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Investigational New Drugs

, Volume 31, Issue 2, pp 293–303 | Cite as

Mechanisms of acquired resistance to insulin-like growth factor 1 receptor inhibitor in MCF-7 breast cancer cell line

  • Roudy Chiminch Ekyalongo
  • Toru MukoharaEmail author
  • Yu Kataoka
  • Yohei Funakoshi
  • Hideo Tomioka
  • Naomi Kiyota
  • Yutaka Fujiwara
  • Hironobu Minami
PRECLINICAL STUDIES

Summary

The purpose of this study was to clarify the mechanism of acquired resistance to the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor NVP-AEW541. We developed an acquired resistant model by continuously exposing MCF-7 breast cancer cells to NVP-AEW541 (MCF-7-NR). MCF-7 and MCF-7-NR were comparatively analyzed for cell signaling and cell growth. While phosphorylation of Akt was completely inhibited by 3 μM NVP-AEW541 in both MCF-7 and MCF-7-NR, phosphorylation of S6K remained high only in MCF-7-NR, suggesting a disconnection between Akt and S6K in MCF-7-NR. Consistently, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of both lines for phosphorylation of 42 receptor tyrosine kinases with and without NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR. Protein expression of Tyro3 was found to be higher in MCF-7-NR than in MCF-7. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth and cyclin D1 expression in both lines. While Tyro3 expression was inhibited by NVP-AEW541 and everolimus in MCF-7, it was reduced only by everolimus in MCF-7-NR. These findings suggested that cyclin D1 expression was regulated in a S6K/Tyro3-dependent manner in both MCF-7 and MCF-7-NR, and that the disconnection between IGF-1R/Akt and S6K may enable MCF-7-NR to keep cyclin D1 high in the presence of NVP-AEW541. In summary, acquired resistance to NVP-AEW541 appears to result from IGF-1R/Akt-independent activation of S6K and expression of Tyro3 and cyclin D1.

Keywords

Breast cancer Insulin-like growth factor-1 receptor NVP-AEW541 Resistance Tyro3 

Notes

Acknowledgment

This study was supported by the Global Centers of Excellence Program (H.M.), Grant-in-Aid for Scientific Research (C) (T.M.) and Grant-in-Aid for Young Scientists (B) (T.M) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Research Grant from Takeda Science Foundation (T.M).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10637_2012_9855_MOESM1_ESM.ppt (628 kb)
Supplemental Fig. 1 Effect of IRS-2 knock-down on cell signaling and cell growth in MCF-7-NR lines. On Day -1, MCF-7-NR cells were treated with IRS-2 siRNA. (a) On Day 0, cells were lysed and immunoblotted for phosphorylated Akt, S6K, and ERK1/2. Blots were then stripped and re-probed for β-actin as a loading control. (b) On Days 0 through 4, cells were subjected to serial MTS assay. Daily OD values are shown on the y-axis. Each data point represents the mean value and standard deviation of 6–12 replicate wells. (PPT 628 kb)
10637_2012_9855_MOESM2_ESM.ppt (508 kb)
Supplemental Fig. 2 Effects of NVP-AEW541 on phosphorylation of TSC-2 and PRAS-40, and Rheb expression in MCF-7 and MCF-7-NR lines. Cells grown in 10 % FBS-containing media with and without 3 μM NVP-AEW541 for 24 h were lysed and immunoblotted for phosphorylated TSC-2, TSC-2, phosphorylated PRAS40, and Rheb. Blots were stripped and re-probed for β-actin as a loading control. (PPT 508 kb)
10637_2012_9855_MOESM3_ESM.ppt (466 kb)
Supplemental Fig. 3 Baseline expression of Tyro3 in NVP-AEW541 resistant breast cancer cell lines. Cells grown in 10 % FBS-containing media for 24 h were lysed and immunoblotted for Tyro3. Blots were stripped and re-probed for β-actin as a loading control. (PPT 465 kb)

References

  1. 1.
    Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792PubMedCrossRefGoogle Scholar
  2. 2.
    Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500PubMedCrossRefGoogle Scholar
  3. 3.
    Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139PubMedCrossRefGoogle Scholar
  4. 4.
    Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, Heinrich MC, Tuveson DA, Singer S, Janicek M, Fletcher JA, Silverman SG, Silberman SL, Capdeville R, Kiese B, Peng B, Dimitrijevic S, Druker BJ, Corless C, Fletcher CD, Joensuu H (2002) Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 347:472–480PubMedCrossRefGoogle Scholar
  5. 5.
    Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352:786–792PubMedCrossRefGoogle Scholar
  6. 6.
    Tamborini E, Bonadiman L, Greco A, Albertini V, Negri T, Gronchi A, Bertulli R, Colecchia M, Casali PG, Pierotti MA, Pilotti S (2004) A new mutation in the KIT ATP pocket causes acquired resistance to imatinib in a gastrointestinal stromal tumor patient. Gastroenterology 127:294–299PubMedCrossRefGoogle Scholar
  7. 7.
    Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo F, Mok T, Lee C, Johnson BE, Cantley LC, Janne PA (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316:1039–1043PubMedCrossRefGoogle Scholar
  8. 8.
    Pollak MN, Schernhammer ES, Hankinson SE (2004) Insulin-like growth factors and neoplasia. Nat Rev Cancer 4:505–518PubMedCrossRefGoogle Scholar
  9. 9.
    Maiso P, Ocio EM, Garayoa M, Montero JC, Hofmann F, Garcia-Echeverria C, Zimmermann J, Pandiella A, San Miguel JF (2008) The insulin-like growth factor-I receptor inhibitor NVP-AEW541 provokes cell cycle arrest and apoptosis in multiple myeloma cells. Br J Haematol 141:470–482PubMedCrossRefGoogle Scholar
  10. 10.
    Scotlandi K, Manara MC, Nicoletti G, Lollini PL, Lukas S, Benini S, Croci S, Perdichizzi S, Zambelli D, Serra M, Garcia-Echeverria C, Hofmann F, Picci P (2005) Antitumor activity of the insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors. Cancer Res 65:3868–3876PubMedCrossRefGoogle Scholar
  11. 11.
    Jin Q, Esteva FJ (2008) Cross-talk between the ErbB/HER family and the type I insulin-like growth factor receptor signaling pathway in breast cancer. J Mammary Gland Biol Neoplasia 13:485–498PubMedCrossRefGoogle Scholar
  12. 12.
    Huang X, Gao L, Wang S, McManaman JL, Thor AD, Yang X, Esteva FJ, Liu B. Heterotrimerization of the growth factor receptors erbB2, erbB3, and insulin-like growth factor-i receptor in breast cancer cells resistant to herceptin. Cancer Res 70:1204–1214Google Scholar
  13. 13.
    Ibrahim YH, Yee D (2005) Insulin-like growth factor-I and breast cancer therapy. Clin Cancer Res 11:944s–950sPubMedGoogle Scholar
  14. 14.
    Shimizu C, Hasegawa T, Tani Y, Takahashi F, Takeuchi M, Watanabe T, Ando M, Katsumata N, Fujiwara Y (2004) Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis. Hum Pathol 35:1537–1542PubMedCrossRefGoogle Scholar
  15. 15.
    Chang Q, Li Y, White MF, Fletcher JA, Xiao S (2002) Constitutive activation of insulin receptor substrate 1 is a frequent event in human tumors: therapeutic implications. Cancer Res 62:6035–6038PubMedGoogle Scholar
  16. 16.
    Rocha RL, Hilsenbeck SG, Jackson JG, VanDenBerg CL, Weng C, Lee AV, Yee D (1997) Insulin-like growth factor binding protein-3 and insulin receptor substrate-1 in breast cancer: correlation with clinical parameters and disease-free survival. Clin Cancer Res 3:103–109PubMedGoogle Scholar
  17. 17.
    Mukohara T, Shimada H, Ogasawara N, Wanikawa R, Shimomura M, Nakatsura T, Ishii G, Park JO, Janne PA, Saijo N, Minami H (2009) Sensitivity of breast cancer cell lines to the novel insulin-like growth factor-1 receptor (IGF-1R) inhibitor NVP-AEW541 is dependent on the level of IRS-1 expression. Cancer Lett 282:14–24Google Scholar
  18. 18.
    Liu P, Cheng H, Roberts TM, Zhao JJ (2009) Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov 8:627–644PubMedCrossRefGoogle Scholar
  19. 19.
    Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M (2001) Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 93:1852–1857PubMedCrossRefGoogle Scholar
  20. 20.
    Liu L, Greger J, Shi H, Liu Y, Greshock J, Annan R, Halsey W, Sathe GM, Martin AM, Gilmer TM (2009) Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: activation of AXL. Cancer Res 69:6871–6878PubMedCrossRefGoogle Scholar
  21. 21.
    Arvisais EW, Romanelli A, Hou X, Davis JS (2006) AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2alpha. J Biol Chem 281:26904–26913PubMedCrossRefGoogle Scholar
  22. 22.
    Gonzalez-Garcia A, Garrido E, Hernandez C, Alvarez B, Jimenez C, Cantrell DA, Pullen N, Carrera AC (2002) A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation. J Biol Chem 277:1500–1508PubMedCrossRefGoogle Scholar
  23. 23.
    Markova B, Albers C, Breitenbuecher F, Melo JV, Brummendorf TH, Heidel F, Lipka D, Duyster J, Huber C, Fischer T. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway. Oncogene 29:739–751Google Scholar
  24. 24.
    Engelman JA (2009) Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer 9:550–562PubMedCrossRefGoogle Scholar
  25. 25.
    Kataoka Y, Mukohara T, Shimada H, Saijo N, Hirai M, Minami H. Association between gain-of-function mutations in PIK3CA and resistance to HER2-targeted agents in HER2-amplified breast cancer cell lines. Ann Oncol 21:255–262Google Scholar
  26. 26.
    Nahta R, O’Regan RM. Evolving Strategies for Overcoming Resistance to HER2-Directed Therapy: Targeting the PI3K/Akt/mTOR Pathway. Clin Breast Cancer 10:S72–78Google Scholar
  27. 27.
    Shiozawa Y, Pedersen EA, Patel LR, Ziegler AM, Havens AM, Jung Y, Wang J, Zalucha S, Loberg RD, Pienta KJ, Taichman RS. GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche. Neoplasia 12:116–127Google Scholar
  28. 28.
    Weinger JG, Gohari P, Yan Y, Backer JM, Varnum B, Shafit-Zagardo B (2008) In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation. J Neurochem 106:134–146PubMedCrossRefGoogle Scholar
  29. 29.
    Gjerdrum C, Tiron C, Hoiby T, Stefansson I, Haugen H, Sandal T, Collett K, Li S, McCormack E, Gjertsen BT, Micklem DR, Akslen LA, Glackin C, Lorens JB. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc Natl Acad Sci U S A 107:1124–1129Google Scholar
  30. 30.
    Li-Ping Z, Da-Lei Z, Jian H, Liang-Quan X, Ai-Xia X, Xiao-Yu D, Dan-Feng T, Yue-Hui Z. Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways. Reprod Biol Endocrinol 8:66Google Scholar
  31. 31.
    Scutera S, Fraone T, Musso T, Cappello P, Rossi S, Pierobon D, Orinska Z, Paus R, Bulfone-Paus S, Giovarelli M (2009) Survival and migration of human dendritic cells are regulated by an IFN-alpha-inducible Axl/Gas6 pathway. J Immunol 183:3004–3013PubMedCrossRefGoogle Scholar
  32. 32.
    O’Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, Espinosa R 3rd, Le Beau MM, Earp HS, Liu ET (1991) axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 11:5016–5031PubMedGoogle Scholar
  33. 33.
    Hong CC, Lay JD, Huang JS, Cheng AL, Tang JL, Lin MT, Lai GM, Chuang SE (2008) Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia. Cancer Lett 268:314–324PubMedCrossRefGoogle Scholar
  34. 34.
    Li Y, Ye X, Tan C, Hongo JA, Zha J, Liu J, Kallop D, Ludlam MJ, Pei L (2009) Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis. Oncogene 28:3442–3455PubMedCrossRefGoogle Scholar
  35. 35.
    Lay JD, Hong CC, Huang JS, Yang YY, Pao CY, Liu CH, Lai YP, Lai GM, Cheng AL, Su IJ, Chuang SE (2007) Sulfasalazine suppresses drug resistance and invasiveness of lung adenocarcinoma cells expressing AXL. Cancer Res 67:3878–3887PubMedCrossRefGoogle Scholar
  36. 36.
    Zhu S, Wurdak H, Wang Y, Galkin A, Tao H, Li J, Lyssiotis CA, Yan F, Tu BP, Miraglia L, Walker J, Sun F, Orth A, Schultz PG, Wu X (2009) A genomic screen identifies TYRO3 as a MITF regulator in melanoma. Proc Natl Acad Sci U S A 106:17025–17030PubMedCrossRefGoogle Scholar
  37. 37.
    Sun WS, Fujimoto J, Tamaya T (2003) Clinical implications of coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine leiomyoma. Mol Hum Reprod 9:701–707PubMedCrossRefGoogle Scholar
  38. 38.
    Sun WS, Fujimoto J, Tamaya T (2003) Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine endometrial cancers. Ann Oncol 14:898–906PubMedCrossRefGoogle Scholar
  39. 39.
    Hagerstrand D, Lindh MB, Pena C, Garcia-Echeverria C, Nister M, Hofmann F, Ostman A. PI3K/PTEN/Akt pathway status affects the sensitivity of high-grade glioma cell cultures to the insulin-like growth factor-1 receptor inhibitor NVP-AEW541. Neuro Oncol 12:967–975Google Scholar
  40. 40.
    Guerreiro AS, Boller D, Shalaby T, Grotzer MA, Arcaro A (2006) Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin-like growth factor receptor inhibition. Int J Cancer 119:2527–2538PubMedCrossRefGoogle Scholar
  41. 41.
    Attias-Geva Z, Bentov I, Fishman A, Werner H, Bruchim. I Insulin-like growth factor-I receptor inhibition by specific tyrosine kinase inhibitor NVP-AEW541 in endometrioid and serous papillary endometrial cancer cell lines. Gynecol OncolGoogle Scholar
  42. 42.
    Weroha SJ, Haluska P (2008) IGF-1 receptor inhibitors in clinical trials–early lessons. J Mammary Gland Biol Neoplasia 13:471–483PubMedCrossRefGoogle Scholar
  43. 43.
    Molloy CA, May FE, Westley BR (2000) Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line. J Biol Chem 275:12565–12571PubMedCrossRefGoogle Scholar
  44. 44.
    Maor S, Mayer D, Yarden RI, Lee AV, Sarfstein R, Werner H, Papa MZ (2006) Estrogen receptor regulates insulin-like growth factor-I receptor gene expression in breast tumor cells: involvement of transcription factor Sp1. J Endocrinol 191:605–612PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Roudy Chiminch Ekyalongo
    • 1
  • Toru Mukohara
    • 1
    • 2
    Email author
  • Yu Kataoka
    • 1
  • Yohei Funakoshi
    • 1
  • Hideo Tomioka
    • 1
  • Naomi Kiyota
    • 1
  • Yutaka Fujiwara
    • 1
    • 3
  • Hironobu Minami
    • 1
    • 2
  1. 1.Division of Medical Oncology/Hematology, Department of Internal MedicineKobe University Graduate School of MedicineKobeJapan
  2. 2.Cancer CenterKobe University HospitalKobeJapan
  3. 3.Division of Internal Medicine and Thoracic OncologyNational Cancer Center HospitalTokyoJapan

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