Clinical & Experimental Metastasis

, Volume 22, Issue 3, pp 237–246 | Cite as

Expression of MMP2, MMP9 and MMP3 in Breast Cancer Brain Metastasis in a Rat Model

  • Odete Mendes
  • Hun-Taek Kim
  • George StoicaEmail author


In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.

Key words

animal model brain metastasis breast cancer MMP2 MMP3 MMP9 



breast cancer






matrix metalloproteinases


phosphate buffered saline


reverse transcriptase polymerase chain reaction


tissue inhibitors of metalloproteinases


Western blotting


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Stark AM, Tongers K, Maass N et al. Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2004; Dec 8 (E-Pub):1432–35Google Scholar
  2. 2.
    Rusciano, D, Burger, MM 1992Why do cancer cells metastasize into particular organs?Bioessays1418594CrossRefPubMedGoogle Scholar
  3. 3.
    Hall, DG, Stoica, G 1994Characterization of brain and bone-metastasizing clones selected from an ethylnitrosurea-induced rat mammary carcinomaClin Exp Metastasis1228395CrossRefPubMedGoogle Scholar
  4. 4.
    Nishizuka A I et al. Analysis of gene expression involved in brain metastasis from breast cancer using cDNA microarray Breast Cancer. 2002; 9(1): 26–32.Google Scholar
  5. 5.
    Leppa, S, Saarto, T, Vehmanen, L,  et al. 2004A high serum matrix metalloproteinase-2 level is associated with an adverse prognosis in node-positive breast carcinomaClin Cancer Res10105763PubMedGoogle Scholar
  6. 6.
    Stetler-Stevenson, WG, Aznavoorian, S, Liotta, LA 1993Tumor cell interactions with the extracellular matrix during invasion and metastasisAnnu Rev Cell Biol954173CrossRefPubMedGoogle Scholar
  7. 7.
    Levicar, N, Nuttall, RK, Lah, TT 2003Proteases in brain tumor progressionActa Neurochir14582538Google Scholar
  8. 8.
    Menter, DG, Herrmann, JL, Nicolson, GL 1995The role of trophic factors and autocrine/paracrine growth factors in brain metastasisClin Exp Metastasis136788CrossRefPubMedGoogle Scholar
  9. 9.
    Fujimaki, T, Price, JE, Fan, D, Bucana, CD, Itoh, K, Kirino, T, Fidler, IJ 1996Selective growth of human melanoma cells in the brain parenchyma of nude miceMelanoma Res636371PubMedGoogle Scholar
  10. 10.
    Nie, J, Pei, D 2003Direct activation of pro-matrix metallopreoteinase-2 by Leukolysin/membrane-type 6 Matrix metalloproteinase/Matrix metalloproteinase 25 at the Asn109-Tyr bondCancer Res63675862PubMedGoogle Scholar
  11. 11.
    Rocca, G, Pucci-Minafra, I, Marrazo, A,  et al. 2004Zymographic detection and clinical correlations of MMP-2 and MMP-9 in breast cancer seraBr J Cancer90141421CrossRefPubMedGoogle Scholar
  12. 12.
    Djonov, V, Cresto, N, Aebersold, DM,  et al. 2002Tumor cell specific expression of MMP-2 correlates with tumor vascularisation in breast cancerInt J Oncol212530PubMedGoogle Scholar
  13. 13.
    Talvensaari-Mattila, A, Paakko, P, Blanco-Sequeiros, G,  et al. 2001Matrix metalloproteinase-2 (MMP-2) is associated with the risk for a relapse in postmenopausal patients with node-positive breast carcinoma treated with antiestrogen adjuvant therapyBreast Cancer Res Treat655561CrossRefPubMedGoogle Scholar
  14. 14.
    Duffy, MJ, Maguire, TM, Hill, A,  et al. 2000Metalloproteinases: role in breast carcinogenesis, invasion and metastasisBreast Cancer Res225257CrossRefPubMedGoogle Scholar
  15. 15.
    Hynes, RO 2003Metastatic potential: generic predisposition of the primary tumor or rare metastatic variants-or both?Cell11382123CrossRefPubMedGoogle Scholar
  16. 16.
    Balduyck, M, Zerimech, F, Gouyer, V,  et al. 2000Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitroClin Exp Metastasis1817178CrossRefPubMedGoogle Scholar
  17. 17.
    Lebeau, A, Nerlich, AG, Sauer, U,  et al. 1999Tissue distribution of major matrix metalloproteinases and their transcripts in human breast carcinomasAnticancer Res19425764PubMedGoogle Scholar
  18. 18.
    Heppner, KJ, Matrisian, LM, Jensen, RA,  et al. 1996Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host responseAm J Pathol1492738PubMedGoogle Scholar
  19. 19.
    Yoneda, T 2000Cellular and molecular basis of preferential metastasis of breast cancer to boneJ Orthop Sci57581CrossRefPubMedGoogle Scholar
  20. 20.
    Oshiba, T, Miyaura, C, Inada, M,  et al. 2003Role of RANKL-induced osteoclast formation and MMP-dependent matrix degerdation in bone destruction by breast cancer metastasisBritish J Cancer8813181326Google Scholar
  21. 21.
    Scott, KA, Holdsworth, H, Balkwill, FR,  et al. 2000Exploiting changes in the tumour microenvironment with sequential cytokine and matrix metalloprotease inhibitor treatment in a murine breast cancer modelBr J Cancer83153843CrossRefPubMedGoogle Scholar
  22. 22.
    Tester, AM, Waltham, M, Oh, SJ,  et al. 2004Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude miceCancer Res646528CrossRefPubMedGoogle Scholar
  23. 23.
    Nozaki, S, Sissons, S, Chien, DS, Sledge, GW,Jr 2003Activity of biphenyl matrix metalloproteinase inhibitor BAY 12–9566 in a human breast cancer orthotopic modelClin Exp Metastasis2040712CrossRefPubMedGoogle Scholar
  24. 24.
    Lee, SJ, Sakurai, H, Oshima, K, Kim, SH, Saiki, I 2003 JulAnti-metastatic and anti-angiogenic activities of a new matrix metalloproteinase inhibitor, TN-6bEur J Cancer39163241CrossRefGoogle Scholar
  25. 25.
    Saad, S, Bendall, LJ, James, A,  et al. 2000Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblastsBreast Cancer Res Treat6310515CrossRefPubMedGoogle Scholar
  26. 26.
    Lafleur, MA, Tester, AM, Thompson, EW 2003Selective involvement of TIMP-2 in the second activational cleavage of pro-MMP-2: refinement of the pro-MMP-2 activation mechanismFEBS Lett55345763CrossRefPubMedGoogle Scholar
  27. 27.
    Yan SJ, Blomme EAG In situ zymography: A molecular pathology technique to localize endogenous protease activity in tissue sections Veterinary Pathology 2003; 40: 227–36Google Scholar
  28. 28.
    Lee, P, Hwang, J, Murphy, G, Io, M 2000Functional significance of MMP-9 in tumor necrosis factor-induced proliferation and branching morphogenesis of mammary epithelial cellsEndocrinology141376473CrossRefPubMedGoogle Scholar
  29. 29.
    Lu, W, Zhou, X, Hong, B, Liu, J, Yue, Z 2004Suppression of invasion in human U87 glioma cells by adenovirus-mediated co-transfer of TIMP2 and PTEN geneCancer Lett21420513CrossRefPubMedGoogle Scholar
  30. 30.
    Chapman, RE, Scott, AA, Deschamps, AM, Lowry, AS, Stroud, RE, Ikonomidis, JS, Spinale, FG 2003Matrix metalloproteinase abundance in human myocardial fibroblasts: effects of sustained pharmacologic matrix metalloproteinase inhibitionJ Mol Cell Cardiol3553948CrossRefPubMedGoogle Scholar
  31. 31.
    Chancey, AL, Brower, GL, Peterson, JT, Janicki, JS 2002Effects of matrix metalloproteinase inhibition on ventricular remodeling due to volume overloadCirculation105198388CrossRefPubMedGoogle Scholar
  32. 32.
    A Yu Q, Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated invasion Genes &Development 1999; 13: 35–48Google Scholar
  33. 33.
    Muir, EM, Adcock, KH, Morgesnten, DA,  et al. 2002Matrix Metalloproteinases and their inhibitors are produced by overlapping populations of brain astrocytesMol Brain Res10010317CrossRefPubMedGoogle Scholar
  34. 34.
    Wang, M, Liu, YE, Greene, J,  et al. 1997Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4Oncogene14276774CrossRefPubMedGoogle Scholar
  35. 35.
    Jones, JL, Glynn, P, Walker, RA 1999Expression of MMP-2 and MMP-9, their inhibitors, and the activator MT1-MMP in primary breast carcinomasJ Pathol1891618CrossRefPubMedGoogle Scholar
  36. 36.
    Caudroy, S, Polette, M, Tournier, JM,  et al. 1999Expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) and the matrix metalloproteinase-2 in bronchopulmonary and breast lesionsJ Histochem Cytochem47157580PubMedGoogle Scholar
  37. 37.
    Bartsch, JE, Staren, ED, Appert, HE 2003Matrix metalloproteinase expression in breast cancerJ Surg Res11038392CrossRefPubMedGoogle Scholar
  38. 38.
    Brummer, O, Athar, S, Riethdorf, L,  et al. 1999Matrix-metalloproteinases 1, 2, and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization studyVirchows Arch43556673CrossRefPubMedGoogle Scholar
  39. 39.
    Rudolph-Owen, LA, Matrisian, LM 1998Matrix metalloproteinases in remodeling of the normal and neoplastic mammary glandJ Mammary Gland Biol Neoplasia317789CrossRefPubMedGoogle Scholar
  40. 40.
    Shekhar, MP, Werdell, J, Santner, SJ,  et al. 2001Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progressionCancer Res61132026PubMedGoogle Scholar
  41. 41.
    Mellick, AS, Blackmore, D, Weinstein, SR,  et al. 2003An Assessment of MMP and TIMP Gene Expression in Cell Lines and Stroma-Tumour Differences in Microdissected Breast Cancer BiopsiesTumour Biol2425862PubMedGoogle Scholar
  42. 42.
    Nabeshima, A 2002K Matrix metalloproteinases in tumor invasion: Role for cell migrationPathol Int5225564CrossRefPubMedGoogle Scholar
  43. 43.
    Arai, K, Lee, S, Loo, EH 2003Essential role for ERK mitogen-activated protein kinase in matrix mettaloproteinase-9 regulation in rat cortical astrocytesGlia4325464CrossRefPubMedGoogle Scholar
  44. 44.
    Rosenberg, GA, Cunningham, LA, Wallace, J,  et al. 2001Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglial in cell culturesBrain Res82310412Google Scholar
  45. 45.
    Rivera, S, Ogier, C, Jourquin, J,  et al. 2002Gelatinase B and TIMP-1 are regulated in a cell- and time-dependent manner in association with neuronal death and glial reactivity after global forebrain ischemiaEur J Neurosci151932CrossRefPubMedGoogle Scholar
  46. 46.
    Le, DM, Besson, A, Fogg, DK,  et al. 2003Exploitation of astrocytes by glioma cells to facilitate invasivemess: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascadeThe J of Neurosci23403443Google Scholar
  47. 47.
    Sierra, A, Price, JE, Garcia-Ramirez, M,  et al. 1997Astrocyte-derived cytokines contribute to the metastatic brain specificity of breast cancer cellsLab Invest7735768PubMedGoogle Scholar
  48. 48.
    Le, DM, Besson, A, Fogg, DK,  et al. 2003Exploitation of astrocytes by glioma cells to facilitate invasivemess: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascadeThe J of Neurosci23403443Google Scholar
  49. 49.
    O’Brien, PM, Ortwine, DF, Pavlovsky, AG, Picard, JA, Sliskovic, DR, Roth, BD, Dyer, RD, Johnson, LL, Man, CF, Hallak, H 2000Structure-activity relationships and pharmacokinetic analysis for a series of potent, systemically available biphenylsulfonamide matrix metalloproteinase inhibitorsJ Med Chem4315666PubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Department of Pathobiology, College of Veterinary MedicineTexas A&M UniversityCollege StationUSA

Personalised recommendations