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.

  • Original Article
  • Published:

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia

Oncogene volume 36, pages 2589–2598 (2017)Cite this article

Subjects

Abstract

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1− cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Goyama S, Wunderlich M, Mulloy JC . Xenograft models for normal and malignant stem cells. Blood 2015; 125: 2630–2640.

    Article  CAS  PubMed  Google Scholar 

  2. Somervaille TC, Cleary ML . Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 2006; 10: 257–268.

    Article  CAS  PubMed  Google Scholar 

  3. Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 2006; 442: 818–822.

    Article  CAS  PubMed  Google Scholar 

  4. Ye M, Zhang H, Yang H, Koche R, Staber PB, Cusan M et al. Hematopoietic differentiation is required for initiation of acute myeloid leukemia. Cell Stem Cell 2015; 17: 611–623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jan M, Snyder TM, Corces-Zimmerman MR, Vyas P, Weissman IL, Quake SR et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci Transl Med 2012; 4: 149ra118.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Corces-Zimmerman MR, Hong WJ, Weissman IL, Medeiros BC, Majeti R . Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proc Natl Acad Sci USA 2014; 111: 2548–2553.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 2014; 506: 328–333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang Y, Krivtsov AV, Sinha AU, North TE, Goessling W, Feng Z et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science 2010; 327: 1650–1653.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Goyama S, Schibler J, Cunningham L, Zhang Y, Rao Y, Nishimoto N et al. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. J Clin Invest 2013; 123: 3876–3888.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhong JF, Zhao Y, Sutton S, Su A, Zhan Y, Zhu L et al. Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells. Proc Natl Acad Sci USA 2005; 102: 2448–2453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Aronovich A, Nur Y, Shezen E, Rosen C, Zlotnikov Klionsky Y, Milman I et al. A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure. Blood 2013; 122: 2562–2571.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Baumer N, Krause A, Kohler G, Lettermann S, Evers G, Hascher A et al. Proteinase-activated receptor 1 (PAR1) regulates leukemic stem cell functions. PLoS One 2014; 9: e94993.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Song SJ, Pagel CN, Campbell TM, Pike RN, Mackie EJ . The role of protease-activated receptor-1 in bone healing. Am J Pathol 2005; 166: 857–868.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pagel CN, Song SJ, Loh LH, Tudor EM, Murray-Rust TA, Pike RN et al. Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids. Bone 2009; 44: 813–821.

    Article  CAS  PubMed  Google Scholar 

  15. Yue R, Li H, Liu H, Li Y, Wei B, Gao G et al. Thrombin receptor regulates hematopoiesis and endothelial-to-hematopoietic transition. Dev Cell 2012; 22: 1092–1100.

    Article  CAS  PubMed  Google Scholar 

  16. Degen JL, Palumbo JS . Hemostatic factors, innate immunity and malignancy. Thromb Res 2012; 129 (Suppl 1): S1–S5.

    Article  CAS  PubMed  Google Scholar 

  17. Veiga Cde S, Carneiro-Lobo TC, Coelho CJ, Carvalho SM, Maia RC, Vasconcelos FC et al. Increased expression of protease-activated receptor 1 (PAR-1) in human leukemias. Blood Cells Mol Dis 2011; 46: 230–234.

    Article  PubMed  Google Scholar 

  18. Bower M, Parry P, Carter M, Lillington DM, Amess J, Lister TA et al. Prevalence and clinical correlations of MLL gene rearrangements in AML-M4/5. Blood 1994; 84: 3776–3780.

    CAS  PubMed  Google Scholar 

  19. Cohen SG, Itkin T, Chakrabarty S, Graf C, Kollet O, Ludin A et al. PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells. Nat Med 2015; 21: 1307–1317.

    Article  CAS  Google Scholar 

  20. Tanaka Y, Joshi A, Wilson NK, Kinston S, Nishikawa S, Gottgens B . The transcriptional programme controlled by Runx1 during early embryonic blood development. Dev Biol 2012; 366: 404–419.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mendler JH, Maharry K, Radmacher MD, Mrozek K, Becker H, Metzeler KH et al. RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and MicroRNA expression signatures. J Clin Oncol 2012; 30: 3109–3118.

    PubMed  PubMed Central  Google Scholar 

  22. Wei J, Wunderlich M, Fox C, Alvarez S, Cigudosa JC, Wilhelm JS et al. Microenvironment determines lineage fate in a human model of MLL-AF9 leukemia. Cancer Cell 2008; 13: 483–495.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Martin CB, Mahon GM, Klinger MB, Kay RJ, Symons M, Der CJ et al. The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways. Oncogene 2001; 20: 1953–1963.

    Article  CAS  PubMed  Google Scholar 

  24. Nierodzik ML, Plotkin A, Kajumo F, Karpatkin S . Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991; 87: 229–236.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Heider I, Schulze B, Oswald E, Henklein P, Scheele J, Kaufmann R . PAR1-type thrombin receptor stimulates migration and matrix adhesion of human colon carcinoma cells by a PKCepsilon-dependent mechanism. Oncol Res 2004; 14: 475–482.

    Article  CAS  PubMed  Google Scholar 

  26. Kanemaru M, Maehara N, Iwamura T, Chijiiwa K . Thrombin stimulates integrin beta1-dependent adhesion of human pancreatic cancer cells to vitronectin through protease-activated receptor (PAR)-1. Hepatogastroenterology 2012; 59: 1614–1620.

    CAS  PubMed  Google Scholar 

  27. Tellez C, Bar-Eli M . Role and regulation of the thrombin receptor (PAR-1) in human melanoma. Oncogene 2003; 22: 3130–3137.

    Article  CAS  PubMed  Google Scholar 

  28. Even-Ram S, Uziely B, Cohen P, Grisaru-Granovsky S, Maoz M, Ginzburg Y et al. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med 1998; 4: 909–914.

    Article  CAS  PubMed  Google Scholar 

  29. Yang E, Cisowski J, Nguyen N, O'Callaghan K, Xu J, Agarwal A et al. Dysregulated protease activated receptor 1 (PAR1) promotes metastatic phenotype in breast cancer through HMGA2. Oncogene 2016; 35: 1529–1540.

    Article  CAS  PubMed  Google Scholar 

  30. Auvergne R, Wu C, Connell A, Au S, Cornwell A, Osipovitch M et al. PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo. Oncogene 2016; 35: 3817–3828.

    Article  CAS  PubMed  Google Scholar 

  31. Austin KM, Covic L, Kuliopulos A . Matrix metalloproteases and PAR1 activation. Blood 2013; 121: 431–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nakahara F, Kitaura J, Uchida T, Nishida C, Togami K, Inoue D et al. Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells. Blood 2014; 123: 3932–3942.

    Article  CAS  PubMed  Google Scholar 

  33. Huang YQ, Li JJ, Karpatkin S . Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway. J Biol Chem 2000; 275: 6462–6468.

    Article  CAS  PubMed  Google Scholar 

  34. Ishii K, Hein L, Kobilka B, Coughlin SR . Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling. J Biol Chem 1993; 268: 9780–9786.

    CAS  PubMed  Google Scholar 

  35. Morita S, Kojima T, Kitamura T . Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Therapy 2000; 7: 1063–1066.

    Article  CAS  PubMed  Google Scholar 

  36. Wunderlich M, Krejci O, Wei J, Mulloy JC . Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability. Blood 2006; 108: 1690–1697.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chou FS, Griesinger A, Wunderlich M, Lin S, Link KA, Shrestha M et al. The THPO/MPL/Bcl-xL pathway is essential for survival and self-renewal in human pre-leukemia induced by AML1-ETO. Blood 2012; 120: 709–719.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Moore MA, Nimer SD . The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells. Blood 2002; 99: 15–23.

    Article  CAS  PubMed  Google Scholar 

  39. Mulloy JC, Cammenga J, Berguido FJ, Wu K, Zhou P, Comenzo RL et al. Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element. Blood 2003; 102: 4369–4376.

    Article  CAS  PubMed  Google Scholar 

  40. Goyama S, Schibler J, Gasilina A, Shrestha M, Lin S, Link KA et al. UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO. Leukemia 2016; 30: 728–739.

    Article  CAS  PubMed  Google Scholar 

  41. Wunderlich M, Mulloy JC . Model systems for examining effects of leukemia-associated oncogenes in primary human CD34+ cells via retroviral transduction. Methods Mol Biol 2009; 538: 263–285.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mulloy JC, Wunderlich M, Zheng Y, Wei J . Transforming human blood stem and progenitor cells: a new way forward in leukemia modeling. Cell Cycle 2008; 7: 3314–3319.

    Article  CAS  PubMed  Google Scholar 

  43. Darrow AL, Fung-Leung WP, Ye RD, Santulli RJ, Cheung WM, Derian CK et al. Biological consequences of thrombin receptor deficiency in mice. Thromb Haemost 1996; 76: 860–866.

    Article  CAS  PubMed  Google Scholar 

  44. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013; 6: pl1.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012; 2: 401–404.

    Article  PubMed  Google Scholar 

  46. Hu Y, Smyth GK . ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 2009; 347: 70–78.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr J Trejo and Dr M Onodera for plasmids. We also thank the Flow Cytometry Core and the Mouse Core at Cincinnati Children's Hospital Medical Center for their help. This work was supported by an Institutional Clinical and Translational Science Award, NIH/NCRR Grant Number 1UL1RR026314-01, Translational Trials Development and Support Laboratory award (USPHS Grant Number MO1 RR 08084), CA176907 (JCM) and a Center of Excellence in Molecular Hematology P30 award (DK090971). JCM is a Leukemia and Lymphoma Society Scholar.

Author contributions

SG conceived the project, designed and performed the research, analyzed the data and wrote the paper. MS, JS, LR, WM, EO assisted with experiments. BM and TK participated in analyzing the data, JSP and JCM conceived the project, secured funding, designed the research, analyzed the data and wrote the paper.

Author information

Authors and Affiliations

  1. Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, OH, USA

    S Goyama, M Shrestha, J Schibler, L Rosenfeldt, W Miller, E O’Brien, B Mizukawa, J S Palumbo & J C Mulloy

  2. Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    S Goyama & T Kitamura

Authors
  1. S Goyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Shrestha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Schibler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L Rosenfeldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. W Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. E O’Brien
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. B Mizukawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T Kitamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J S Palumbo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J C Mulloy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to J S Palumbo or J C Mulloy.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Goyama, S., Shrestha, M., Schibler, J. et al. Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia. Oncogene 36, 2589–2598 (2017). https://doi.org/10.1038/onc.2016.416

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2016.416

This article is cited by

Search

Advanced search

Quick links