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. 2001 Apr;84(7):936–945. doi: 10.1054/bjoc.2000.1678

Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

M Silvy 1, C Giusti 1, P-M Martin 1, Y Berthois 1
PMCID: PMC2363843  PMID: 11286474

Abstract

Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10−8M EGF stimulated AR expression in HMECs to 140–300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5–10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. © 2001 Cancer Research Campaign http://www.bjcancer.com

Keywords: amphiregulin, epidermal growth factor, proteases, breast cancer cells

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Selected References

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  1. Andreasen P. A., Kjøller L., Christensen L., Duffy M. J. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 1997 Jul 3;72(1):1–22. doi: 10.1002/(sici)1097-0215(19970703)72:1<1::aid-ijc1>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  2. Bouchet C., Spyratos F., Martin P. M., Hacène K., Gentile A., Oglobine J. Prognostic value of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas. Br J Cancer. 1994 Feb;69(2):398–405. doi: 10.1038/bjc.1994.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ciardiello F., Kim N., Liscia D. S., Bianco C., Lidereau R., Merlo G., Callahan R., Greiner J., Szpak C., Kidwell W. mRNA expression of transforming growth factor alpha in human breast carcinomas and its activity in effusions of breast cancer patients. J Natl Cancer Inst. 1989 Aug 2;81(15):1165–1171. doi: 10.1093/jnci/81.15.1165. [DOI] [PubMed] [Google Scholar]
  4. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  5. Foekens J. A., Schmitt M., van Putten W. L., Peters H. A., Bontenbal M., Jänicke F., Klijn J. G. Prognostic value of urokinase-type plasminogen activator in 671 primary breast cancer patients. Cancer Res. 1992 Nov 1;52(21):6101–6105. [PubMed] [Google Scholar]
  6. Fontanini G., De Laurentiis M., Vignati S., Chinè S., Lucchi M., Silvestri V., Mussi A., De Placido S., Tortora G., Bianco A. R. Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clin Cancer Res. 1998 Jan;4(1):241–249. [PubMed] [Google Scholar]
  7. Fry C. J., Farnham P. J. Context-dependent transcriptional regulation. J Biol Chem. 1999 Oct 15;274(42):29583–29586. doi: 10.1074/jbc.274.42.29583. [DOI] [PubMed] [Google Scholar]
  8. Higashiyama S., Abraham J. A., Miller J., Fiddes J. C., Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991 Feb 22;251(4996):936–939. doi: 10.1126/science.1840698. [DOI] [PubMed] [Google Scholar]
  9. Johnson G. R., Kannan B., Shoyab M., Stromberg K. Amphiregulin induces tyrosine phosphorylation of the epidermal growth factor receptor and p185erbB2. Evidence that amphiregulin acts exclusively through the epidermal growth factor receptor at the surface of human epithelial cells. J Biol Chem. 1993 Feb 5;268(4):2924–2931. [PubMed] [Google Scholar]
  10. Johnson G. R., Prigent S. A., Gullick W. J., Stromberg K. Characterization of high and low molecular weight forms of amphiregulin that differ in glycosylation and peptide core length. Evidence that the NH2-terminal region is not critical for bioactivity. J Biol Chem. 1993 Sep 5;268(25):18835–18843. [PubMed] [Google Scholar]
  11. Johnson G. R., Saeki T., Gordon A. W., Shoyab M., Salomon D. S., Stromberg K. Autocrine action of amphiregulin in a colon carcinoma cell line and immunocytochemical localization of amphiregulin in human colon. J Cell Biol. 1992 Aug;118(3):741–751. doi: 10.1083/jcb.118.3.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kenney N. J., Huang R. P., Johnson G. R., Wu J. X., Okamura D., Matheny W., Kordon E., Gullick W. J., Plowman G., Smith G. H. Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland. Mol Reprod Dev. 1995 Jul;41(3):277–286. doi: 10.1002/mrd.1080410302. [DOI] [PubMed] [Google Scholar]
  13. Kenney N. J., Smith G. H., Rosenberg K., Cutler M. L., Dickson R. B. Induction of ductal morphogenesis and lobular hyperplasia by amphiregulin in the mouse mammary gland. Cell Growth Differ. 1996 Dec;7(12):1769–1781. [PubMed] [Google Scholar]
  14. Kondapaka S. B., Fridman R., Reddy K. B. Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells. Int J Cancer. 1997 Mar 17;70(6):722–726. doi: 10.1002/(sici)1097-0215(19970317)70:6<722::aid-ijc15>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
  15. LeJeune S., Leek R., Horak E., Plowman G., Greenall M., Harris A. L. Amphiregulin, epidermal growth factor receptor, and estrogen receptor expression in human primary breast cancer. Cancer Res. 1993 Aug 1;53(15):3597–3602. [PubMed] [Google Scholar]
  16. Li S., Plowman G. D., Buckley S. D., Shipley G. D. Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells. J Cell Physiol. 1992 Oct;153(1):103–111. doi: 10.1002/jcp.1041530114. [DOI] [PubMed] [Google Scholar]
  17. Ma L., Gauvillé C., Berthois Y., Millot G., Johnson G. R., Calvo F. Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line. Oncogene. 1999 Nov 11;18(47):6513–6520. doi: 10.1038/sj.onc.1203042. [DOI] [PubMed] [Google Scholar]
  18. Martínez-Lacaci I., Saceda M., Plowman G. D., Johnson G. R., Normanno N., Salomon D. S., Dickson R. B. Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell lines. Endocrinology. 1995 Sep;136(9):3983–3992. doi: 10.1210/endo.136.9.7649107. [DOI] [PubMed] [Google Scholar]
  19. Normanno N., Ciardiello F., Brandt R., Salomon D. S. Epidermal growth factor-related peptides in the pathogenesis of human breast cancer. Breast Cancer Res Treat. 1994 Jan;29(1):11–27. doi: 10.1007/BF00666178. [DOI] [PubMed] [Google Scholar]
  20. Normanno N., Selvam M. P., Bianco C., Damiano V., de Angelis E., Grassi M., Magliulo G., Tortora G., Salomon D. S., Ciardiello F. Amphiregulin anti-sense oligodeoxynucleotides inhibit growth and transformation of a human colon carcinoma cell line. Int J Cancer. 1995 Sep 15;62(6):762–766. doi: 10.1002/ijc.2910620619. [DOI] [PubMed] [Google Scholar]
  21. Normanno N., Selvam M. P., Qi C. F., Saeki T., Johnson G., Kim N., Ciardiello F., Shoyab M., Plowman G., Brandt R. Amphiregulin as an autocrine growth factor for c-Ha-ras- and c-erbB-2-transformed human mammary epithelial cells. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2790–2794. doi: 10.1073/pnas.91.7.2790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Panico L., D'Antonio A., Salvatore G., Mezza E., Tortora G., De Laurentiis M., De Placido S., Giordano T., Merino M., Salomon D. S. Differential immunohistochemical detection of transforming growth factor alpha, amphiregulin and CRIPTO in human normal and malignant breast tissues. Int J Cancer. 1996 Jan 3;65(1):51–56. doi: 10.1002/(SICI)1097-0215(19960103)65:1<51::AID-IJC9>3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]
  23. Qi C. F., Liscia D. S., Normanno N., Merlo G., Johnson G. R., Gullick W. J., Ciardiello F., Saeki T., Brandt R., Kim N. Expression of transforming growth factor alpha, amphiregulin and cripto-1 in human breast carcinomas. Br J Cancer. 1994 May;69(5):903–910. doi: 10.1038/bjc.1994.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Riese D. J., Kim E. D., Elenius K., Buckley S., Klagsbrun M., Plowman G. D., Stern D. F. The epidermal growth factor receptor couples transforming growth factor-alpha, heparin-binding epidermal growth factor-like factor, and amphiregulin to Neu, ErbB-3, and ErbB-4. J Biol Chem. 1996 Aug 16;271(33):20047–20052. doi: 10.1074/jbc.271.33.20047. [DOI] [PubMed] [Google Scholar]
  25. Rosenthal E. L., Johnson T. M., Allen E. D., Apel I. J., Punturieri A., Weiss S. J. Role of the plasminogen activator and matrix metalloproteinase systems in epidermal growth factor- and scatter factor-stimulated invasion of carcinoma cells. Cancer Res. 1998 Nov 15;58(22):5221–5230. [PubMed] [Google Scholar]
  26. Salomon D. S., Normanno N., Ciardiello F., Brandt R., Shoyab M., Todaro G. J. The role of amphiregulin in breast cancer. Breast Cancer Res Treat. 1995;33(2):103–114. doi: 10.1007/BF00682718. [DOI] [PubMed] [Google Scholar]
  27. Sehgal I., Bailey J., Hitzemann K., Pittelkow M. R., Maihle N. J. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells. Mol Biol Cell. 1994 Mar;5(3):339–347. doi: 10.1091/mbc.5.3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shoyab M., McDonald V. L., Bradley J. G., Todaro G. J. Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6528–6532. doi: 10.1073/pnas.85.17.6528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Silvy M., Martin P. M., Chajry N., Berthois Y. Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation. Endocrinology. 1998 May;139(5):2382–2391. doi: 10.1210/endo.139.5.5981. [DOI] [PubMed] [Google Scholar]
  30. Souttou B., Hamelin R., Crépin M. FGF2 as an autocrine growth factor for immortal human breast epithelial cells. Cell Growth Differ. 1994 Jun;5(6):615–623. [PubMed] [Google Scholar]
  31. Sundareshan P., Nagle R. B., Bowden G. T. EGF induces the expression of matrilysin in the human prostate adenocarcinoma cell line, LNCaP. Prostate. 1999 Aug 1;40(3):159–166. doi: 10.1002/(sici)1097-0045(19990801)40:3<159::aid-pros3>3.0.co;2-w. [DOI] [PubMed] [Google Scholar]
  32. Visscher D. W., Sarkar F. H., Kasunic T. C., Reddy K. B. Clinicopathologic analysis of amphiregulin and heregulin immunostaining in breast neoplasia. Breast Cancer Res Treat. 1997 Aug;45(1):75–80. doi: 10.1023/a:1005845512804. [DOI] [PubMed] [Google Scholar]

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