BRMS1 suppresses breast cancer metastasis in multiple experimental models of metastasis by reducing solitary cell survival and inhibiting growth initiation

  • Benjamin D. Hedley
  • Kedar S. Vaidya
  • Pushar Phadke
  • Lisa MacKenzie
  • David W. Dales
  • Carl O. Postenka
  • Ian C. MacDonald
  • Ann F. Chambers
Research Paper


The majority of breast cancer related deaths occur as a result of metastasis. The failure of effective treatments for metastasis is the underlying cause for this. Much remains unknown about the process of metastasis and how best to prevent or treat metastatic breast cancer. Therefore, a better understanding of the metastatic process is needed in order to determine effective therapeutic interventions to either eradicate, or slow down metastatic outgrowth of breast cancer. Metastasis is an inefficient process, however the ability of only a small number of cells to complete this process may have serious, life-threatening consequences. Little is known about whether expression of the metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) can suppress metastatic outgrowth in different organs in multiple experimental models of metastasis, or what effect BRMS1 expression has on the various steps in metastatic cascade. In this study we investigated the effect of BRMS1 expression on organ-specific metastasis. In addition, the steps in metastasis that are inhibited by BRMS1-expression were determined. In vivo, BRMS1 expression reduced metastatic burden to liver, bone, brain, and lung in mice by at least 75% (P < 0.05). Detailed quantitative analysis of the metastatic process in lung showed that BRMS1 expression significantly reduced the numbers of solitary single cells that survive after initial arrest within the lung microvasculature, and also inhibited the initiation of growth subsequent to arrest. In vitro, BRMS1 expression decreased cancer cell survival under stress conditions (hypoxia), increased anoikis, and decreased the ability of cancer cells to adhere. These novel findings demonstrate that BRMS1 is a potent suppressor of metastasis in multiple organs, and identify two steps in the metastatic process that are sensitive to inhibition by BRMS1.


Adhesion Apoptosis Breast cancer Growth suppression Metastasis Metastasis suppressor 



Breast cancer metastasis suppressor gene 1


Haematoxylin and eosin


Hank’s buffered salt solution


Red green blue


Serum free media


Phosphate buffer


Phosphate buffered saline




  1. 1.
    Parkin DM, Bray F, Ferlay J et al (2005) Global cancer statistics, 2002. CA Cancer J Clin 55(2):74–108PubMedGoogle Scholar
  2. 2.
    Jemal A, Siegel R, Ward E et al (2007) Cancer statistics 2007. CA Cancer J Clin 57(1):43–66PubMedCrossRefGoogle Scholar
  3. 3.
    DeVita VT, Rosenberg SA (2004) Principles and practice of oncology. Lippincott Williams & Wilkins, PhiladelphiaGoogle Scholar
  4. 4.
    Bundred NJ (2001) Prognostic and predictive factors in breast cancer. Cancer Treat Rev 27(3):137–142. doi:10.1053/ctrv.2000.0207 PubMedCrossRefGoogle Scholar
  5. 5.
    Carter CL, Allen C, Henson DE (1989) Relation of tumor size, lymph node status, and survival in 24, 740 breast cancer cases. Cancer 63(1):181–187. doi:10.1002/1097-0142(19890101)63:1<181::AID-CNCR2820630129>3.0.CO;2-HGoogle Scholar
  6. 6.
    Elledge RM, McGuire WL, Osborne CK (1992) Prognostic factors in breast cancer. Semin Oncol 19(3):244–253PubMedGoogle Scholar
  7. 7.
    Weiss L (1992) Comments on hematogenous metastatic patterns in humans as revealed by autopsy. Clin Exp Metastasis 10(3):191–199. doi:10.1007/BF00132751 PubMedCrossRefGoogle Scholar
  8. 8.
    Smith I, Procter M, Gelber RD et al (2007) 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet 369(9555):29–36. doi:10.1016/S0140-6736(07)60028-2 PubMedCrossRefGoogle Scholar
  9. 9.
    Aduvant Breast Cancer Trials Collborative Group (2007) Polychemotherapy for early breast cancer: results from the international adjuvant breast cancer chemotherapy randomized trial. J Natl Cancer Inst 99(7):506–515CrossRefGoogle Scholar
  10. 10.
    Clarke M, Collins R, Darby S, Davies C et al (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365(9472):1687–1717. doi:10.1016/S0140-6736(05)66544-0 CrossRefGoogle Scholar
  11. 11.
    Cole BF, Gelber RD, Gelber S et al (2001) Polychemotherapy for early breast cancer: an overview of the randomised clinical trials with quality-adjusted survival analysis. Lancet 358(9278):277–286. doi:10.1016/S0140-6736(01)05483-6 PubMedCrossRefGoogle Scholar
  12. 12.
    Baselga J, Perez EA, Pienkowski T et al (2006) Adjuvant trastuzumab: a milestone in the treatment of HER-2-positive early breast cancer. Oncologist 11(Suppl 1):4–12. doi:10.1634/theoncologist.11-90001-4 PubMedCrossRefGoogle Scholar
  13. 13.
    Stemmler HJ, Kahlert S, Siekiera W et al (2006) Characteristics of patients with brain metastases receiving trastuzumab for HER2 overexpressing metastatic breast cancer. Breast 15(2):219–225. doi:10.1016/j.breast.2005.04.017 PubMedCrossRefGoogle Scholar
  14. 14.
    Kauffman EC, Robinson VL, Stadler WM et al (2003) Metastasis suppression: the evolving role of metastasis suppressor genes for regulating cancer cell growth at the secondary site. J Urol 169(3):1122–1133. doi:10.1097/01.ju.0000051580.89109.4b PubMedCrossRefGoogle Scholar
  15. 15.
    Steeg PS, Bevilacqua G, Kopper L et al (1988) Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 80(3):200–204. doi:10.1093/jnci/80.3.200 PubMedCrossRefGoogle Scholar
  16. 16.
    Welch DR, Steeg PS, Rinker-Schaeffer CW (2000) Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis. Breast Cancer Res 2(6):408–416. doi:10.1186/bcr87 PubMedCrossRefGoogle Scholar
  17. 17.
    Yoshida BA, Sokoloff MM, Welch DR et al (2000) Metastasis-suppressor genes: a review and perspective on an emerging field. J Natl Cancer Inst 92(21):1717–1730. doi:10.1093/jnci/92.21.1717 PubMedCrossRefGoogle Scholar
  18. 18.
    Hunter KW, Broman KW, Voyer TL et al (2001) Predisposition to efficient mammary tumor metastatic progression is linked to the breast cancer metastasis suppressor gene Brms1. Cancer Res 61(24):8866–8872PubMedGoogle Scholar
  19. 19.
    Samant RS, Debies MT, Shevde LA et al (2002) Identification and characterization of the murine ortholog (brms1) of breast-cancer metastasis suppressor 1 (BRMS1). Int J Cancer 97(1):15–20. doi:10.1002/ijc.1569 PubMedCrossRefGoogle Scholar
  20. 20.
    Seraj MJ, Samant RS, Verderame MF et al (2000) Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13. Cancer Res 60(11):2764–2769PubMedGoogle Scholar
  21. 21.
    Shevde LA, Samant RS, Goldberg SF et al (2002) Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1. Exp Cell Res 273(2):229–239. doi:10.1006/excr.2001.5452 PubMedCrossRefGoogle Scholar
  22. 22.
    Cicek M, Fukuyama R, Welch DR et al (2005) Breast cancer metastasis suppressor 1 inhibits gene expression by targeting nuclear factor-kappaB activity. Cancer Res 65(9):3586–3595. doi:10.1158/0008-5472.CAN-04-3139 PubMedCrossRefGoogle Scholar
  23. 23.
    Samant RS, Clark DW, Fillmore RA et al (2007) Breast cancer metastasis suppressor 1 BRMS1 inhibits osteopontin transcription by abrogating NFkappaB activation. Mol Cancer 6:6. doi:10.1186/1476-4598-6-6 Google Scholar
  24. 24.
    DeWald DB, Torabinejad J, Samant RS et al (2005) Metastasis suppression by breast cancer metastasis suppressor 1 involves reduction of phosphoinositide signaling in MDA-MB-435 breast carcinoma cells. Cancer Res 65(3):713–717PubMedGoogle Scholar
  25. 25.
    Saunders MM, Seraj MJ, Li Z et al (2001) Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication. Cancer Res 61(5):1765–1767PubMedGoogle Scholar
  26. 26.
    Meehan WJ, Samant RS, Hopper JE et al (2004) Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription. J Biol Chem 279(2):1562–1569. doi:10.1074/jbc.M307969200 PubMedCrossRefGoogle Scholar
  27. 27.
    Phadke PA, Vaidya KS, Nash KT et al (2008) BRMS1 suppresses breast cancer experimental metastasis to multiple organs by inhibiting several steps of the metastatic process. Am J Pathol 172(3):809–817. doi:10.2353/ajpath.2008.070772 PubMedCrossRefGoogle Scholar
  28. 28.
    Hicks DG, Yoder BJ, Short S et al (2006) Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients. Clin Cancer Res 12(22):6702–6708. doi:10.1158/1078-0432.CCR-06-0635 PubMedCrossRefGoogle Scholar
  29. 29.
    Price JE, Polyzos A, Zhang RD et al (1990) Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 50(3):717–721PubMedGoogle Scholar
  30. 30.
    Price JE, Zhang RD (1990) Studies of human breast cancer metastasis using nude mice. Cancer Metastasis Rev 8(4):285–297. doi:10.1007/BF00052605 PubMedCrossRefGoogle Scholar
  31. 31.
    Rae JM, Creighton CJ, Meck JM et al (2007) MDA-MB-435 cells are derived from M14 melanoma cells—a loss for breast cancer, but a boon for melanoma research. Breast Cancer Res Treat 104(1):13–19. doi:10.1007/s10549-006-9392-8 PubMedCrossRefGoogle Scholar
  32. 32.
    Graham KC, Wirtzfeld LA, MacKenzie LT et al (2005) Three-dimensional high-frequency ultrasound imaging for longitudinal evaluation of liver metastases in preclinical models. Cancer Res 65(12):5231–5237. doi:10.1158/0008-5472.CAN-05-0440 PubMedCrossRefGoogle Scholar
  33. 33.
    Yoneda T, Williams PJ, Hiraga 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):1486–1495. doi:10.1359/jbmr.2001.16.8.1486 PubMedCrossRefGoogle Scholar
  34. 34.
    Heyn C, Ronald JA, Ramadan SS et al (2006) In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain. Magn Reson Med 56(5):1001–1010. doi:10.1002/mrm.21029 PubMedCrossRefGoogle Scholar
  35. 35.
    Chekmareva MA, Kadkhodaian MM, Hollowell CM et al (1998) Chromosome 17-mediated dormancy of AT6 1 prostate cancer micrometastases. Cancer Res 58(21):4963–4969PubMedGoogle Scholar
  36. 36.
    Allan AL, George R, Vantyghem SA et al (2006) Role of the integrin-binding protein osteopontin in lymphatic metastasis of breast cancer. Am J Pathol 169(1):233–246. doi:10.2353/ajpath.2006.051152 PubMedCrossRefGoogle Scholar
  37. 37.
    Hou Y, Wong E, Martin J et al (2006) A role for cyclic-GMP dependent protein kinase in anoikis. Cell Signal 18(6):882–888. doi:10.1016/j.cellsig.2005.07.015 PubMedCrossRefGoogle Scholar
  38. 38.
    Luzzi KJ, MacDonald IC, Schmidt EE et al (1998) Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases. Am J Pathol 153(3):865–873PubMedGoogle Scholar
  39. 39.
    Heyn C, Ronald JA, Mackenzie LT et al (2006) In vivo magnetic resonance imaging of single cells in mouse brain with optical validation. Magn Reson Med 55(1):23–29. doi:10.1002/mrm.20747 PubMedCrossRefGoogle Scholar
  40. 40.
    Cameron MD, Schmidt EE, Kerkvliet N et al (2000) Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Res 60(9):2541–2546PubMedGoogle Scholar
  41. 41.
    Chambers AF, Wilson S (1988) Use of NeoR B16F1 murine melanoma cells to assess clonality of experimental metastases in the immune-deficient chick embryo. Clin Exp Metastasis 6(2):171–182PubMedCrossRefGoogle Scholar
  42. 42.
    McCarthy RP, Wang M, Jones TD et al (2006) Molecular evidence for the same clonal origin of multifocal papillary thyroid carcinomas. Clin Cancer Res 12(8):2414–2418. doi:10.1158/1078-0432.CCR-05-2818 PubMedCrossRefGoogle Scholar
  43. 43.
    Hedley BD, Welch DR, Allan AL et al. (2008) Downregulation of osteopontin contributes to metastasis suppression by breast cancer metastasis suppressor 1. Int J Cancer. doi:10.1002/ijc.23542 PubMedGoogle Scholar
  44. 44.
    Koop S, MacDonald IC, Luzzi K et al (1995) Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate. Cancer Res 55(12):2520–2523PubMedGoogle Scholar
  45. 45.
    Wyckoff JB, Jones JG, Condeelis JS et al (2000) A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res 60(9):2504–2511PubMedGoogle Scholar
  46. 46.
    Glinskii OV, Huxley VH, Glinsky GV et al (2005) Mechanical entrapment is insufficient and intercellular adhesion is essential for metastatic cell arrest in distant organs. Neoplasia 7(5):522–527. doi:10.1593/neo.04646 PubMedCrossRefGoogle Scholar
  47. 47.
    Schweinitz A, Steinmetzer T, Banke IJ et al (2004) Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents. J Biol Chem 279(32):33613–33622. doi:10.1074/jbc.M314151200 PubMedCrossRefGoogle Scholar
  48. 48.
    MacDonald IC, Groom AC, Chambers AF (2002) Cancer spread and micrometastasis development: quantitative approaches for in vivo models. BioEssays 24(10):885–893. doi:10.1002/bies.10156 PubMedCrossRefGoogle Scholar
  49. 49.
    Minn AJ, Kang Y, Serganova I et al (2005) Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest 115(1):44–55PubMedGoogle Scholar
  50. 50.
    Hedley BD, Allan AL, Chambers AF (2006) Tumor dormancy and the role of metastasis suppressor genes in regulating ectopic growth. Future Oncol 2(5):627–641. doi:10.2217/14796694.2.5.627 PubMedCrossRefGoogle Scholar
  51. 51.
    Goldberg SF, Harms JF, Quon K et al (1999) Metastasis-suppressed C8161 melanoma cells arrest in lung but fail to proliferate. Clin Exp Metastasis 17(7):601–607. doi:10.1023/A:1006718800891 PubMedCrossRefGoogle Scholar
  52. 52.
    Al-Mehdi AB, Tozawa K, Fisher AB et al (2000) Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat Med 6(1):100–102. doi:10.1038/71429 PubMedCrossRefGoogle Scholar
  53. 53.
    Wong CW, Song C, Grimes MM et al (2002) Intravascular location of breast cancer cells after spontaneous metastasis to the lung. Am J Pathol 161(3):749–753PubMedGoogle Scholar
  54. 54.
    Rinker-Schaeffer CW, O’Keefe JP, Welch DR et al (2006) Metastasis suppressor proteins: discovery, molecular mechanisms, and clinical application. Clin Cancer Res 12(13):3882–3889. doi:10.1158/1078-0432.CCR-06-1014 PubMedCrossRefGoogle Scholar
  55. 55.
    Yoon SY, Lee YJ, Seo JH et al (2006) uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line. Cell Res 16(1):75–81. doi:10.1038/ PubMedCrossRefGoogle Scholar
  56. 56.
    Graham CH, Forsdike J, Fitzgerald CJ et al (1999) Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression. Int J Cancer 80(4):617–623. doi:10.1002/(SICI)1097-0215(19990209)80:4<617::AID-IJC22>3.0.CO;2-CGoogle Scholar
  57. 57.
    Cogswell PC, Guttridge DC, Funkhouser WK et al (2000) Selective activation of NF-kappa B subunits in human breast cancer: potential roles for NF-kappa B2/p52 and for Bcl-3. Oncogene 19(9):1123–1131. doi:10.1038/sj.onc.1203412 PubMedCrossRefGoogle Scholar
  58. 58.
    Helbig G, Christopherson KW 2nd, Bhat-Nakshatri P et al (2003) NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem 278(24):21631–21638. doi:10.1074/jbc.M300609200 PubMedCrossRefGoogle Scholar
  59. 59.
    Nakshatri H, Goulet RJ Jr (2002) NF-kappaB and breast cancer. Curr Probl Cancer 26(5):282–309. doi:10.1067/mcn.2002.129977 PubMedCrossRefGoogle Scholar
  60. 60.
    Sovak MA, Bellas RE, Kim DW et al (1997) Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 100(12):2952–2960. doi:10.1172/JCI119848 PubMedCrossRefGoogle Scholar
  61. 61.
    Janicke F, Schmitt M, Ulm K et al (1998) Urokinase type plasminogen activator antigen and early relapse in breast cancer. Lancet 2(8670):1049. doi:10.1016/S0140-6736(89)91070-2 Google Scholar
  62. 62.
    Harbeck N, Kates RE, Schmitt M et al (2004) Urokinase-type plasminogen activator and its inhibitor type 1 predict disease outcome and therapy response in primary breast cancer. Clin Breast Cancer 5(5):348–352PubMedCrossRefGoogle Scholar
  63. 63.
    Zhou Y, Eppenberger-Castori S, Marx C et al (2005) Activation of nuclear factor-kappaB (NFkappaB) identifies a high-risk subset of hormone-dependent breast cancers. Int J Biochem Cell Biol 37(5):1130–1144. doi:10.1016/j.biocel.2004.09.006 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Benjamin D. Hedley
    • 1
    • 2
  • Kedar S. Vaidya
    • 3
  • Pushar Phadke
    • 3
  • Lisa MacKenzie
    • 2
  • David W. Dales
    • 1
  • Carl O. Postenka
    • 1
  • Ian C. MacDonald
    • 2
    • 4
  • Ann F. Chambers
    • 1
    • 2
    • 4
    • 5
  1. 1.London Regional Cancer ProgramLondon Health Sciences CentreLondonCanada
  2. 2.Department of Medical Biophysics, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada
  3. 3.Department of Pathology and Comprehensive Cancer CentreUniversity of Alabama at BirminghamBirminghamUSA
  4. 4.Department of Oncology, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada
  5. 5.Department of Pathology, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada

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