Clinical & Experimental Metastasis

, Volume 31, Issue 2, pp 233–245 | Cite as

RANK expression on breast cancer cells promotes skeletal metastasis

  • Michelle L. Blake
  • Mark Tometsko
  • Robert Miller
  • Jon C. Jones
  • William C. Dougall
Research Paper


RANK ligand (RANKL), acting through its cognate receptor RANK, is a key factor for bone remodeling and metastasis by regulating the differentiation, survival and activation of osteoclasts. RANKL is also crucial for the development of mouse mammary glands during pregnancy and has been recently linked to the etiology of breast cancer via its direct activity on RANK-expressing normal or transformed breast epithelial cells, leading to increased mitogenesis, enhanced regenerative potential of mammary stem cells, and increased invasion and migration. We demonstrate that higher RANK expression in MDA-MB-231 breast cancer cells (MDA-231-RANK cells) is sufficient to confer a significantly greater metastatic growth rate in the bone compared with MDA-MB-231 cells which do not express high levels of RANK. Blockade of osteoclastic bone resorption, achieved with treatment by either RANKL inhibition or zoledronic acid, did reduce skeletal tumor progression of MDA-231-RANK cells suggesting that the vicious cycle contributes to metastatic growth. However, RANKL inhibition reduced skeletal growth of MDA-231-RANK tumors to a significantly greater extent than zoledronic acid, indicating that skeletal growth of RANK-positive tumors is also driven by direct RANKL effects. RANKL stimulated the expression of multiple genes associated with cell invasive behavior, including several matrix metalloproteinases and other genes previously defined as part of a bone metastasis gene signature. These data indicate that RANKL provokes breast cancer bone metastases via two distinct, but potentially overlapping mechanisms: stimulation of tumor-associated osteoclastogenesis and stimulation of RANK-expressing tumor cells.


Breast cancer Bone metastases RANKL RANK 



RANK ligand


Mammary stem cell


Matrix metalloproteinase


American Type Culture Collection




Bioluminescent imaging


Epithelial-to-mesenchymal transition





This work was supported by Amgen Inc. We thank Dan Branstetter and Martine Roudier for pathology support, Ryan Erwert for technical support, Winnie Weng for expert assistance with statistical analysis, Albert Rhee for editorial assistance, and Shobana Ganesan, CACTUS Communications for formatting assistance. We would also like to thank Tony Polverino and Allison Jacob for critical reading of the manuscript in preparation.

Conflict of interest

Michelle Blake, Robert Miller, and Jon Jones are former employees and shareholders of Amgen Inc. Mark Tometsko and William C. Dougall are employees and shareholders of Amgen Inc.

Supplementary material

10585_2013_9624_MOESM1_ESM.eps (1.5 mb)
Fig. 1 Effect of RANKL treatment on MDA-231 ATCC and RANK transduced MDA-231 cells. Activation of cell signaling pathways downstream of RANK in MDA-231-ATCC, MDA-231-RANK and MDA-231-RANKΔ339 transduced cells following RANKL stimulation. Cells were treated for the indicated times with 1 μg/mL RANKL following overnight serum starvation. Whole cell lysates were immunoblotted for phosphorylated forms of proteins, followed by blot stripping and re-probing for total protein to verify equivalent loading (EPS 1533 kb)
10585_2013_9624_MOESM2_ESM.eps (1.4 mb)
Fig. 2 Correlation of hindlimb BLI, tumor area (histology) and lytic lesion area (X-ray) in mice with bone metastases from MDA-231-RANK cells. A strong correlation between lytic lesion (X-ray) and tumor area (histology) was observed (Spearman correlation coefficient [ρ] = 0.9427). Correlation between BLI and tumor area or lytic lesion and BLI are also evident (ρ = 0.8542 or 0.7803, respectively) (EPS 1441 kb)


  1. 1.
    Solomayer EF et al (2000) Metastatic breast cancer: clinical course, prognosis and therapy related to the first site of metastasis. Breast Cancer Res Treat 59(3):271–278PubMedCrossRefGoogle Scholar
  2. 2.
    Mundy GR (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2(8):584–593PubMedCrossRefGoogle Scholar
  3. 3.
    Lacey DL et al (2012) Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab. Nat Rev Drug Discov 11(5):401–419PubMedCrossRefGoogle Scholar
  4. 4.
    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):326–335PubMedCrossRefGoogle Scholar
  5. 5.
    Canon JR et al (2008) Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis. Clin Exp Metastasis 25(2):119–129PubMedCrossRefGoogle Scholar
  6. 6.
    Canon J et al (2012) RANKL inhibition combined with tamoxifen treatment increases anti-tumor efficacy and prevents tumor-induced bone destruction in an estrogen receptor-positive breast cancer bone metastasis model. Breast Cancer Res Treat 135(3):771–780PubMedCrossRefGoogle Scholar
  7. 7.
    Stopeck AT et al (2010) Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: a randomized, double-blind study. J Clin Oncol 28(35):5132–5139PubMedCrossRefGoogle Scholar
  8. 8.
    Smith MR et al (2012) Denosumab and bone-metastasis-free survival in men with castration-resistant prostate cancer: results of a phase 3, randomised, placebo-controlled trial. Lancet 379(9810):39–46PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Fata JE et al (2000) The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103(1):41–50PubMedCrossRefGoogle Scholar
  10. 10.
    Gonzalez-Suarez E et al (2007) RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini. Mol Cell Biol 27(4):1442–1454PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Cao Y et al (2001) IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 107(6):763–775PubMedCrossRefGoogle Scholar
  12. 12.
    Gonzalez-Suarez E (2011) RANKL inhibition: a promising novel strategy for breast cancer treatment. Clin Transl Oncol 13(4):222–228PubMedCrossRefGoogle Scholar
  13. 13.
    Beleut M et al (2010) Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland. Proc Natl Acad Sci USA 107(7):2989–2994PubMedCrossRefGoogle Scholar
  14. 14.
    Gonzalez-Suarez E et al (2010) RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468(7320):103–107PubMedCrossRefGoogle Scholar
  15. 15.
    Tanos T et al (2013) Progesterone/RANKL is a major regulatory axis in the human breast. Sci Transl Med 5(182):182ra55PubMedCrossRefGoogle Scholar
  16. 16.
    Joshi PA et al (2010) Progesterone induces adult mammary stem cell expansion. Nature 465(7299):803–807PubMedCrossRefGoogle Scholar
  17. 17.
    Asselin-Labat ML et al (2010) Control of mammary stem cell function by steroid hormone signalling. Nature 465(7299):798–802PubMedCrossRefGoogle Scholar
  18. 18.
    Schramek D et al (2010) Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468(7320):98–102PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Jones DH et al (2006) Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440(7084):692–696PubMedCrossRefGoogle Scholar
  20. 20.
    Armstrong AP et al (2008) RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes. Prostate 68(1):92–104PubMedCrossRefGoogle Scholar
  21. 21.
    Tan W et al (2011) Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling. Nature 470(7335):548–553PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Santini D et al (2011) Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients. PLoS ONE 6(4):e19234PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Yoneda T et al (2001) A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res 16(8):1486–1495PubMedCrossRefGoogle Scholar
  24. 24.
    Kang Y et al (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3(6):537–549PubMedCrossRefGoogle Scholar
  25. 25.
    Armstrong AP et al (2002) A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function. J Biol Chem 277(46):44347–44356PubMedCrossRefGoogle Scholar
  26. 26.
    Wilson TJ, Singh RK (2008) Proteases as modulators of tumor-stromal interaction: primary tumors to bone metastases. Biochim Biophys Acta 1785(2):85–95PubMedCentralPubMedGoogle Scholar
  27. 27.
    Coleman RE (1997) Skeletal complications of malignancy. Cancer 80(8 Suppl):1588–1594PubMedCrossRefGoogle Scholar
  28. 28.
    Coleman RE (2006) Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin Cancer Res 12(20 Pt 2):6243s–6249sPubMedCrossRefGoogle Scholar
  29. 29.
    Zheng Y et al (2007) Inhibition of bone resorption, rather than direct cytotoxicity, mediates the anti-tumour actions of ibandronate and osteoprotegerin in a murine model of breast cancer bone metastasis. Bone 40(2):471–478PubMedCrossRefGoogle Scholar
  30. 30.
    Martin TJ, Gillespie MT (2001) Receptor activator of nuclear factor kappa B ligand (RANKL): another link between breast and bone. Trends Endocrinol Metab 12(1):2–4PubMedCrossRefGoogle Scholar
  31. 31.
    Casimiro S et al (2013) RANKL/RANK/MMP-1 molecular triad contributes to the metastatic phenotype of breast and prostate cancer cells in vitro. PLoS ONE 8(5):e63153PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Palafox M et al (2012) RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Cancer Res 72(11):2879–2888PubMedCrossRefGoogle Scholar
  33. 33.
    Ithimakin S et al (2013) HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res 73(5):1635–1646PubMedCrossRefGoogle Scholar
  34. 34.
    Korkaya H et al (2012) Activation of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer by expanding the cancer stem cell population. Mol Cell 47(4):570–584PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Roodman GD (2002) Role of the bone marrow microenvironment in multiple myeloma. J Bone Miner Res 17(11):1921–1925PubMedCrossRefGoogle Scholar
  36. 36.
    Li J (2012) Correlation of receptor activator of nuclear factor kappa b (RANK) expression in breast cancer (BC) at the time of diagnosis with recurrence-free survival (RFS) and risk of bone-dominant metastases (BDM) in the ISPY1 trial. J Clin Oncol 30 (27 Suppl):2Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Michelle L. Blake
    • 1
  • Mark Tometsko
    • 2
  • Robert Miller
    • 1
  • Jon C. Jones
    • 1
  • William C. Dougall
    • 2
  1. 1.Oncology ResearchAmgen WashingtonSeattleUSA
  2. 2.Therapeutic Innovation UnitAmgen Inc.SeattleUSA

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