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


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”.


Acquired resistance Addiction Gastric cancer MET inhibitor 



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)


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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

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