Skip to main content

Advertisement

SpringerLink
Log in

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Massagué J (2008) TGFβ in cancer. Cell 134:215–230. doi:10.1016/j.cell.2008.07.001

    Article  PubMed  Google Scholar 

  2. Akhurst RJ, Derynck R (2001) TGF-β signaling in cancer—a double-edged sword. Trends Cell Biol 11:S44–S51. doi:10.1016/S0962-8924(01)02130-4

    PubMed  CAS  Google Scholar 

  3. Ghellal A, Li C, Hayes M, Byrne G, Bundred N, Kumar S (2000) Prognostic significance of TGFβ1 and TGFβ3 in human breast carcinoma. Anticancer Res 20:4413–4418

    PubMed  CAS  Google Scholar 

  4. Sheen-Chen SM, Chen HS, Sheen CW, Eng HL, Chen WJ (2001) Serum levels of transforming growth factor β1 in patients with breast cancer. Arch Surg 136:937–940

    Article  PubMed  CAS  Google Scholar 

  5. Ivanovic V, Todorovic-Rakovic N, Demajo M, Neskovic-Konstantinovic Z, Subota V, Ivanisevic-Milovanovic O, Nikolic-Vukosavljevic D (2003) Elevated plasma levels of transforming growth factor-β1 (TGF-β1) in patients with advanced breast cancer: association with disease progression. Eur J Cancer 39:454–461. doi:10.1016/S0959-8049(02)00502-6

    Article  PubMed  CAS  Google Scholar 

  6. Desruisseau S, Palmari J, Giusti C, Romain S, Martin PM, Berthois Y (2006) Determination of TGFβ1 protein level in human primary breast cancers and its relationship with survival. Br J Cancer 94:239–246. doi:10.1038/sj.bjc.6602920

    Article  PubMed  CAS  Google Scholar 

  7. ten Dijke P, Hill CS (2004) New insights into TGF-β-Smad signalling. Trends Biochem Sci 29:265–273. doi:10.1016/j.tibs.2004.03.008

    Article  PubMed  CAS  Google Scholar 

  8. Moustakas A, Heldin CH (2009) The regulation of TGFβ signal transduction. Development 136:3699–3714. doi:10.1242/dev.030338

    Article  PubMed  CAS  Google Scholar 

  9. Chen CR, Kang Y, Massagué J (2001) Defective repression of c-myc in breast cancer cells: a loss at the core of the transforming growth factor β growth arrest program. Proc Natl Acad Sci USA 98:992–999

    Article  PubMed  CAS  Google Scholar 

  10. Hannon GJ, Beach D (1994) p15INK4B is a potential effector of TGF-β-induced cell cycle arrest. Nature 371:257–261. doi:10.1038/371257a0

    Article  PubMed  CAS  Google Scholar 

  11. Datto MB, Li Y, Panus JF, Howe DJ, Xiong Y, Wang XF (1995) Transforming growth factor β induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc Natl Acad Sci USA 92:5545–5549

    Article  PubMed  CAS  Google Scholar 

  12. Levy L, Hill CS (2006) Alterations in components of the TGF-β superfamily signaling pathways in human cancer. Cytokine Growth Factor Rev 17:41–58. doi:10.1016/j.cytogfr.2005.09.009

    Article  PubMed  CAS  Google Scholar 

  13. Gomis RR, Alarcon C, Nadal C, Van PC, Massagué J (2006) C/EBPβ at the core of the TGFβ cytostatic response and its evasion in metastatic breast cancer cells. Cancer Cell 10:203–214. doi:10.1016/j.ccr.2006.07.019

    Article  PubMed  CAS  Google Scholar 

  14. Wakefield LM, Piek E, Bottinger EP (2001) TGF-beta signaling in mammary gland development and tumorigenesis. J Mammary Gland Biol Neoplasia 6:67–82. doi:10.1023/A:1009568532177

    Article  PubMed  CAS  Google Scholar 

  15. Dumont N, Arteaga CL (2000) Transforming growth factor-β and breast cancer: tumor promoting effects of transforming growth factor-β. Breast Cancer Res 2:125–132. doi:10.1186/bcr44

    Article  PubMed  CAS  Google Scholar 

  16. ten Dijke P, Goumans MJ, Itoh F, Itoh S (2002) Regulation of cell proliferation by Smad proteins. J Cell Physiol 191:1–16. doi:10.1002/jcp.10066

    Article  PubMed  CAS  Google Scholar 

  17. Deckers M, van DM, Buijs J, Que I, Lowik C, van der Pluijm G, ten Dijke P (2006) The tumor suppressor Smad4 is required for transforming growth factor β-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. Cancer Res 66:2202–2209. doi:10.1158/0008-5472.CAN-05-3560

    Article  PubMed  CAS  Google Scholar 

  18. Viloria-Petit AM, David L, Jia JY, Erdemir T, Bane AL, Pinnaduwage D, Roncari L, Narimatsu M, Bose R, Moffat J, Wong JW, Kerbel RS, O’Malley FP, Andrulis IL, Wrana JL (2009) A role for the TGFβ-Par6 polarity pathway in breast cancer progression. Proc Natl Acad Sci USA 106:14028–14033. doi:10.1073/pnas.0906796106

    Article  PubMed  CAS  Google Scholar 

  19. Xu J, Lamouille S, Derynck R (2009) TGF-β-induced epithelial to mesenchymal transition. Cell Res 19:156–172. doi:10.1038/cr.2009.5

    Article  PubMed  CAS  Google Scholar 

  20. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S (1980) Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67–68. doi:10.1038/284067a0

    Article  PubMed  CAS  Google Scholar 

  21. Weigelt B, Peterse JL, van’t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602. doi:10.1038/nrc1670

    Article  PubMed  CAS  Google Scholar 

  22. Overall CM, Kleifeld O (2006) Tumour microenvironment—opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 6:227–239. doi:10.1038/nrc1821

    Article  PubMed  CAS  Google Scholar 

  23. Soule HD, Maloney TM, Wolman SR, Peterson WD Jr, Brenz R, McGrath CM, Russo J, Pauley RJ, Jones RF, Brooks SC (1990) Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50:6075–6086

    PubMed  CAS  Google Scholar 

  24. Strickland LB, Dawson PJ, Santner SJ, Miller FR (2000) Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers. Breast Cancer Res Treat 64:235–240. doi:10.1023/A:1026562720218

    Article  PubMed  CAS  Google Scholar 

  25. Santner SJ, Dawson PJ, Tait L, Soule HD, Eliason J, Mohamed AN, Wolman SR, Heppner GH, Miller FR (2001) Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat 65:101–110. doi:10.1023/A:1006461422273

    Article  PubMed  CAS  Google Scholar 

  26. Kim ES, Kim MS, Moon A (2004) TGF-β-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells. Int J Oncol 25:1375–1382

    PubMed  CAS  Google Scholar 

  27. Miyazono K (2009) Transforming growth factor-β signaling in epithelial-mesenchymal transition and progression of cancer. Proc Jpn Acad Ser B Phys Biol Sci 85:314–323. doi:10.2183/pjab.85.314

    Article  PubMed  CAS  Google Scholar 

  28. Smalley KS, Lioni M, Herlyn M (2006) Life isn’t flat: taking cancer biology to the next dimension. In Vitro Cell Dev Biol Anim 42:242–247. doi:10.1290/0604027.1

    Article  PubMed  CAS  Google Scholar 

  29. Lin RZ, Chang HY (2008) Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J 3:1172–1184. doi:10.1002/biot.200700228

    Article  PubMed  CAS  Google Scholar 

  30. Ohmori T, Yang JL, Price JO, Arteaga CL (1998) Blockade of tumor cell transforming growth factor-βs enhances cell cycle progression and sensitizes human breast carcinoma cells to cytotoxic chemotherapy. Exp Cell Res 245:350–359. doi:10.1006/excr.1998.4261

    Article  PubMed  CAS  Google Scholar 

  31. Graham CH, Kobayashi H, Stankiewicz KS, Man S, Kapitain SJ, Kerbel RS (1994) Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents. J Natl Cancer Inst 86:975–982

    Article  PubMed  CAS  Google Scholar 

  32. Petersen M, Pardali E, van der Horst G, Cheung H, van den Hoogen C, van der Pluijm G, ten Dijke P (2010) Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis. Oncogene 29:1351–1361. doi:10.1038/onc.2009.426

    Article  PubMed  CAS  Google Scholar 

  33. Tang B, Vu M, Booker T, Santner SJ, Miller FR, Anver MR, Wakefield LM (2003) TGF-β switches from tumor suppressor to prometastatic factor in a model of breast cancer progression. J Clin Invest 112:1116–1124. doi:10.1172/JCI18899

    PubMed  CAS  Google Scholar 

  34. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62:65–74. doi:10.1124/mol.62.1.65

    Article  PubMed  CAS  Google Scholar 

  35. Kang Y, He W, Tulley S, Gupta GP, Serganova I, Chen CR, Manova-Todorova K, Blasberg R, Gerald WL, Massagué J (2005) Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway. Proc Natl Acad Sci USA 102:13909–13914. doi:10.1073/pnas.0506517102

    Article  PubMed  CAS  Google Scholar 

  36. Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161–174. doi:10.1038/nrc745

    Article  PubMed  CAS  Google Scholar 

  37. van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415:530–536. doi:10.1038/415530a

    Article  Google Scholar 

  38. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massagué J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436:518–524. doi:10.1038/nature03799

    Article  PubMed  CAS  Google Scholar 

  39. Tamura Y, Watanabe F, Nakatani T, Yasui K, Fuji M, Komurasaki T, Tsuzuki H, Maekawa R, Yoshioka T, Kawada K, Sugita K, Ohtani M (1998) Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives. J Med Chem 41:640–649. doi:10.1021/jm9707582

    Article  PubMed  CAS  Google Scholar 

  40. Cowell JK, Laduca J, Rossi MR, Burkhardt T, Nowak NJ, Matsui S (2005) Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line. Cancer Genet Cytogenet 163:23–29. doi:10.1016/j.cancergencyto.2005.04.019

    Article  PubMed  CAS  Google Scholar 

  41. Kadota M, Yang HH, Gomez B, Sato M, Clifford RJ, Meerzaman D, Dunn BK, Wakefield LM, Lee MP (2010) Delineating genetic alterations for tumor progression in the MCF10A series of breast cancer cell lines. PLoS One 5:e9201. doi:10.1371/journal.pone.0009201

    Article  PubMed  Google Scholar 

  42. Provenzano PP, Inman DR, Eliceiri KW, Knittel JG, Yan L, Rueden CT, White JG, Keely PJ (2008) Collagen density promotes mammary tumor initiation and progression. BMC Med 6:11. doi:10.1186/1741-7015-6-11

    Article  PubMed  Google Scholar 

  43. Ramaswamy S, Ross KN, Lander ES, Golub TR (2003) A molecular signature of metastasis in primary solid tumors. Nat Genet 33:49–54. doi:10.1038/ng1060

    Article  PubMed  CAS  Google Scholar 

  44. Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ (1992) Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci USA 89:9064–9068

    Article  PubMed  CAS  Google Scholar 

  45. Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30:256–268. doi:10.1016/S1046-2023(03)00032-X

    Article  PubMed  CAS  Google Scholar 

  46. Li Q, Mullins SR, Sloane BF, Mattingly RR (2008) p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer. Neoplasia 10:314–329. doi:10.1593/neo.07970

    PubMed  CAS  Google Scholar 

  47. Li Q, Chow AB, Mattingly RR (2010) Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors. J Pharmacol Exp Ther 332:821–828. doi:10.1124/jpet.109.160390

    Article  PubMed  CAS  Google Scholar 

  48. Garamszegi N, Garamszegi SP, Samavarchi-Tehrani P, Walford E, Schneiderbauer MM, Wrana JL, Scully SP (2010) Extracellular matrix-induced transforming growth factor-beta receptor signaling dynamics. Oncogene 29:2368–2380. doi:10.1038/onc.2009.514

    Article  PubMed  CAS  Google Scholar 

  49. Incorvaia L, Badalamenti G, Rini G, Arcara C, Fricano S, Sferrazza C, Di TD, Gebbia N, Leto G (2007) MMP-2, MMP-9 and activin A blood levels in patients with breast cancer or prostate cancer metastatic to the bone. Anticancer Res 27:1519–1525

    PubMed  CAS  Google Scholar 

  50. Strizzi L, Postovit LM, Margaryan NV, Seftor EA, Abbott DE, Seftor RE, Salomon DS, Hendrix MJ (2008) Emerging roles of nodal and Cripto-1: from embryogenesis to breast cancer progression. Breast Dis 29:91–103

    PubMed  Google Scholar 

  51. Adkins HB, Bianco C, Schiffer SG, Rayhorn P, Zafari M, Cheung AE, Orozco O, Olson D, De LA, Chen LL, Miatkowski K, Benjamin C, Normanno N, Williams KP, Jarpe M, LePage D, Salomon D, Sanicola M (2003) Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo. J Clin Invest 112:575–587. doi:10.1172/JCI17788

    PubMed  CAS  Google Scholar 

  52. Dzwonek J, Preobrazhenska O, Cazzola S, Conidi A, Schellens A, van DM, Stubbs A, Klippel A, Huylebroeck D, ten Dijke P, Verschueren K (2009) Smad3 is a key nonredundant mediator of transforming growth factor β signaling in Nme mouse mammary epithelial cells. Mol Cancer Res 7:1342–1353. doi:10.1158/1541-7786.MCR-08-0558

    Article  PubMed  CAS  Google Scholar 

  53. Tian F, Byfield SD, Parks WT, Stuelten CH, Nemani D, Zhang YE, Roberts AB (2004) Smad-binding defective mutant of transforming growth factor β type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Cancer Res 64:4523–4530. doi:10.1158/0008-5472.CAN-04-0030

    Article  PubMed  CAS  Google Scholar 

  54. Tian F, DaCosta BS, Parks WT, Yoo S, Felici A, Tang B, Piek E, Wakefield LM, Roberts AB (2003) Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Cancer Res 63:8284–8292

    PubMed  CAS  Google Scholar 

  55. Stetler-Stevenson WG (1994) Progelatinase A activation during tumor cell invasion. Invasion Metastasis 14:259–268

    PubMed  CAS  Google Scholar 

  56. Coussens LM, Tinkle CL, Hanahan D, Werb Z (2000) MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103:481–490. doi:10.1016/S0092-8674(00)00139-2

    Article  PubMed  CAS  Google Scholar 

  57. Safina A, Vandette E, Bakin AV (2007) ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells. Oncogene 26:2407–2422. doi:10.1038/sj.onc.1210046

    Article  PubMed  CAS  Google Scholar 

  58. Tester AM, Waltham M, Oh SJ, Bae SN, Bills MM, Walker EC, Kern FG, Stetler-Stevenson WG, Lippman ME, Thompson EW (2004) Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice. Cancer Res 64:652–658

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank our colleagues, in particular Lukas Hawinkels, for valuable discussion and help during experiments. We are grateful to Niek Henriquez, Petra van Overveld, and Geertje van der Horst for their help to set up the invasion assay. We thank Ed Leof (Mayo Clinic, Minnesota, USA) and Ken Iwata (OSI Pharmaceuticals, New York, USA), for reagents; Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA) for the cell lines. This study was supported by the Dutch Cancer Society (RUL 2005-3371), European Union Framework 6 grant BRECOSM (5032240) and Tumor Host Genomics (518198), Centre for Biomedical Genetics, Swedish Cancerfonden (09 0773), and Ludwig Institute for Cancer Research.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands

    Eliza Wiercinska, Hildegonda P. H. Naber, Evangelia Pardali, Hans van Dam & Peter ten Dijke

  2. Department of Urology and Endocrinology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands

    Gabri van der Pluijm

  3. Ludwig Institute for Cancer Research and Uppsala University, Box 595, 75124, Uppsala, Sweden

    Peter ten Dijke

Authors
  1. Eliza Wiercinska
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hildegonda P. H. Naber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evangelia Pardali
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gabri van der Pluijm
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans van Dam
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter ten Dijke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter ten Dijke.

Additional information

Eliza Wiercinska, Hildegonda P. H. Naber authors contributed equally.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wiercinska, E., Naber, H.P.H., Pardali, E. et al. The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system. Breast Cancer Res Treat 128, 657–666 (2011). https://doi.org/10.1007/s10549-010-1147-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-010-1147-x

Keywords

Search

Navigation