified proteins and networks are those important for the malignant phenotype.
Figure 5: Hsp90 facilitates an enhanced STAT5 activity in CML.

Similar content being viewed by others

References

  1. Ley, T.J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008).

    Article  CAS  Google Scholar 

  2. Parsons, D.W. et al. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807–1812 (2008).

    Article  CAS  Google Scholar 

  3. Hanash, S. & Taguchi, A. The grand challenge to decipher the cancer proteome. Nat. Rev. Cancer 10, 652–660 (2010).

    Article  CAS  Google Scholar 

  4. Kolch, W. & Pitt, A. Functional proteomics to dissect tyrosine kinase signalling pathways in cancer. Nat. Rev. Cancer 10, 618–629 (2010).

    Article  CAS  Google Scholar 

  5. Nomura, D.K., Dix, M.M. & Cravatt, B.F. Activity-based protein profiling for biochemical pathway discovery in cancer. Nat. Rev. Cancer 10, 630–638 (2010).

    Article  CAS  Google Scholar 

  6. Brehme, M. et al. Charting the molecular network of the drug target Bcr-Abl. Proc. Natl. Acad. Sci. USA 106, 7414–7419 (2009).

    Article  CAS  Google Scholar 

  7. Ashman, K. & Villar, E.L. Phosphoproteomics and cancer research. Clin. Transl. Oncol. 11, 356–362 (2009).

    Article  CAS  Google Scholar 

  8. Zuehlke, A. & Johnson, J.L. Hsp90 and co-chaperones twist the functions of diverse client proteins. Biopolymers 93, 211–217 (2010).

    Article  CAS  Google Scholar 

  9. Workman, P., Burrows, F., Neckers, L. & Rosen, N. Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann. NY Acad. Sci. 1113, 202–216 (2007).

    Article  CAS  Google Scholar 

  10. Kamal, A. et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 425, 407–410 (2003).

    Article  CAS  Google Scholar 

  11. Janin, Y.L. ATPase inhibitors of heat-shock protein 90, second season. Drug Discov. Today 15, 342–353 (2010).

    Article  CAS  Google Scholar 

  12. Tsaytler, P.A., Krijgsveld, J., Goerdayal, S.S., Rudiger, S. & Egmond, M.R. Novel Hsp90 partners discovered using complementary proteomic approaches. Cell Stress Chaperones 14, 629–638 (2009).

    Article  CAS  Google Scholar 

  13. da Rocha Dias, S. et al. Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 65, 10686–10691 (2005).

    Article  CAS  Google Scholar 

  14. Grbovic, O.M. et al. V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc. Natl. Acad. Sci. USA 103, 57–62 (2006).

    Article  CAS  Google Scholar 

  15. Taldone, T. & Chiosis, G. Purine-scaffold hsp90 inhibitors. Curr. Top. Med. Chem. 9, 1436–1446 (2009).

    Article  CAS  Google Scholar 

  16. Dezwaan, D.C. & Freeman, B.C. HSP90: the Rosetta stone for cellular protein dynamics? Cell Cycle 7, 1006–1012 (2008).

    Article  CAS  Google Scholar 

  17. Ren, R. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat. Rev. Cancer 5, 172–183 (2005).

    Article  CAS  Google Scholar 

  18. Burke, B.A. & Carroll, M. BCR-ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia. Leukemia 24, 1105–1112 (2010).

    Article  CAS  Google Scholar 

  19. McClellan, A.J. et al. Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell 131, 121–135 (2007).

    Article  CAS  Google Scholar 

  20. Carayol, N. et al. Critical roles for mTORC2- and rapamycin-insensitive mTORC1-complexes in growth and survival of BCR-ABL-expressing leukemic cells. Proc. Natl. Acad. Sci. USA 107, 12469–12474 (2010).

    Article  CAS  Google Scholar 

  21. Nobukuni, T., Kozma, S.C. & Thomas, G. hvps34, an ancient player, enters a growing game: mTOR Complex1/S6K1 signaling. Curr. Opin. Cell Biol. 19, 135–141 (2007).

    Article  CAS  Google Scholar 

  22. McCubrey, J.A. et al. Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy. Leukemia 22, 708–722 (2008).

    Article  CAS  Google Scholar 

  23. Häcker, H. & Karin, M. Regulation and function of IKK and IKK-related kinases. Sci. STKE 2006, re13 (2006).

    Article  Google Scholar 

  24. Mihailovic, T. et al. Protein kinase D2 mediates activation of nuclear factor kappaB by Bcr-Abl in Bcr-Abl+ human myeloid leukemia cells. Cancer Res. 64, 8939–8944 (2004).

    Article  CAS  Google Scholar 

  25. Hendriks, R.W. & Kersseboom, R. Involvement of SLP-65 and Btk in tumor suppression and malignant transformation of pre-B cells. Semin. Immunol. 18, 67–76 (2006).

    Article  CAS  Google Scholar 

  26. Mahajan, S. et al. Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells. J. Biol. Chem. 276, 31216–31228 (2001).

    Article  CAS  Google Scholar 

  27. Oda, A., Wakao, H. & Fujita, H. Calpain is a signal transducer and activator of transcription (STAT) 3 and STAT5 protease. Blood 99, 1850–1852 (2002).

    Article  CAS  Google Scholar 

  28. Si, J. & Collins, S.J. Activated Ca2+/calmodulin-dependent protein kinase IIgamma is a critical regulator of myeloid leukemia cell proliferation. Cancer Res. 68, 3733–3742 (2008).

    Article  CAS  Google Scholar 

  29. Salgia, R. et al. Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL. Oncogene 11, 1149–1155 (1995).

    CAS  PubMed  Google Scholar 

  30. Le, Y. et al. FAK silencing inhibits leukemogenesis in BCR/ABL-transformed hematopoietic cells. Am. J. Hematol. 84, 273–278 (2009).

    Article  CAS  Google Scholar 

  31. Sawyers, C.L. The role of myc in transformation by Bcr-Abl. Leuk. Lymphoma 11, 45–46 (1993).

    Article  Google Scholar 

  32. Naka, K. et al. TGF-beta-FOXO signaling maintains leukemia-initiating cells in chronic myeloid leukemia. Nature 463, 676–680 (2010).

    Article  CAS  Google Scholar 

  33. Bedford, M.T. & Clarke, S.G. Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1–13 (2009).

    Article  CAS  Google Scholar 

  34. Maloney, A. et al. Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 67, 3239–3253 (2007).

    Article  CAS  Google Scholar 

  35. Jaganathan, S., Yue, P. & Turkson, J. Enhanced sensitivity of pancreatic cancer cells to concurrent inhibition of aberrant signal transducer and activator of transcription 3 and epidermal growth factor receptor or Src. J. Pharmacol. Exp. Ther. 333, 373–381 (2010).

    Article  CAS  Google Scholar 

  36. Apsel, B. et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat. Chem. Biol. 4, 691–699 (2008).

    Article  CAS  Google Scholar 

  37. Deininger, M.W. & Druker, B.J. Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol. Rev. 55, 401–423 (2003).

    Article  CAS  Google Scholar 

  38. Katzav, S. Flesh and blood: the story of Vav1, a gene that signals in hematopoietic cells but can be transforming in human malignancies. Cancer Lett. 255, 241–254 (2007).

    Article  CAS  Google Scholar 

  39. Pratt, W.B., Morishima, Y. & Osawa, Y. The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts. J. Biol. Chem. 283, 22885–22889 (2008).

    Article  CAS  Google Scholar 

  40. de Groot, R.P., Raaijmakers, J.A., Lammers, J.W., Jove, R. & Koenderman, L. STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells. Blood 94, 1108–1112 (1999).

    CAS  PubMed  Google Scholar 

  41. An, W.G., Schulte, T.W. & Neckers, L.M. The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src proteins before their degradation by the proteasome. Cell Growth Differ. 11, 355–360 (2000).

    CAS  PubMed  Google Scholar 

  42. Klejman, A. et al. The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells. EMBO J. 21, 5766–5774 (2002).

    Article  CAS  Google Scholar 

  43. Xu, D. & Qu, C.K. Protein tyrosine phosphatases in the JAK/STAT pathway. Front. Biosci. 13, 4925–4932 (2008).

    Article  CAS  Google Scholar 

  44. Lim, C.P. & Cao, X. Structure, function and regulation of STAT proteins. Mol. Biosyst. 2, 536–550 (2006).

    Article  CAS  Google Scholar 

  45. Paukku, K. & Silvennoinen, O. STATs as critical mediators of signal transduction and transcription: lessons learned from STAT5. Cytokine Growth Factor Rev. 15, 435–455 (2004).

    Article  CAS  Google Scholar 

  46. Rix, U. & Superti-Furga, G. Target profiling of small molecules by chemical proteomics. Nat. Chem. Biol. 5, 616–624 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Geoffrey Beene Cancer Research Center of the Memorial Sloan-Kettering Cancer Center (G.C.), Leukemia and Lymphoma Society (G.C., M.L.G., S.D.N., X.Z. and R.L.), Breast Cancer Research Fund (G.C.), the SPORE Pilot Award and Research & Therapeutics Program in Prostate Cancer (G.C.), the Hirshberg Foundation for Pancreatic Cancer (G.C.), the Byrne Fund (G.C.), 1U01 AG032969-01A1 (G.C.), 1R01 CA155226-01 (G.C. and A.M.) and US National Cancer Institute (NCI) Cancer Center Support Grant P30 CA08748 (H.E.B.). K.B. and L.N. were supported by funds from the Intramural Program of the NCI. S.M.L. and P.M.S.-J. are supported by the Ludwig Center for Cancer Immunotherapy at MSKCC and by NCI Grant P50-CA86483. M.L.G. is funded by the US National Institutes of Health (NIH) through the NIH Director's New Innovator Award Program, 1 DP2 OD007399-01 and the V foundation. F.P. is funded by the American Italian Cancer Foundation. We thank D. Toft (Mayo Clinic) and M. Cox (University of Texas) for the gifts of H9010 Hsp90-specific antibodies, L.A. Fabrizio, A.M. Morrishow, H. Deng and J. Fernandez for help with MS analysis, A. Perl, C.T. Jordan, M. Becker and J. Nicoll for providing the primary CML samples or suggestions on their use, and B. Clarkson, J. Bromberg and P. Gregor for suggestions with the manuscript.

Author information

Author notes

  1. Kamalika Moulick, James H Ahn and Hongliang Zong: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, New York, New York, USA

    Kamalika Moulick, James H Ahn, Anna Rodina, Erica M Gomes DaGama, Eloisi Caldas-Lopes, Fabiana Perna, Ly P Vu, Xinyang Zhao, Danuta Zatorska, Tony Taldone, Mary Alpaugh, Stephen D Nimer & Gabriela Chiosis

  2. Division of Hematology and Oncology, Weill Cornell Medical College, New York, New York, USA

    Hongliang Zong, Leandro Cerchietti, Katerina Hatzi, Monica L Guzman & Ari Melnick

  3. Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

    Kristin Beebe & Len Neckers

  4. Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Peter Smith-Jones, Nagavarakishore Pillarsetty, Thomas Ku, Jason S Lewis & Steven M Larson

  5. Department of Pharmacology, Weill Medical College of Cornell University, New York, New York, USA

    Steven S Gross

  6. Department of Medicine, Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Ross Levine

  7. Microchemistry and Proteomics Core, Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Hediye Erdjument-Bromage

Authors
  1. Kamalika Moulick
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James H Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hongliang Zong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Rodina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Leandro Cerchietti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Erica M Gomes DaGama
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eloisi Caldas-Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kristin Beebe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fabiana Perna
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Katerina Hatzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ly P Vu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Xinyang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Danuta Zatorska
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Tony Taldone
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Peter Smith-Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Mary Alpaugh
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Steven S Gross
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Nagavarakishore Pillarsetty
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Thomas Ku
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Jason S Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Steven M Larson
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Ross Levine
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Hediye Erdjument-Bromage
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Monica L Guzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Stephen D Nimer
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Ari Melnick
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Len Neckers
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Gabriela Chiosis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.M., J.H.A., H.Z., L.C., A.R., K.B., P.S.-J., F.P., K.H., L.P.V., X.Z., H.E.-B., N.P. and T.K. performed experiments and D.Z., T.T., A.R., R.L., S.M.L., M.L.G. and S.S.G. provided reagents. All authors participated in the design and analysis of various experiments and G.C., A.M., S.D.N., M.L.G. and L.N. wrote the paper.

Corresponding authors

Correspondence to Stephen D Nimer, Ari Melnick, Len Neckers or Gabriela Chiosis.

Ethics declarations

Competing interests

Memorial Sloan-Kettering Cancer Center holds the intellectual rights to PU-H71. Samus Therapeutics, of which G.C. has partial ownership, has licensed PU-H71.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 3530 kb)

Supplementary Data Set 1

Dataset 1 (XLS 312 kb)

Supplementary Data Set Legend

Dataset 1 Legend (PDF 51 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Moulick, K., Ahn, J., Zong, H. et al. Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90. Nat Chem Biol 7, 818–826 (2011). https://doi.org/10.1038/nchembio.670

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.670

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research