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

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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.

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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.

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Author notes

  1. Yutaka Fujiwara

    Present address: Division of Internal Medicine and Thoracic Oncology, National Cancer Center Hospital, 5-1-1, Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

Authors and Affiliations

  1. Division of Medical Oncology/Hematology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

    Roudy Chiminch Ekyalongo, Toru Mukohara, Yu Kataoka, Yohei Funakoshi, Hideo Tomioka, Naomi Kiyota, Yutaka Fujiwara & Hironobu Minami

  2. Cancer Center, Kobe University Hospital, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

    Toru Mukohara & Hironobu Minami

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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)

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)

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)

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Ekyalongo, R.C., Mukohara, T., Kataoka, Y. et al. Mechanisms of acquired resistance to insulin-like growth factor 1 receptor inhibitor in MCF-7 breast cancer cell line. Invest New Drugs 31, 293–303 (2013). https://doi.org/10.1007/s10637-012-9855-1

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