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Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells

  • Preclinical Study
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Abstract

Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

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Acknowledgments

We would like to thank Birgit Reiter for excellent technical assistance and Dr. Monika Marine Mortensen for help with FACS analysis. This work was supported by Grants from the Danish Cancer Society, Danish Cancer Research Foundation, Astrid Thaysen’s Foundation, Wedell-Wedellsborg’s Foundation, Harboe Foundation, and A Race Against Breast Cancer.

Conflict of interest

The authors declare that they have no conflict of interest.

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    Authors and Affiliations

    1. Unit of Cell Death and Metabolism, Breast Cancer Group, Danish Cancer Society Research Center, Strandboulevarden 49, 2100, Copenhagen Ø, Denmark

      Stine Hole, Astrid M. Pedersen, Anne E. Lykkesfeldt & Christina W. Yde

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    1. Stine Hole
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    2. Astrid M. Pedersen
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    3. Anne E. Lykkesfeldt
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    4. Christina W. Yde
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    Corresponding author

    Correspondence to Christina W. Yde.

    Additional information

    Stine Hole and Astrid M. Pedersen have contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material. Online Resource 1. Data for hits identified in the kinase inhibitor screen. Triplicate samples of parental MCF-7 cells and AI-resistant cells (LetR-1, LetR-3, ExeR-1 and ExeR-3) were treated with 1.0 µM of the indicated kinase inhibitors for five days. The effect of each compound was determined by a cell viability assay. Hits were defined as compounds exerting at least two-fold statistically significant growth inhibition of one or more AI-resistant cell line compared with MCF-7 cells (indicated in red for the individual cell lines). Inhibitory effect (± SD) and P-values are shown.Online Resource 2. Cell division defects induced by barasertib. a) MCF-7, LetR-1, and ExeR-1 were treated for 48 days with 0.1 % DMSO or 0.05 µM barasertib, and cell cycle analyses were done by flow cytometry. Gating of single cells was done, and the DNA content was measured as the intensity of the propidium iodide signal. Percentage of cells with > 4 N DNA content was quantified using FlowJo Software and indicated in the figure. b ) Upon treatment of cells for 96 h with 0.1 % DMSO or 0.05 µM barasertib, 1 μg/ml Hoechst 33342 was added to the medium and fluorescence pictures of live cells were captured using an Olympus IX71 microscope. Scale bar; 10 µm.Online Resource 3. Dose – response growth experiments with MCF - 7 and AI - resistant cell lines with AI in the presence and absence of JNJ - 7706621 or alisertib. Aromatase inhibitor was withdrawn from the medium of the resistant cell lines one week before start of the experiment. MCF-7, LetR-1, and ExeR-1 cells were treated with increasing concentrations of letrozole or exemestane as indicated with or without a) 0.5 μM JNJ-7706621 or b) 0.05 µM alisertib. Cell number was determined after five days by a colorimetric assay and expressed as percentage of the control cells (0.1 % EtOH treated). Error bars represent SD of the mean of at least three replicate values. Asterisks (*) indicate significant growth inhibition compared with cells grown without aromatase inhibitor (p < 0.05).

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    Hole, S., Pedersen, A.M., Lykkesfeldt, A.E. et al. Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells. Breast Cancer Res Treat 149, 715–726 (2015). https://doi.org/10.1007/s10549-015-3284-8

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    • DOI: https://doi.org/10.1007/s10549-015-3284-8

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