Breast Cancer Research and Treatment

, Volume 81, Issue 1, pp 81–93 | Cite as

Oestrogen Receptor-Mediated Modulation of the EGFR/MAPK Pathway in Tamoxifen-Resistant MCF-7 Cells

  • Iain R. Hutcheson
  • Janice M. Knowlden
  • Tracie-Ann Madden
  • Denise Barrow
  • Julia M.W. Gee
  • Alan E. Wakeling
  • Robert I. Nicholson


Oestrogen receptor (ER) levels are usually maintained on acquisition of tamoxifen resistance in the clinic, however, tumour re-growth is associated with increased expression of epidermal growth factor receptor (EGFR) and activation of the mitogen activated protein kinase (MAPK) pathway. In the present study we have used the ER down-regulator fulvestrant ('Faslodex') to investigate the influence of the ER on growth of a tamoxifen-resistant (TAM-R) human breast cancer cell line. Expression levels of ER mRNA and protein were equivalent in parental wild-type MCF-7 (WT) and TAM-R cells. Fulvestrant eliminated ER protein expression and inhibited proliferation in both cell lines. The growth inhibitory effects of fulvestrant were associated with a decrease in basal EGFR, c-erbB2 and ERK1/2 activity in TAM-R but not WT cells. ER functionality as determined by oestrogen response element (ERE)-luciferase reporter activity and expression of PgR, pS2 and transforming growth factor alpha (TGFα) was significantly reduced in TAM-R compared to WT cells and was further decreased by fulvestrant treatment in both cell lines. Epidermal growth factor (EGF) and TGFα significantly increased EGFR/MAPK pathway activity in both cell lines. Ligand-induced EGFR/MAPK activation promoted TAM-R cell growth in both the absence and presence of fulvestrant, whereas no proliferative activity was observed under the same conditions in WT cells. These results suggest that the ER modulates EGFR/MAPK signalling efficiency in TAM-R cells possibly through the regulation of TGFα availability. This effect may be overcome by the action of exogenous EGFR ligands, which strengthen EGFR/MAPK signalling activity to generate endocrine-insensitive cell growth.

c-erbB2 epidermal growth factor receptor fulvestrant mitogen-activated protein kinase oestrogen receptor tamoxifen-resistance transforming growth factor alpha 


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  1. 1.
    Howell A, Defriend D, Anderson E: Mechanisms of response and resistance to endocrine therapy for breast cancer and the development of new treatments. Rev Endocr-Related Cancer 43: 5–21, 1993Google Scholar
  2. 2.
    Osborne CK, Fuqua SA:Mechanisms of tamoxifen resistance. Breast Cancer Res Treat 32: 49–55, 1994Google Scholar
  3. 3.
    Clarke R, Leonessa F, Welch JN, Skaar TC: Cellular and molecular pharmacology of antiestrogen action and resistance. Pharmacol Rev 53: 25–71, 2001Google Scholar
  4. 4.
    Wright C, Nicholson S, Angus B, Sainsbury JR, Farndon J, Cairns J, Harris AL, Horne CH: Relationship between cerbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br J Cancer 65: 118–121, 1992Google Scholar
  5. 5.
    Nicholson RI, McClelland RA, Finlay P, Eaton CL, Gullick WJ, Dixon AR, Robertson JF, Ellis IO, Blamey RW: Relationship between EGF-R, c-erbB2 protein expression and Ki67 immunostaining in breast cancer and hormone sensitivity. Eur J Cancer 29A: 1018–1023, 1993Google Scholar
  6. 6.
    Nicholson RI, McClelland RA, Gee JMW, Manning DL, Cannon P, Robertson JF, Ellis IO, Blamey RW: Epidermal growth factor receptor expression in breast cancer: Association with response to endocrine therapy. Breast Cancer Res Treat 29: 117–125, 1994Google Scholar
  7. 7.
    Daly RJ: Take your partners, please-signal diversification by the erbB family of receptor tyrosine kinases. Growth Factors 16: 255–263, 1999Google Scholar
  8. 8.
    Olayioye MA, Neve RM, Lane HA, Hynes N: The erbB signaling network: receptor heterodimerization in development and cancer. EMBO J 19: 3159–3167, 2000Google Scholar
  9. 9.
    Long B, McKibben BM, Lynch M, van den Berg HW: Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line. Br J Cancer 65: 865–869, 1992Google Scholar
  10. 10.
    El-Zarruk AA, van den Berg HW: The anti-proliferative effects of tyrosine kinase inhibitors towards tamoxifen-sensitive and tamoxifen-resistant human breast cancer cell lines in relation to the expression of epidermal growth factor receptors (EGF-R) and the inhibition of EGF-R tyrosine kinase. Cancer Lett 142: 185–193, 1999Google Scholar
  11. 11.
    van Agthoven TT, van Agthoven TL, Portengen H, Foekens JA, Dorssers LC: Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells. Cancer Res 52: 5082–5088, 1992Google Scholar
  12. 12.
    Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK: Estrogen-dependent tamoxifen-resistant tumorigenic growth of MCF7 cells transfected with HER2/neu. Breast Cancer Res Treat 24: 85–95, 1992Google Scholar
  13. 13.
    Miller DL, El-Ashry D, Cheville AL, Liu Y, McLeskey SW, Kern FG: Emergence of MCF7 cells overexpressing a transfected epidermal growth factor receptor (EGFR) under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression. Cell Growth Diff 5: 1263–1274, 1994Google Scholar
  14. 14.
    Liu Y, El-Ashry D, Chen D, Ding IY, Kern FG: MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo. Breast Cancer Res Treat 34: 97–117, 1995Google Scholar
  15. 15.
    Kurokawa H, Lenferink AEG, Simpson JF, Piscane PI, Sliwkowski MX, Forbes JT, Arteaga CL: Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res 60: 5887–5894, 2000Google Scholar
  16. 16.
    Schlessinger J: Cell Signaling by receptor tyrosine kinases. Cell 103: 211–225, 2000Google Scholar
  17. 17.
    Mueller H, Flury N, Eppenberger-Castori S, Kueng W, David F, Eppenberger U: Potential prognostic value of mitogenactivated protein kinase activity for disease-free survival of primary breast cancer patients. Int J Cancer (Pred Oncol) 89: 384–388, 2000Google Scholar
  18. 18.
    Gee JMW, Robertson JFR, Ellis IO, Nicholson RI: Phosphorylation of ERK1/2 mitogen activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer. Int J Cancer 95: 247–254, 2001Google Scholar
  19. 19.
    Coutts AS, Murphy LC: Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells. Cancer Res 58: 4071–4074, 1998Google Scholar
  20. 20.
    Shim W-S, Conaway M, Masamura S, Yue W, Wang J-P, Kumar R, Santen RJ: Estradiol hypersensitivity and mitogenactivated protein kinase expression in long-term estrogen deprived human breast cancer cells in vivo. Endocrinol 141: 396–405, 2000Google Scholar
  21. 21.
    El-Ashry D, Miller DL, Kharbanda S, Lippman ME, Kern FG: Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis. Oncogene 15: 423–435, 1997Google Scholar
  22. 22.
    Donovan JCH, Milic A, Slingerland JM: Constitutive MEK/MAPK activation leads to p27Kip1 deregulation and antiestrogen resistance in human breast cancer cells. J Biol Chem 276: 40888–40895, 1997Google Scholar
  23. 23.
    McClelland RA, Barrow D, Madden T, Dutkowski CM, Pamment J, Knowlden J, Gee JMW, Nicholson RI: Enhanced epidermal growth factor receptor signalling in MCF7 breast cancer cells following long-term culture in the presence of the pure antioestrogen ICI 182,780 (Faslodex). Endocrinol 142: 2776–2788, 2001Google Scholar
  24. 24.
    Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JMW, Harper ME, Barrow D, Wakeling AE, Nicholson RI: Elevated Levels of Epidermal Growth Factor Receptor/c-erbB2 Heterodimers Mediate an Autocrine Growth Regulatory Pathway in Tamoxifen-Resistant MCF-7 Cells. Endocrinol 144: 1032–1044, 2003Google Scholar
  25. 25.
    Encarnacion CA, Ciocca DR, McGuire WL, Clark GM, Fuqua SAW, Osborne CK: Measurement of steroid hormone receptors in breast cancer patients on tamoxifen. Breast Cancer Res Treat 26: 237–246, 1993Google Scholar
  26. 26.
    Brunner N, Frandesen TL, Holst-Hansen C, Bei M, Thompson EW, Wakeling AE, Lippman ME, Clarke R: MCF7/LCC2: a 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182,780. Cancer Res 53: 3229–3232, 1993Google Scholar
  27. 27.
    Lykkesfeldt AE, Mogens MW, Briand P: Altered expression of estrogen-regulated genes in a tamoxifen-resistant and ICI 164,384 and ICI 182,780 sensitive human breast cancer cell line, MCF-7/TAM R-1. Cancer Res 54: 1587–1595, 1994Google Scholar
  28. 28.
    Robertson JF: Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer 73: 5–12, 1996Google Scholar
  29. 29.
    Dauvois S, Danielian PS, White R, Parker MG: Antiestrogen ICI164384 reduces cellular oestrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89: 4037–4041, 1992Google Scholar
  30. 30.
    Howell A, Osborne K, Wakeling A: Faslodex (ICI 182,780). Development of a novel ‘pure’ antiestrogen. Cancer 89: 817–825, 2000Google Scholar
  31. 31.
    Wakeling AE: Similarities and distinctions in the mode of action of different classes of antiestrogens. Endocr-Related Cancer 7: 17–28, 2000Google Scholar
  32. 32.
    Howell A, Robertson J: Response to a specific antioestrogen (ICI 182780) in tamoxifen-resistant breast cancer. Lancet 345: 989–990, 1995Google Scholar
  33. 33.
    Howell A, DeFriend DJ, Robertson JF, Blamey RW, Anderson L, Anderson E, Sutcliffe FA, Walton P: Pharmacokinetics, pharmacological and anti-tumor effects of the specific antiestrogen ICI 182,780 in women with advanced breast cancer. Br J Cancer 74: 300–308, 1996Google Scholar
  34. 34.
    Howell A, Robertson JF, Quaresma Albano J, Aschermannova A, Mauriac L, Kleeberg UR, Vergote I, Erikstein B, Webster A, Morris C: Fulvestrant, formerly ICI 182,780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment. J Clin Oncol 20: 3396–3403, 2002Google Scholar
  35. 35.
    Coopman P, Garcia M, Brunner N, Derocq D, Clarke R, Rochefort H: Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifenOestrogen receptor and tamoxifen resistance 93 resistant human breast-cancer cells. Int J Cancer 56: 295–300, 1994Google Scholar
  36. 36.
    Hu XF, Veroni M, De Luise M, Wakeling A, Sutherland R, Watts CK, Zalcberg JR: Circumvention of tamoxifen resistance by the pure anti-estrogen ICI 182,780. Int J Cancer 55: 873–876, 1993Google Scholar
  37. 37.
    Knowlden JM, Gee JMW, Bryant S, McClelland RA, Manning DL, Mansel R, Ellis IO, Blamey RW, Robertson JF, Nicholson RI: Use of reverse transcription-polymerase chain reaction methodology to detect estrogen-regulated gene expression in small breast cancer specimens. Clin Cancer Res 3: 2165–2172,1997Google Scholar
  38. 38.
    Gee JMW, Robertson JF, Ellis IO, Willsher P, McClelland RA, Hoyle HB, Kyme SR, Finlay P, Blamey RW, Nicholson RI: Immunocytochemical localization of BCL-2 protein in human breast cancers and its relationship to a series of prognostic markers and response to endocrine therapy. Int J Cancer 59: 619–628, 1994Google Scholar
  39. 39.
    Johnston SR, Lu B, Dowsett M, Liang X, Kaufmann M, Scott GK, Osborne CK, Benz CC: Comparison of estrogen receptor DNA binding in untreated and acquired antiestrogenresistant human breast tumors. Cancer Res 57: 3723–3727, 1997Google Scholar
  40. 40.
    Katzenellenbogen BS, Montano MM, Ekena K, Herman ME, Melnerney EM: William M. McGuire Memorial Lecture. Antiestrogens: mechanisms of action and resistance in breast cancer. Breast Cancer Res Treat 44: 23–38, 1997Google Scholar
  41. 41.
    Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P: Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270: 1491–1494, 1995Google Scholar
  42. 42.
    Arnold SF, Obourn JD, Jaffe H, Notides AC: Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro. Mol Endocrinol 9: 24–33, 1995Google Scholar
  43. 43.
    Bunone G, Briand P-A, Miksicek RJ, Picard D: Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15: 2174–2183, 1996Google Scholar
  44. 44.
    Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P: Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74: 311–317, 2000Google Scholar
  45. 45.
    Webb P, Lopez GN, Uht RM, Kushner PJ: Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9: 443–456, 1995Google Scholar
  46. 46.
    Dumont JA, Bitonti AJ, Wallace CD, Baumann RJ, Cashman EA, Cross-Doersen DE: Progression of MCF7 breast cancer cells to antiestrogen-resistant phenotype is accompanied by elevated levels of AP-1 DNA-binding activity. Cell Growth Diff 7: 351–359, 1996Google Scholar
  47. 47.
    Johnston SRD, Lu B, Scott GK, Kushner PJ, Smith IE, Dowsett M, Benz CC: Increased activator protein-1 DNA binding and c-Jun NH2-terminal kinase activity in human breast tumours with acquired tamoxifen resistance. Clin Cancer Res 5: 251–256, 1999Google Scholar
  48. 48.
    Porter W, Saville B, Hoivik D, Safe S: Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11: 1569–1580, 1997Google Scholar
  49. 49.
    McPherson LA, Weigel RJ: AP2α and AP2γ: a comparison of binding site specificity and trans-activation of the estrogen receptor promoter and single site constructs. Nucl Acid Res 27: 4040–4049, 1999Google Scholar
  50. 50.
    Nicholson RI, McClelland RA, Robertson JFR, Gee JMW: Involvement of steroid hormone and growth factor cross-talk in endocrine response in breast cancer. Endocr-Related Cancer 6: 373–387, 1999Google Scholar
  51. 51.
    Nicholson RI, Gee JMW: Oestrogen and growth factor crosstalk and endocrine insensitivity and acquired resistance in breast cancer. Br J Cancer 82: 501–513, 2000Google Scholar
  52. 52.
    Berthois Y, Dong XF, Martin PM: Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7. Biochem Biophys Res Commun 159: 126–131, 1989Google Scholar
  53. 53.
    Read LD, Keith Jr D, Slamon DJ, Katzenellenbogen BS: Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines. Cancer Res 50: 3947–3951, 1990Google Scholar
  54. 54.
    De Bortoli M, Dati C, Antoniotti S, Maggiora P, Sapei ML: Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells. J Steroid Biochem Mol Biol 43: 21–25, 1992Google Scholar
  55. 55.
    Bates SE, Davidson NE, Valverius EM, Freter CE, Dickson RB, Tam JP, Kudlow JE, Lippman ME, Salomon DS: Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance. Mol Endocrinol 2: 543–555, 1988Google Scholar
  56. 56.
    Saeki T, Cristiano A, Lynch MJ, Brattain M, Kim N, Normanno N, Kenney N, Ciardiello F, Salomon DS: Regulation by estrogen through the 5-flanking region of the transforming growth factor alpha gene. Mol Endocrinol 5: 1955–1963, 1991Google Scholar
  57. 57.
    MacGregor Schafer J, Liu H, Levenson AS, Horiguchi J, Chen Z, Jordan VC: Estrogen receptor alpha mediated induction of the transforming growth factor alpha gene by estradiol and 4-hydroxytamoxifen inMDA-MB-231 breast cancer cells. J Steroid Biochem Mol Biol 78: 41–50, 2001Google Scholar
  58. 58.
    Kumar V, Bustin SA, McKay IA: Transforming growth factor alpha. Cell Biol Int 19: 373–388, 1995Google Scholar
  59. 59.
    El-Ashry D, Chrysogelos SA, Lippman ME, Kern FG: Estrogen induction of TGF-alpha is mediated by an estrogen response element composed of two imperfect palindromes. J Steroid Biochem Mol Biol 59: 261–269, 1996Google Scholar
  60. 60.
    Wang D, Shin TH, Kudlow JE: Transcription factor AP-2 controls transcription of the human transforming growth factor-alpha gene. J Biol Chem 272: 14244–14250, 1997Google Scholar
  61. 61.
    Bjorge JD, Paterson AJ, Kudlow JE: Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in breast cancer cell line. J Biol Chem 264: 4021–4027, 1989Google Scholar
  62. 62.
    Angel P, Karin M: The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129–157, 1991Google Scholar
  63. 63.
    Nicholson RI, McClelland RA, Gee JMW, Manning DL, Cannon P, Robertson JF, Ellis IO, Blamey RW: Transforming growth factor-alpha and endocrine sensitivity in breast cancer. Cancer Res 54: 1684–1689, 1994Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Iain R. Hutcheson
    • 1
  • Janice M. Knowlden
    • 1
  • Tracie-Ann Madden
    • 1
  • Denise Barrow
    • 1
  • Julia M.W. Gee
    • 1
  • Alan E. Wakeling
    • 2
  • Robert I. Nicholson
    • 1
  1. 1.Tenovus Centre for Cancer Research, Welsh School of PharmacyCardiff UniversityCardiffUK
  2. 2.AstraZeneca PharmaceuticalsCheshireUK

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