Advertisement

Investigational New Drugs

, Volume 31, Issue 5, pp 1158–1168 | Cite as

Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line

  • Yohei Funakoshi
  • Toru MukoharaEmail author
  • Hideo Tomioka
  • Roudy Chiminch Ekyalongo
  • Yu Kataoka
  • Yumiko Inui
  • Yuriko Kawamori
  • Masanori Toyoda
  • Naomi Kiyota
  • Yutaka Fujiwara
  • Hironobu Minami
PRECLINICAL STUDIES

Summary

The clinical efficacy of MET tyrosine kinase inhibitors (MET-TKIs) is hindered by the emergence of acquired resistance, presenting an obstacle to drug discovery. To clarify the mechanisms underlying acquired resistance to MET-TKIs, we established resistance models by continuous exposure of the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR). Baseline expression and phosphorylation of MET were elevated in MKN45-PR and MKN45-GR compared to MKN45 cells, and higher concentrations of MET-TKIs were required to inhibit MET phosphorylation compared to parental cells. Alterations in MET previously associated with resistance to MET-TKIs were observed in resistant cells, including elevated MET copy number, observed in both resistant lines compared to MKN45 cells, and the Y1230H mutation, detected in MKN45-PR cells. Notably, the growth of resistant lines was lower in the absence of MET-TKIs, suggesting “addiction” to inhibitors. While MKN45-PR cells exhibited a higher S-phase fraction in the absence of PHA665752, bromodeoxyuridine (BrdU) uptake was identical. Baseline phosphorylation of ATR, Chk1 and p53 and p21waf1/Cip1 expression was higher in MKN45-PR compared to MKN45 cells, and levels were reduced to those observed in untreated MKN45 cells following PHA665752 treatment. Furthermore, targeted knockdown of MET enhanced the growth of MKN45-PR cells. These findings suggest that alterations in MET leading to acquired MET-TKI resistance, may cause excessive MET signaling, subsequent replication stress and DNA damage response, and intra-S-phase arrest in the absence of MET-TKIs. Thus, partial MET inhibition is necessary for resistant cells to proliferate, a phenomenon we refer to as MET-TKI “addiction”.

Keywords

Acquired resistance Addiction Gastric cancer MET inhibitor 

Notes

Acknowledgments

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 a Research Grant from the Takeda Science Foundation (T.M).

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

10637_2013_9959_MOESM1_ESM.pdf (51 kb)
Suppl. Figure S1 STAT3 knock-down is associated with increased phosphorylation of Akt and ERK1/2. Day 0: MKN45-PR cells were treated with STAT3 siRNA. Day 1: cells were lysed and immunoblotted for phospho- and total-Akt and ERK1/2 using β-actin as a loading control. (PDF 43 kb)
10637_2013_9959_MOESM2_ESM.pdf (181 kb)
Suppl. Figure S2 Changes in BrdU uptake in MKN45 and MKN45-PR cells following PHA665752 treatment. Representative plots showing BrdU uptake in cells after treatment with PHA665752 (0 and 0.5 μM) for 8 h are shown. The population of cells incorporating BrdU is shown in the top right corner. (PDF 171 kb)

References

  1. 1.
    Vlahovic G, Crawford J (2003) Activation of tyrosine kinases in cancer. Oncologist 8(6):531–538CrossRefGoogle Scholar
  2. 2.
    Weinstein IB (2002) Cancer. Addiction to oncogenes–the Achilles heal of cancer. Science 297(5578):63–64. doi: https://doi.org/10.1126/science.1073096 CrossRefGoogle Scholar
  3. 3.
    Weinstein IB, Joe A (2008) Oncogene addiction. Cancer Res 68(9):3077–3080. doi: https://doi.org/10.1158/0008-5472.CAN-07-3293, discussion 3080CrossRefGoogle Scholar
  4. 4.
    Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos D, Campone M, Davidson N, Berger M, Oliva C, Rubin SD, Stein S, Cameron D (2006) Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 355(26):2733–2743. doi: https://doi.org/10.1056/NEJMoa064320 CrossRefGoogle Scholar
  5. 5.
    Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, Gemma A, Harada M, Yoshizawa H, Kinoshita I, Fujita Y, Okinaga S, Hirano H, Yoshimori K, Harada T, Ogura T, Ando M, Miyazawa H, Tanaka T, Saijo Y, Hagiwara K, Morita S, Nukiwa T (2010) Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 362(25):2380–2388. doi: https://doi.org/10.1056/NEJMoa0909530 CrossRefGoogle Scholar
  6. 6.
    Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, Maoleekoonpiroj S, Smylie M, Martins R, van Kooten M, Dediu M, Findlay B, Tu D, Johnston D, Bezjak A, Clark G, Santabarbara P, Seymour L (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353(2):123–132. doi: https://doi.org/10.1056/NEJMoa050753 CrossRefGoogle Scholar
  7. 7.
    Sierra JR, Tsao MS (2011) c-MET as a potential therapeutic target and biomarker in cancer. Ther Adv Med Oncol 3(1 Suppl):S21–S35. doi: https://doi.org/10.1177/1758834011422557 CrossRefGoogle Scholar
  8. 8.
    Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, Chitale D, Motoi N, Szoke J, Broderick S, Balak M, Chang WC, Yu CJ, Gazdar A, Pass H, Rusch V, Gerald W, Huang SF, Yang PC, Miller V, Ladanyi M, Yang CH, Pao W (2007) MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A 104(52):20932–20937. doi: https://doi.org/10.1073/pnas.0710370104 CrossRefGoogle Scholar
  9. 9.
    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(5827):1039–1043. doi: https://doi.org/10.1126/science.1141478 CrossRefGoogle Scholar
  10. 10.
    McDermott U, Pusapati RV, Christensen JG, Gray NS, Settleman J (2010) Acquired resistance of non-small cell lung cancer cells to MET kinase inhibition is mediated by a switch to epidermal growth factor receptor dependency. Cancer Res 70(4):1625–1634. doi: https://doi.org/10.1158/0008-5472.CAN-09-3620 CrossRefGoogle Scholar
  11. 11.
    Cepero V, Sierra JR, Corso S, Ghiso E, Casorzo L, Perera T, Comoglio PM, Giordano S (2010) MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors. Cancer Res 70(19):7580–7590. doi: https://doi.org/10.1158/0008-5472.CAN-10-0436 CrossRefGoogle Scholar
  12. 12.
    Qi J, McTigue MA, Rogers A, Lifshits E, Christensen JG, Janne PA, Engelman JA (2011) Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors. Cancer Res 71(3):1081–1091. doi: https://doi.org/10.1158/0008-5472.CAN-10-1623 CrossRefGoogle Scholar
  13. 13.
    Rege-Cambrin G, Scaravaglio P, Carozzi F, Giordano S, Ponzetto C, Comoglio PM, Saglio G (1992) Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene. Cancer Genet Cytogenet 64(2):170–173CrossRefGoogle Scholar
  14. 14.
    Mukohara T, Civiello G, Davis IJ, Taffaro ML, Christensen J, Fisher DE, Johnson BE, Janne PA (2005) Inhibition of the met receptor in mesothelioma. Clin Cancer Res 11(22):8122–8130. doi: https://doi.org/10.1158/1078-0432.CCR-05-1191 CrossRefGoogle Scholar
  15. 15.
    Kosaka T, Yatabe Y, Endoh H, Yoshida K, Hida T, Tsuboi M, Tada H, Kuwano H, Mitsudomi T (2006) Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res 12(19):5764–5769. doi: https://doi.org/10.1158/1078-0432.CCR-06-0714 CrossRefGoogle Scholar
  16. 16.
    Engelman JA, Settleman J (2008) Acquired resistance to tyrosine kinase inhibitors during cancer therapy. Curr Opin Genet Dev 18(1):73–79. doi: https://doi.org/10.1016/j.gde.2008.01.004 CrossRefGoogle Scholar
  17. 17.
    Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, Eisenberg BL, von Mehren M, Fletcher CD, Sandau K, McDougall K, Ou WB, Chen CJ, Fletcher JA (2006) Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol Off J Am Soc Clin Oncol 24(29):4764–4774. doi: https://doi.org/10.1200/JCO.2006.06.2265 CrossRefGoogle Scholar
  18. 18.
    Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL (2002) Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2(2):117–125CrossRefGoogle Scholar
  19. 19.
    Engelman JA, Mukohara T, Zejnullahu K, Lifshits E, Borras AM, Gale CM, Naumov GN, Yeap BY, Jarrell E, Sun J, Tracy S, Zhao X, Heymach JV, Johnson BE, Cantley LC, Janne PA (2006) Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J Clin Invest 116(10):2695–2706. doi: https://doi.org/10.1172/JCI28656 CrossRefGoogle Scholar
  20. 20.
    Suda K, Tomizawa K, Osada H, Maehara Y, Yatabe Y, Sekido Y, Mitsudomi T (2012) Conversion from the “oncogene addiction” to “drug addiction” by intensive inhibition of the EGFR and MET in lung cancer with activating EGFR mutation. Lung Cancer 76(3):292–299. doi: https://doi.org/10.1016/j.lungcan.2011.11.007 CrossRefGoogle Scholar
  21. 21.
    Tsantoulis PK, Kotsinas A, Sfikakis PP, Evangelou K, Sideridou M, Levy B, Mo L, Kittas C, Wu XR, Papavassiliou AG, Gorgoulis VG (2008) Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study. Oncogene 27(23):3256–3264. doi: https://doi.org/10.1038/sj.onc.1210989 CrossRefGoogle Scholar
  22. 22.
    Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere M, Ditullio RA Jr, Kastrinakis NG, Levy B, Kletsas D, Yoneta A, Herlyn M, Kittas C, Halazonetis TD (2005) Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434(7035):907–913. doi: https://doi.org/10.1038/nature03485 CrossRefGoogle Scholar
  23. 23.
    Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J (2005) DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434(7035):864–870. doi: https://doi.org/10.1038/nature03482 CrossRefGoogle Scholar
  24. 24.
    Fridman JS, Lowe SW (2003) Control of apoptosis by p53. Oncogene 22(56):9030–9040. doi: https://doi.org/10.1038/sj.onc.1207116 CrossRefGoogle Scholar
  25. 25.
    Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ (2002) Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol 22(6):1834–1843CrossRefGoogle Scholar
  26. 26.
    Lu X, Nannenga B, Donehower LA (2005) PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev 19(10):1162–1174. doi: https://doi.org/10.1101/gad.1291305 CrossRefGoogle Scholar
  27. 27.
    Myers K, Gagou ME, Zuazua-Villar P, Rodriguez R, Meuth M (2009) ATR and Chk1 suppress a caspase-3-dependent apoptotic response following DNA replication stress. PLoS Genet 5(1):e1000324. doi: https://doi.org/10.1371/journal.pgen.1000324 CrossRefGoogle Scholar
  28. 28.
    Shenberger JS, Dixon PS (1999) Oxygen induces S-phase growth arrest and increases p53 and p21(WAF1/CIP1) expression in human bronchial smooth-muscle cells. Am J Respir Cell Mol Biol 21(3):395–402CrossRefGoogle Scholar
  29. 29.
    Yao J, Duan L, Fan M, Yuan J, Wu X (2007) Overexpression of BLCAP induces S phase arrest and apoptosis independent of p53 and NF-kappaB in human tongue carcinoma: BLCAP overexpression induces S phase arrest and apoptosis. Mol Cell Biochem 297(1–2):81–92. doi: https://doi.org/10.1007/s11010-006-9332-2 CrossRefGoogle Scholar
  30. 30.
    Blay JY, Le Cesne A, Ray-Coquard I, Bui B, Duffaud F, Delbaldo C, Adenis A, Viens P, Rios M, Bompas E, Cupissol D, Guillemet C, Kerbrat P, Fayette J, Chabaud S, Berthaud P, Perol D (2007) Prospective multicentric randomized phase III study of imatinib in patients with advanced gastrointestinal stromal tumors comparing interruption versus continuation of treatment beyond 1 year: the French Sarcoma Group. J Clin Oncol Off J Am Soc Clin Oncol 25(9):1107–1113. doi: https://doi.org/10.1200/JCO.2006.09.0183 CrossRefGoogle Scholar
  31. 31.
    Riely GJ, Kris MG, Zhao B, Akhurst T, Milton DT, Moore E, Tyson L, Pao W, Rizvi NA, Schwartz LH, Miller VA (2007) Prospective assessment of discontinuation and reinitiation of erlotinib or gefitinib in patients with acquired resistance to erlotinib or gefitinib followed by the addition of everolimus. Clin Cancer Res 13(17):5150–5155. doi: https://doi.org/10.1158/1078-0432.CCR-07-0560 CrossRefGoogle Scholar
  32. 32.
    Blanke CD, Rankin C, Demetri GD, Ryan CW, von Mehren M, Benjamin RS, Raymond AK, Bramwell VH, Baker LH, Maki RG, Tanaka M, Hecht JR, Heinrich MC, Fletcher CD, Crowley JJ, Borden EC (2008) Phase III randomized, intergroup trial assessing imatinib mesylate at two dose levels in patients with unresectable or metastatic gastrointestinal stromal tumors expressing the kit receptor tyrosine kinase: S0033. J Clin Oncol Off J Am Soc Clin Oncol 26(4):626–632. doi: https://doi.org/10.1200/JCO.2007.13.4452 CrossRefGoogle Scholar
  33. 33.
    Zalcberg JR, Verweij J, Casali PG, Le Cesne A, Reichardt P, Blay JY, Schlemmer M, Van Glabbeke M, Brown M, Judson IR (2005) Outcome of patients with advanced gastro-intestinal stromal tumours crossing over to a daily imatinib dose of 800 mg after progression on 400 mg. Eur J Cancer 41(12):1751–1757. doi: https://doi.org/10.1016/j.ejca.2005.04.034 CrossRefGoogle Scholar
  34. 34.
    Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E, Nicaise C, Sawyers CL (2008) Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 14(6):485–493. doi: https://doi.org/10.1016/j.ccr.2008.11.001 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

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

Personalised recommendations