Breast Cancer Research and Treatment

, Volume 164, Issue 1, pp 57–67 | Cite as

Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression

  • Monica E. Reyes
  • Takeo Fujii
  • Daniel Branstetter
  • Savitri Krishnamurthy
  • Hiroko Masuda
  • Xiaoping Wang
  • James M. Reuben
  • Wendy A. Woodward
  • Beatrice J. Edwards
  • Gabriel N. Hortobagyi
  • Debu Tripathy
  • William C. Dougall
  • Bedrich L. EckhardtEmail author
  • Naoto T. UenoEmail author
Preclinical study



As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC.


RANK mRNA and protein expression was evaluated in TNBC using genomic databases, cell lines and in a tissue microarray of curated primary tumor samples derived from 87 patients with TNBC. RANK expression was evaluated either by Mann–Whitney U test on log-normalized gene expression data or by Student’s t test on FACS data. Analysis of RANK and RANKL immunostaining was calculated by H-score, and correlations to clinical factors performed using χ 2 or Fisher’s exact test. Associations with RFS and OS were assessed using univariate and multivariate Cox proportional hazard models. Survival estimates were generated using the Kaplan–Meier method.


In three distinct datasets spanning 684 samples, RANK mRNA expression was higher in primary tumors derived from TNBC patients than from those with other molecular subtypes (P < 0.01). Cell surface-localized RANK protein was consistently higher in TNBC cell lines (P = 0.037). In clinical samples, TNBC patients that expressed both RANK and RANKL proteins had significantly worse RFS (P = 0.0032) and OS (P = 0.004) than patients with RANK-positive, RANKL-negative tumors. RANKL was an independent, poor prognostic factor for RFS (P = 0.04) and OS (P = 0.01) in multivariate analysis in samples that expressed both RANK and RANKL.


RANK and RANKL co-expression is associated with poor RFS and OS in patients with TNBC.


Triple-negative breast cancer RANK RANKL Relapse-free survival Overall survival 



Estrogen receptor


Fluorescence-activated cell sorting


Human epidermal growth factor receptor 2


Overall survival


Progesterone receptor


Receptor activator of nuclear factor kappa B


RANK ligand


Relapse-free survival


Triple-negative breast cancer


Tissue microarray



We thank Stephanie Deming for providing editorial support on behalf of Scientific Publications at MD Anderson Cancer Center, and Albert Rhee for providing medical writing support on behalf of Amgen, Inc.

Author’s contribution

Amgen, Inc. funded the study and supported the authors in the development of the study design, and in the collection, analysis, and interpretation of data. MER, TF, DB, HM, BLE, WCD, and NU contributed to the study conception and design; MER, TF, DB, BLE, SK, XW, JR, WW, GH, DT, WCD, and NU contributed to the collection, and analysis of data; MER, TF, DB, BLE, HM, BJE, SK, XW, JMR, WAW, GNH, DT, WCD, and NTU contributed to the interpretation of the data and development of the manuscript. Medical writing support was provided by Amgen, Inc. The decision to submit was taken jointly by all authors and Amgen, Inc. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Funding support for this study was provided by Amgen, Inc. Technical support for generation of flow cytometry data was provided by The University of Texas MD Anderson Cancer Center Flow Cytometry and Cellular Imaging Core Facility, funded by the National Cancer Institute Cancer Support Grant P30CA16672.

Compliance with ethical standards

Conflict of interest

Author NU is principal investigator of the above-mentioned study of denosumab (NCT01952054), which is supported by Amgen, Inc. Authors WCD and DB are former employees and shareholders of Amgen, Inc. Authors MER, TF, SK, HM, XW, JMR, WAW, BJE, GNH, DT, BLE declare no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

Informed consent

This study adhered to ethical guidelines at MD Anderson Cancer Center and the IRB study protocol (LAB05-0530).


  1. 1.
    Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N (2009) Triple-negative breast cancer–current status and future directions. Ann Oncol 20(12):1913–1927. doi: 10.1093/annonc/mdp492 CrossRefPubMedGoogle Scholar
  2. 2.
    Gelmon K, Dent R, Mackey JR, Laing K, McLeod D, Verma S (2012) Targeting triple-negative breast cancer: optimising therapeutic outcomes. Ann Oncol 23(9):2223–2234. doi: 10.1093/annonc/mds067 CrossRefPubMedGoogle Scholar
  3. 3.
    Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13(15 Pt 1):4429–4434. doi: 10.1158/1078-0432.CCR-06-3045 CrossRefPubMedGoogle Scholar
  4. 4.
    Dent R, Hanna WM, Trudeau M, Rawlinson E, Sun P, Narod SA (2009) Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat 115(2):423–428. doi: 10.1007/s10549-008-0086-2 CrossRefPubMedGoogle Scholar
  5. 5.
    Gonzalez-Suarez E, Jacob AP, Jones J, Miller R, Roudier-Meyer MP, Erwert R, Pinkas J, Branstetter D, Dougall WC (2010) RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468(7320):103–107. doi: 10.1038/nature09495 CrossRefPubMedGoogle Scholar
  6. 6.
    Dougall WC (2012) Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res 18(2):326–335. doi: 10.1158/1078-0432.CCR-10-2507 CrossRefPubMedGoogle Scholar
  7. 7.
    Walsh MC, Choi Y (2014) Biology of the RANKL-RANK-OPG System in immunity, bone, and beyond. Front Immunol 5:511. doi: 10.3389/fimmu.2014.00511 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Inoue J (1997) The TRAF family protein-mediated B cell proliferation signal and the mechanism of LMP1-induced B cell transformation. Nihon Rinsho 55(2):299–304PubMedGoogle Scholar
  9. 9.
    Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, Yasuda H, Smyth GK, Martin TJ, Lindeman GJ, Visvader JE (2010) Control of mammary stem cell function by steroid hormone signalling. Nature 465(7299):798–802. doi: 10.1038/nature09027 CrossRefPubMedGoogle Scholar
  10. 10.
    Azim HA Jr, Peccatori FA, Brohee S, Branstetter D, Loi S, Viale G, Piccart M, Dougall WC, Pruneri G, Sotiriou C (2015) RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy. Breast Cancer Res 17:24. doi: 10.1186/s13058-015-0538-7 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tang ZN, Zhang F, Tang P, Qi XW, Jiang J (2011) RANKL-induced migration of MDA-MB-231 human breast cancer cells via Src and MAPK activation. Oncol Rep 26(5):1243–1250. doi: 10.3892/or.2011.1368 PubMedGoogle Scholar
  12. 12.
    Joshi PA, Jackson HW, Beristain AG, Di Grappa MA, Mote PA, Clarke CL, Stingl J, Waterhouse PD, Khokha R (2010) Progesterone induces adult mammary stem cell expansion. Nature 465(7299):803–807. doi: 10.1038/nature09091 CrossRefPubMedGoogle Scholar
  13. 13.
    Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, Pasparakis M, Khokha R, Ormandy CJ, Widschwendter M, Schett G, Penninger JM (2010) Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468(7320):98–102. doi: 10.1038/nature09387 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Palafox M, Ferrer I, Pellegrini P, Vila S, Hernandez-Ortega S, Urruticoechea A, Climent F, Soler MT, Munoz P, Vinals F, Tometsko M, Branstetter D, Dougall WC, Gonzalez-Suarez E (2012) RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Cancer Res 72(11):2879–2888. doi: 10.1158/0008-5472.CAN-12-0044 CrossRefPubMedGoogle Scholar
  15. 15.
    Blake ML, Tometsko M, Miller R, Jones JC, Dougall WC (2014) RANK expression on breast cancer cells promotes skeletal metastasis. Clin Exp Metas 31(2):233–245. doi: 10.1007/s10585-013-9624-3 CrossRefGoogle Scholar
  16. 16.
    Pfitzner BM, Branstetter D, Loibl S, Denkert C, Lederer B, Schmitt WD, Dombrowski F, Werner M, Rudiger T, Dougall WC, von Minckwitz G (2014) RANK expression as a prognostic and predictive marker in breast cancer. Breast Cancer Res Treat 145(2):307–315. doi: 10.1007/s10549-014-2955-1 CrossRefPubMedGoogle Scholar
  17. 17.
    Hein A, Bayer CM, Schrauder MG, Haberle L, Heusinger K, Strick R, Ruebner M, Lux MP, Renner SP, Schulz-Wendtland R, Ekici AB, Hartmann A, Beckmann MW, Fasching PA (2014) Polymorphisms in the RANK/RANKL genes and their effect on bone specific prognosis in breast cancer patients. Biomed Res Int 2014:842452. doi: 10.1155/2014/842452 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Park HS, Lee A, Chae BJ, Bae JS, Song BJ, Jung SS (2014) Expression of receptor activator of nuclear factor kappa-B as a poor prognostic marker in breast cancer. J Surg Oncol 110(7):807–812. doi: 10.1002/jso.23737 CrossRefPubMedGoogle Scholar
  19. 19.
    Zhang L, Teng Y, Zhang Y, Liu J, Xu L, Qu J, Hou K, Yang X, Liu Y, Qu X (2012) Receptor activator for nuclear factor kappa B expression predicts poor prognosis in breast cancer patients with bone metastasis but not in patients with visceral metastasis. J Clin Pathol 65(1):36–40. doi: 10.1136/jclinpath-2011-200312 CrossRefPubMedGoogle Scholar
  20. 20.
    Parinyanitikul N, Blumenschein GR, Wu Y, Lei X, Chavez-Macgregor M, Smart M, Gonzalez-Angulo AM (2013) Mesothelin expression and survival outcomes in triple receptor negative breast cancer. Clin Breast Cancer 13(5):378–384. doi: 10.1016/j.clbc.2013.05.001 CrossRefPubMedGoogle Scholar
  21. 21.
    Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC (2010) American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 28(16):2784–2795. doi: 10.1200/JCO.2009.25.6529 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, Hayes DF, American Society of Clinical O, College of American P (2013) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 31(31):3997–4013. doi: 10.1200/JCO.2013.50.9984 CrossRefPubMedGoogle Scholar
  23. 23.
    Wood CE, Branstetter D, Jacob AP, Cline JM, Register TC, Rohrbach K, Huang LY, Borgerink H, Dougall WC (2013) Progestin effects on cell proliferation pathways in the postmenopausal mammary gland. Breast Cancer Res 15(4):62. doi: 10.1186/bcr3456 CrossRefGoogle Scholar
  24. 24.
    Branstetter DG, Nelson SD, Manivel JC, Blay JY, Chawla S, Thomas DM, Jun S, Jacobs I (2012) Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone. Clin Cancer Res 18(16):4415–4424. doi: 10.1158/1078-0432.CCR-12-0578 CrossRefPubMedGoogle Scholar
  25. 25.
    McCarty KS Jr, Miller LS, Cox EB, Konrath J, McCarty KS Sr (1985) Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med 109(8):716–721PubMedGoogle Scholar
  26. 26.
    Hosmer DW Jr, Lemeshow S (2008) Applied survival analysis: regression modeling of time to event data, 2nd edn. Wiley, New YorkCrossRefGoogle Scholar
  27. 27.
    Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121(7):2750–2767. doi: 10.1172/JCI45014 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zheng Y, Chow SO, Boernert K, Basel D, Mikuscheva A, Kim S, Fong-Yee C, Trivedi T, Buttgereit F, Sutherland RL, Dunstan CR, Zhou H, Seibel MJ (2014) Direct crosstalk between cancer and osteoblast lineage cells fuels metastatic growth in bone via auto-amplification of IL-6 and RANKL signaling pathways. J Bone Miner Res 29(9):1938–1949. doi: 10.1002/jbmr.2231 CrossRefPubMedGoogle Scholar
  29. 29.
    Lacey DL, Boyle WJ, Simonet WS, Kostenuik PJ, Dougall WC, Sullivan JK, San Martin J, Dansey R (2012) Bench to bedside: elucidation of the OPG-RANK–RANKL pathway and the development of denosumab. Nat Rev Drug Discov 11(5):401–419. doi: 10.1038/nrd3705 CrossRefPubMedGoogle Scholar
  30. 30.
    Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation. Nature 423(6937):337–342. doi: 10.1038/nature01658 CrossRefPubMedGoogle Scholar
  31. 31.
    Canon JR, Roudier M, Bryant R, Morony S, Stolina M, Kostenuik PJ, Dougall WC (2008) Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis. Clin Exp Metas 25(2):119–129. doi: 10.1007/s10585-007-9127-1 CrossRefGoogle Scholar
  32. 32.
    Holland PM, Miller R, Jones J, Douangpanya H, Piasecki J, Roudier M, Dougall WC (2010) Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis. Cancer Biol Ther 9(7):539–550CrossRefPubMedGoogle Scholar
  33. 33.
    Yoldi G, Pellegrini P, Trinidad EM, Cordero A, Gomez-Miragaya J, Serra-Musach J, Dougall WC, Munoz P, Pujana MA, Planelles L, Gonzalez-Suarez E (2016) RANK signaling blockade reduces breast cancer recurrence by inducing tumor cell differentiation. Cancer Res 76(19):5857–5869. doi: 10.1158/0008-5472.CAN-15-2745 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Monica E. Reyes
    • 1
    • 2
    • 3
  • Takeo Fujii
    • 1
    • 3
    • 8
  • Daniel Branstetter
    • 9
  • Savitri Krishnamurthy
    • 1
    • 4
  • Hiroko Masuda
    • 1
    • 2
    • 3
  • Xiaoping Wang
    • 1
    • 2
    • 3
  • James M. Reuben
    • 1
    • 5
  • Wendy A. Woodward
    • 1
    • 6
  • Beatrice J. Edwards
    • 7
  • Gabriel N. Hortobagyi
    • 1
    • 2
    • 3
  • Debu Tripathy
    • 1
    • 2
    • 3
  • William C. Dougall
    • 10
  • Bedrich L. Eckhardt
    • 1
    • 2
    • 3
    Email author
  • Naoto T. Ueno
    • 1
    • 2
    • 3
    Email author
  1. 1.Morgan Welch Inflammatory Breast Cancer Research Program and ClinicThe University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Section of Translational Breast Cancer ResearchThe University of Texas MD Anderson Cancer CenterHoustonUSA
  3. 3.Department of Breast Medical OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  4. 4.Department of PathologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  5. 5.Department of HematopathologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  6. 6.Department of Radiation OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  7. 7.Department of General Internal MedicineThe University of Texas MD Anderson Cancer CenterHoustonUSA
  8. 8.The University of Texas Health Science Center at Houston School of Public HealthHoustonUSA
  9. 9.Department of PathologyAmgen, Inc.SeattleUSA
  10. 10.Hematology and Oncology ResearchAmgen, Inc.SeattleUSA

Personalised recommendations