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Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling

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Abstract

Although the epidermal growth factor receptor (EGFR) is frequently expressed in human primary breast carcinoma, the majority of breast cancer patients do not respond to treatment with EGFR tyrosine-kinase inhibitors such as gefitinib. We isolated through a stepwise dose escalation of the drug two gefitinib-resistant SK-Br-3 clones, ZD6 and ZD10 (ZD) cells, which showed, respectively, a three- to five-fold increase in the IC50 for gefitinib as compared with parental cells. The levels of expression of EGFR were increased in ZD cells as compared with wild-type SK-Br-3 cells. The phosphorylation of EGFR, ErbB-2, ErbB-3 and Akt was significantly reduced in gefitinib-resistant cells. In contrast, ZD cells showed levels of MAPK phosphorylation similar to untreated wild-type cells when cultured in presence of gefitinib. Persistent activation of MAPK was also observed in gefitinib-resistant clones isolated from MDA-MB-175 and MDA-MB-361 breast cancer cell lines. ZD cells showed an increased sensitivity to the MEK inhibitor PD98059 as compared with SK-Br-3 cells, and a synergistic anti-tumor effect was observed when ZD cells were treated with a combination of gefitinib and PD98059. Overexpression of a constitutively activated form of p42-MAPK in SK-Br-3 cells resulted in an approximately 50% increase in the IC50 to gefitinib. Finally, culture of ZD10 resistant cells in absence of gefitinib led to reversion of the resistant phenotype. These observations suggest that MAPK signaling might play a role in the resistance that develops in breast cancer cells after long-term exposure to gefitinib.

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References

  1. Normanno N, Bianco C, Strizzi L, Mancino M, Maiello MR, De Luca A, Caponigro F, Salomon DS (2005) The ErbB receptors and their ligands in cancer: an overview. Curr Drug Targets 6:243–257

    Article  PubMed  CAS  Google Scholar 

  2. Shankar V, Ciardiello F, Kim N, Derynck R, Liscia DS, Merlo G, Langton BC, Sheer D, Callahan R, Bassin RH et al (1989) Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA. Mol Carcinog 2:1–11

    Article  PubMed  CAS  Google Scholar 

  3. Ciardiello F, McGeady ML, Kim N, Basolo F, Hynes N, Langton BC, Yokozaki H, Saeki T, Elliott JW, Masui H et al (1990) Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation. Cell Growth Differ 1:407–420

    PubMed  CAS  Google Scholar 

  4. Brandt R, Eisenbrandt R, Leenders F, Zschiesche W, Binas B, Juergensen C, Theuring F (2000) Mammary gland specific hEGF receptor transgene expression induces neoplasia and inhibits differentiation. Oncogene 19:2129–2137

    Article  PubMed  CAS  Google Scholar 

  5. Salomon DS, Brandt R, Ciardiello F, Normanno N (1995) Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19:183–232

    Article  PubMed  CAS  Google Scholar 

  6. Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF, Arteaga CL (2001) Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 61:8887–8895

    PubMed  CAS  Google Scholar 

  7. Moasser MM, Basso A, Averbuch SD, Rosen N (2001) The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res 61:7184–7188

    PubMed  CAS  Google Scholar 

  8. Normanno N, Campiglio M, De LA, Somenzi G, Maiello M, Ciardiello F, Gianni L, Salomon DS, Menard S (2002) Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol 13:65–72

    Article  PubMed  CAS  Google Scholar 

  9. Anido J, Matar P, Albanell J, Guzman M, Rojo F, Arribas J, Averbuch S, Baselga J (2003) ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells. Clin Cancer Res 9:1274–1283

    PubMed  CAS  Google Scholar 

  10. Campiglio M, Locatelli A, Olgiati C, Normanno N, Somenzi G, Vigano L, Fumagalli M, Menard S, Gianni L (2004) Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (‘Iressa’) is independent of EGFR expression level. J Cell Physiol 198:259–268

    Article  PubMed  CAS  Google Scholar 

  11. Normanno N, De Luca A, Maiello MR, Mancino M, D’Antonio A, Macaluso M, Caponigro F, Giordano A (2005) Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in breast cancer: current status and future development. Front Biosci 10:2611–2617

    Article  PubMed  CAS  Google Scholar 

  12. Agrawal A, Gutteridge E, Gee JM, Nicholson RI, Robertson JF (2005) Overview of tyrosine kinase inhibitors in clinical breast cancer. Endocr Relat Cancer 12(Suppl 1):S135–S144

    Article  PubMed  CAS  Google Scholar 

  13. Polychronis A, Sinnett HD, Hadjiminas D, Singhal H, Mansi JL, Shivapatham D, Shousha S, Jiang J, Peston D, Barrett N, Vigushin D, Morrison K, Beresford E, Ali S, Slade MJ, Coombes RC (2005) Preoperative gefitinib versus gefitinib and anastrozole in postmenopausal patients with oestrogen-receptor positive and epidermal-growth-factor-receptor-positive primary breast cancer: a double-blind placebo-controlled phase II randomised trial. Lancet Oncol 6:383–391

    Article  PubMed  CAS  Google Scholar 

  14. Hutcheson IR, Knowlden JM, Jones HE, Burmi RS, McClelland RA, Barrow D, Gee JM, Nicholson RI (2006) Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy. Endocr Relat Cancer 13(Suppl 1):S89–S97

    Article  PubMed  CAS  Google Scholar 

  15. Normanno N, Maiello MR, Mancino M, De Luca A (2004) Small molecule epidermal growth factor receptor tyrosine kinase inhibitors: an overview. J Chemother 16(Suppl 4):36–40

    PubMed  CAS  Google Scholar 

  16. Bianco R, Shin I, Ritter CA, Yakes FM, Basso A, Rosen N, Tsurutani J, Dennis PA, Mills GB, Arteaga CL (2003) Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 22:2812–2822

    Article  PubMed  CAS  Google Scholar 

  17. She QB, Solit D, Basso A, Moasser MM (2003) Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3′-kinase/Akt pathway signaling. Clin Cancer Res 9:4340–4346

    PubMed  CAS  Google Scholar 

  18. Jones HE, Goddard L, Gee JM, Hiscox S, Rubini M, Barrow D, Knowlden JM, Williams S, Wakeling AE, Nicholson RI (2004) Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells. Endocr Relat Cancer 11:793–814

    Article  PubMed  CAS  Google Scholar 

  19. Normanno N, De Luca A, Maiello MR, Campiglio M, Napolitano M, Mancino M, Carotenuto A, Viglietto G, Menard S (2006) The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J Cell Physiol 207:420–427

    Article  PubMed  CAS  Google Scholar 

  20. Maiello MR, D’Alessio A, De Luca A, Carotenuto A, Rachiglio AM, Napolitano M, Cito L, Guzzo A, Normanno N (2007) AZD3409 inhibits the growth of breast cancer cells with intrinsic resistance to the EGFR tyrosine kinase inhibitor gefitinib. Breast Cancer Res Treat 102:275–282

    Article  PubMed  CAS  Google Scholar 

  21. Normanno N, Bianco C, De Luca A, Maiello MR, Salomon DS (2003) Target-based agents against ErbB receptors and their ligands: a novel approach to cancer treatment. Endocr Relat Cancer 10:1–21

    Article  PubMed  CAS  Google Scholar 

  22. Baselga J, Albanell J, Ruiz A, Lluch A, Gascon P, Guillem V, Gonzalez S, Sauleda S, Marimon I, Tabernero JM, Koehler MT, Rojo F (2005) Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol 23:5323–5333

    Article  PubMed  CAS  Google Scholar 

  23. Albain KSER, Gradishar WJ, Hayes DF, Rowinsky E, Hudis C, Pusztai L, Tripathy D, Modi S, Rubi S (2002) Open-label, phase II, multicenter trial of ZD1839 (‘Iressa’) in patients with advanced breast cancer. Breast Cancer Res Treat 76:S33

    Google Scholar 

  24. Sergina NV, Rausch M, Wang D, Blair J, Hann B, Shokat KM, Moasser MM (2007) Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 445:437–441

    Article  PubMed  CAS  Google Scholar 

  25. von Minckwitz G, Jonat W, Fasching P, du Bois A, Kleeberg U, Luck HJ, Kettner E, Hilfrich J, Eiermann W, Torode J, Schneeweiss A (2005) A multicentre phase II study on gefitinib in taxane- and anthracycline-pretreated metastatic breast cancer. Breast Cancer Res Treat 89:165–172

    Article  CAS  Google Scholar 

  26. Robinson MJ, Stippec SA, Goldsmith E, White MA, Cobb MH (1998) A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr Biol 8:1141–1150

    Article  PubMed  CAS  Google Scholar 

  27. Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55

    Article  PubMed  CAS  Google Scholar 

  28. Normanno N, De Luca A, Maiello MR, Bianco C, Mancino M, Strizzi L, Arra C, Ciardiello F, Agrawal S, Salomon DS (2004) CRIPTO-1: a novel target for therapeutic intervention in human carcinoma. Int J Oncol 25:1013–1020

    PubMed  CAS  Google Scholar 

  29. Janmaat ML, Kruyt FA, Rodriguez JA, Giaccone G (2003) Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin Cancer Res 9:2316–2326

    PubMed  CAS  Google Scholar 

  30. Magne N, Fischel JL, Dubreuil A, Formento P, Poupon MF, Laurent-Puig P, Milano G (2002) Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 (“Iressa”). Br J Cancer 86:1518–1523

    Article  PubMed  CAS  Google Scholar 

  31. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, Barrow D, Wakeling AE, Nicholson RI (2003) Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 144:1032–1044

    Article  PubMed  CAS  Google Scholar 

  32. Yamasaki F, Johansen MJ, Zhang D, Krishnamurthy S, Felix E, Bartholomeusz C, Aguilar RJ, Kurisu K, Mills GB, Hortobagyi GN, Ueno NT (2007) Acquired resistance to erlotinib in A-431 epidermoid cancer cells requires down-regulation of MMAC1/PTEN and up-regulation of phosphorylated Akt. Cancer Res 67:5779–5788

    Article  PubMed  CAS  Google Scholar 

  33. Xia W, Bacus S, Hegde P, Husain I, Strum J, Liu L, Paulazzo G, Lyass L, Trusk P, Hill J, Harris J, Spector NL (2006) A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer. Proc Natl Acad Sci USA 103:7795–7800

    Article  PubMed  CAS  Google Scholar 

  34. Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, McClelland RA, Jordan N, Wakeling AE, Nicholson RI (2003) The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro. Endocrinology 144:5105–5117

    Article  PubMed  CAS  Google Scholar 

  35. Mellinghoff IK, Wang MY, Vivanco I, Haas-Kogan DA, Zhu S, Dia EQ, Lu KV, Yoshimoto K, Huang JH, Chute DJ, Riggs BL, Horvath S, Liau LM, Cavenee WK, Rao PN, Beroukhim R, Peck TC, Lee JC, Sellers WR, Stokoe D, Prados M, Cloughesy TF, Sawyers CL, Mischel PS (2005) Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 353:2012–2024

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) e Ministero della Salute to N. Normanno. A. De Luca was supported by a fellowship form AIRC. The Authors thank Mr. O. Michele Grassi for technical assistance.

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Authors and Affiliations

  1. Cell Biology and Preclinical Models Unit, INT-Fondazione Pascale, 80131, Naples, Italy

    Nicola Normanno, Monica R. Maiello, Antonella De Luca, Mario Mancino, Marianna Gallo & Amelia D’Alessio

  2. Centro di Ricerche Oncologiche di Mercogliano (CROM), 83013, Mercogliano, AV, Italy

    Nicola Normanno, Monica R. Maiello & Antonella De Luca

  3. Molecular Targeting Unit, Istituto Tumori Milano, 20133, Milan, Italy

    Manuela Campiglio & Sylvie Menard

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  1. Nicola Normanno
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Correspondence to Nicola Normanno.

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Normanno, N., Campiglio, M., Maiello, M.R. et al. Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling. Breast Cancer Res Treat 112, 25–33 (2008). https://doi.org/10.1007/s10549-007-9830-2

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