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Anti-cell growth and anti-cancer stem cell activity of the CDK4/6 inhibitor palbociclib in breast cancer cells

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Abstract

Background

A cyclin-dependent kinase (CDK) 4/6 inhibitor, palbociclib, has been used to treat patients with estrogen receptor (ER)-positive (+) and human epidermal growth factor receptor (HER) 2-negative (−) advanced breast cancer. To investigate the mechanisms underlying the antitumor activity of palbociclib, we conducted a preclinical study on the anti-cell growth and anti-cancer stem cell (CSC) activity of palbociclib in breast cancer cells.

Methods

The effects of palbociclib on Rb phosphorylation, cell growth, cell cycle progression, apoptosis, cell senescence and the proportion of CSCs were investigated in five human breast cancer cell lines of different subtypes. To investigate the mechanisms of the anti-CSC activity of palbociclib, small-interfering RNAs for CDK4 and/or CDK6 were used. Palbociclib dose-dependently reduced Rb phosphorylation and cell growth in association with G1-S cell cycle blockade and the induction of cell senescence, but without increased apoptosis, in all breast cancer cell lines.

Results

The anti-cell growth activity of palbociclib widely differed among the cell lines. Palbociclib also dose-dependently reduced the CSC proportion measured by three different assays in four of five cell lines. The inhibition of CDK4 expression, but not CDK6 expression, reduced the increased proportion of putative CSCs induced by estradiol in ER (+)/HER2 (−) cell lines.

Conclusions

These results suggest that palbociclib exhibits significant anti-cell growth and anti-CSC activity in not only ER (+) breast cancer cell lines but also ER (−) cell lines. CDK4 inhibition induced by palbociclib may be responsible for its anti-CSC activity.

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Acknowledgements

We thank Mrs. Kaoru Tsuboi and Megumi Kuriyama for their technical assistance. All authors read and approved the final manuscript, and agree to be accountable for the integrity of the work.

Funding

This study was partially supported by Research Project Grants from Kawasaki Medical School and MEXT/JSPS KAKENHI Grant number JP17K10566.

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Authors and Affiliations

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

    Emi Kishino, Ryohei Ogata, Wataru Saitoh, Yoshikazu Koike, Yusuke Ohta & Junichi Kurebayashi

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

    Naoki Kanomata

Authors
  1. Emi Kishino
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  2. Ryohei Ogata
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  3. Wataru Saitoh
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  4. Yoshikazu Koike
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  5. Yusuke Ohta
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  6. Naoki Kanomata
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  7. Junichi Kurebayashi
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Corresponding author

Correspondence to Junichi Kurebayashi.

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Conflict of interest

J. Kurebayashi received advisory/consultation fees and research funding from Takeda Pharmaceutical Co. J. Kurebayashi also received research funding from Eisai Co. The other authors declare that they have no conflicts of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Electronic supplementary material

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12282_2019_1035_MOESM1_ESM.pdf

The protein expression levels of Rb and phosphorylated Rb altered by E2 and/or palbociclib in five breast cancer cell lines (a, MCF-7 cells; b, KPL-1 cells; c, BT-474 cells, d, KPL-4 cells and e, MDA-MB-231 cells). Values were analyzed after normalization to the controls and expressed as the mean ± SE. The expression level of each molecule in control cells was defined as 1. White bars, control; lightest grey bars, E2 alone; the second lightest grey bars, E2 plus 50 or 100 nM palbociclib; and the third lightest grey bars, E2 plus 100 or 500 nM palbociclib. * P < 0.05; **P < 0.01 significantly different from the E2-treated cells in ER (+) cells and from control cells in ER (-) cells. (PDF 279 kb)

12282_2019_1035_MOESM2_ESM.pdf

Effects of palbociclib on cell cycle progression in MCF-7 (a), KPL-1 (b), BT-474 (Cc KPL-4 (d) and MDA-MB-231 (e) cell lines. The values are the mean ± SE. White bars, control; lightest grey bars, E2 alone; the second lightest grey bars, E2 plus 50 or 100 nM palbociclib; and the third lightest grey bars, E2 plus 100 or 500 nM palbociclib. * P < 0.05; **P < 0.01 in comparison with cells treated with E2 alone. (PDF 252 kb)

12282_2019_1035_MOESM3_ESM.pdf

Effects of palbociclib on apoptosis in MCF-7 (a), KPL-1 (b), BT-474 (c), KPL-4 (d) and MDA-MB-231 (e) cell lines. Values are the mean ± SE. White bars, control; the lightest grey bars, E2 alone; the second lightest grey bars, E2 plus 50 or 100 nM palbociclib; and the third lightest grey bars, E2 plus 100 or 500 nM palbociclib. No significant difference was observed between palbociclib-treated and control cells. (PDF 233 kb)

12282_2019_1035_MOESM4_ESM.pdf

Effects of siRNA for CDK4 and/or siRNA for CDK6 on mRNA expression of CDK4 and CDK6 (a, b) and the proportion of CD44-high/CD24-low putative CSCs (c) in BT-474 cells. * P < 0.05; **P < 0.01 in comparison with control cells. (PDF 234 kb)

12282_2019_1035_MOESM5_ESM.pdf

Effects of siRNA for CDK4 and/or siRNA for CDK6 on mRNA expression of CDK4 and CDK6 (a, b) and the proportion of CD44-high/CD24-low putative CSCs (c) in KPL-4 cells. * P < 0.05; **P < 0.01 in comparison with control cells. (PDF 227 kb)

12282_2019_1035_MOESM6_ESM.pdf

Effects of siRNA for CDK4 and/or siRNA for CDK6 on mRNA expression of CDK4 and CDK6 (a, b) and the proportion of CD44-high/CD24-low putative CSCs (c) in MDA-MB-231 cells. * P < 0.05; **P < 0.01 in comparison with control cells. (PDF 228 kb)

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Kishino, E., Ogata, R., Saitoh, W. et al. Anti-cell growth and anti-cancer stem cell activity of the CDK4/6 inhibitor palbociclib in breast cancer cells. Breast Cancer 27, 415–425 (2020). https://doi.org/10.1007/s12282-019-01035-5

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  • DOI: https://doi.org/10.1007/s12282-019-01035-5

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