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Spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Purpose

The study aimed to investigate the role of spindle assembly checkpoint (SAC) in cancer cells with compromised genomic integrity. Chromosomal instability (CIN) gives cancer cells an adaptive advantage. However, maintaining the balance of this instability is crucial for the survival of cancer cells as it could lead them to the mitotic catastrophe. Therefore, cancer cells adapt to the detrimental effects of CIN. We hypothesized that changes in SAC might be one such adaptation mechanism. The focus of the study was BUB1B, an integral part of the checkpoint.

Methods

Clinical datasets were analyzed to compare expression levels of SAC genes in normal tissue vs. breast carcinoma. The effects of the reduction of BUB1B expression was examined utilizing RNA interference method with siRNAs. In vitro viability, clonogenicity, apoptosis, and SAC activity levels of a variety of breast cancer (BrCa) cell lines, as well as in vivo tumorigenicity of the triple-negative breast cancer (TNBC) cell line MDA-MB-468, were tested. Additionally, the chromosomal stability of these cells was tested by immunofluorescence staining and flow cytometry.

Results

In clinical breast cancer datasets, SAC genes were elevated in BrCa with BUB1B having the highest fold change. BUB1B overexpression was associated with a decreased probability of overall survival. The knockdown of BUB1B resulted in reduced viability and clonogenicity in BrCa cell lines and a significant increase in apoptosis and cell death. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown also impaired chromosome alignment and resulted in acute chromosomal abnormalities. We also showed that BUB1B knockdown on the MDA-MB-468 cell line decreases tumor growth in mice.

Conclusions

A functional spindle assembly checkpoint is essential for the survival of BrCa cells. BUB1B is a critical factor in SAC, and therefore breast cancer cell survival. Impairment of BUB1B has damaging effects on cancer cell viability and tumorigenicity, especially on the more aggressive variants of BrCa.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BrCa:

Breast cancer

CIN:

Chromosomal instability

ffluc:

Firefly luciferase

MCC:

Mitotic checkpoint complex

NSC:

Non-silencing control

NSG:

NOD scid gamma

PI:

Propidium iodide

SAC:

Spindle assembly checkpoint

TNBC:

Triple-negative breast cancer

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Acknowledgements

We thank Kinga and Edward Scott Lampert for their generous gift to our lab; Sheila and David Fuente Graduate Program in Cancer Biology at the University of Miami for their guidance; Hanna Fiske, Kelsie Medina-Saenz, Jun Sun for their technical support; Patricia Lisette Guevara at the University of Miami SCCC Flow Cytometry Shared Resource and Pete Johnson at Georgetown University LCCC Microscopy & Imaging Shared Resource for their assistance and expertise; Merlyn Donatien for daily administrative assistance.

Funding

This study was funded by the departmental funds of the University of Miami, Sylvester Comprehensive Cancer Center and Georgetown University, Department of Oncology.

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Authors and Affiliations

  1. Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL, USA

    Dilara Koyuncu & Utsav Sharma

  2. Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd NW, Washington, DC, NRB W412, USA

    Utsav Sharma & Marc E. Lippman

  3. Geneyus, LLC, Miami, FL, USA

    Erik T. Goka

Authors
  1. Dilara Koyuncu
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  2. Utsav Sharma
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  3. Erik T. Goka
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  4. Marc E. Lippman
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Contributions

DK and MEL contributed to the study conception and design. All authors contributed to the development of methodology. DK (all experiments) and US (animal studies) contributed to the data collection and analysis. The first draft of the manuscript was written by DK, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Marc E. Lippman.

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The authors declare no conflict of interest.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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10549_2020_5962_MOESM1_ESM.pdf

Supplementary file1 (PDF 73 kb). Supplementary Fig 1 BubR1 protein levels at 60 days after inoculation in tumors transfected with NSC or siBUB1B (61 days after transfection).

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Koyuncu, D., Sharma, U., Goka, E.T. et al. Spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival. Breast Cancer Res Treat 185, 331–341 (2021). https://doi.org/10.1007/s10549-020-05962-2

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  • DOI: https://doi.org/10.1007/s10549-020-05962-2

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