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Anti-cell growth and anti-cancer stem cell activities of the non-canonical hedgehog inhibitor GANT61 in triple-negative breast cancer cells

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Abstract

Background

Triple-negative breast cancer (TNBC) exhibits biologically aggressive behavior and has a poor prognosis. Novel molecular targeting agents are needed to control TNBC. Recent studies revealed that the non-canonical hedgehog (Hh) signaling pathway plays important roles in the regulation of cancer stem cells (CSCs) in breast cancer. Therefore, the anti-cell growth and anti-CSC effects of the non-canonical Hh inhibitor GANT61 were investigated in TNBC cells.

Methods

The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the proportion of CSCs were investigated in three TNBC cell lines. Four ER-positive breast cancer cell lines were also used for comparisons. The expression levels of effector molecules in the Hh pathway: glioma-associated oncogene (GLI) 1 and GLI2, were measured. The combined effects of GANT61 and paclitaxel on anti-cell growth and anti-CSC activities were also investigated.

Results

Basal expression levels of GLI1 and GLI2 were significantly higher in TNBC cells than in ER-positive breast cancer cells. GANT61 dose-dependently decreased cell growth in association with G1–S cell cycle retardation and increased apoptosis. GANT61 significantly decreased the CSC proportion in all TNBC cell lines. Paclitaxel decreased cell growth, but not the CSC proportion. Combined treatments of GANT61 and paclitaxel more than additively enhanced anti-cell growth and/or anti-CSC activities.

Conclusions

The non-canonical Hh inhibitor GANT61 decreased not only cell growth, but also the CSC population in TNBC cells. GANT61 enhanced the anti-cell growth activity of paclitaxel in these cells. These results suggest for the first time that GANT61 has potential as a therapeutic agent in the treatment of patients with TNBC.

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Acknowledgements

We thank Mrs. Kaoru Tsuboi and Ms. Megumi Ogo for their technical assistance. This work was supported by the Research Project Grants from Kawasaki Medical School (26-5 and 27-1) for JK and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (26461964) for JK.

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Authors and Affiliations

  1. Department of Breast and Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan

    Yoshikazu Koike, Yusuke Ohta, Wataru Saitoh, Tetsumasa Yamashita & Junichi Kurebayashi

  2. Department of Pathology 2, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan

    Naoki Kanomata & Takuya Moriya

Authors
  1. Yoshikazu Koike
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  2. Yusuke Ohta
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  3. Wataru Saitoh
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  4. Tetsumasa Yamashita
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  5. Naoki Kanomata
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  6. Takuya Moriya
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  7. Junichi Kurebayashi
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Correspondence to Junichi Kurebayashi.

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Conflict of interest

J. Kurebayashi received advisory/consultation fees and research funding from Takeda Pharmaceutical Co., Limited. J. Kurebayashi received research funding from Takeda Pharmaceutical Co., Esai Co., Chugai Co., and AstraZeneca Pharmaceuticals. The other authors declare that they have no conflict of interest.

Electronic supplementary material

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12282_2017_757_MOESM1_ESM.pptx

Supplementary material 1 (PPTX 144 kb) Figure S1 Basal protein expression levels of GLI1 (A) and GLI2 (B) in three TNBC cell lines and four ER-positive breast cancer cell lines. Expression levels were measured using western blotting as described in the Methods. The deduced molecular weights of GLI1 and Gli2 were approximately 160 kDa and 220 kDa according to the manufacturer’s instructions, respectively

12282_2017_757_MOESM2_ESM.pptx

Supplementary material 2 (PPTX 78 kb) Figure S2 Effects of GANT61 on cell cycle progression in MDA-MB-231 cells (A), MDA-MB-157 cells (B), and HCC1937 cells (C). Cells were treated with GANT61 for 2 days. The percentages of cells at each cell cycle phase were analyzed as described in the Methods. Values are expressed as means ± SEs. White bars, control; the lightest gray bars, 1 μM GANT61; the second lightest gray bars, 5 μM GANT61; the third lightest gray bars, 10-μM GANT61; and the darkest gray bars, 20-μM GANT61. * P < 0.05; **P < 0.01 significantly different from control cells

12282_2017_757_MOESM3_ESM.pptx

Supplementary material 3 (PPTX 54 kb) Figure S3 Effects of GANT61 (0 – 20μM) on the expression levels of survivin in MDA-MB-231 cells. The cells were treated with the indicated concentrations of GANT61 for two days. The expression levels were tested using western blotting as described in the Methods. The deduced molecular weight of surviving was approximately 16 kDa according to the manufacture’s instruction

12282_2017_757_MOESM4_ESM.pptx

Supplementary material 4 (PPTX 67 kb) Figure S4 Time courses of changes induced in mRNA expression levels of GLI1 (A) and GLI2 (B) by GANT61 in MDA-MB-157 cells. The expression levels of GLI1 and GLI2 in controls at the respective time points were defined as 1. In addition, dose-dependent decreases in the mRNA expression levels of GLI1 (C) and GLI2 (D) were investigated in MDA-MB-157 cells. Cells were treated with 1–20-μM GANT61 for 24 h. Values are expressed as means ± SEs. * P < 0.05; **P < 0.01; and #, P < 0.10 significantly different from the respective controls

12282_2017_757_MOESM5_ESM.docx

Supplementary material 5 (DOCX 14 kb) Table S1 Subtypes and IC50s of GANT61 in three TNBC cell lines and four ER-positive breast cancer cell lines. All cell lines were treated with 0 – 20 μM GANT61 for 3 days. Values are expressed as means ± SEs

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Koike, Y., Ohta, Y., Saitoh, W. et al. Anti-cell growth and anti-cancer stem cell activities of the non-canonical hedgehog inhibitor GANT61 in triple-negative breast cancer cells. Breast Cancer 24, 683–693 (2017). https://doi.org/10.1007/s12282-017-0757-0

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  • DOI: https://doi.org/10.1007/s12282-017-0757-0

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