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Cellular and Molecular

AMPK phosphorylates and stabilises copper transporter 1 to synergise metformin and copper chelator for breast cancer therapy

British Journal of Cancer volume 128, pages 1452–1465 (2023)Cite this article

Subjects

Abstract

Background

Predominant roles of copper and its transporter, copper transporter 1 (CTR1), in tumorigenesis have been explored recently; however, the upstream regulation of CTR1 and combinational intervention of copper chelators in malignancies remain largely unclear.

Methods

CRISPR/Cas9-based kinome screening was used to identify the CTR1 upstream kinases. Immunofluorescence assays were utilised to detect CTR1 localisation. In vitro kinase assays and mass spectrometry were performed to detect CTR1 phosphorylation. Ubiquitination assays were performed to validate CTR1 stability. Colony formation, EdU labelling, Annexin V-FITC/PI-based apoptosis assays were carried out to detect the drug effect on cell growth and apoptosis. Xenografted mouse models were employed to investigate drug effects in vivo.

Results

We identify that CTR1 undergoes AMPK-mediated phosphorylation, which enhances CTR1 stabilisation and membrane translocation by affecting Nedd4l interaction, resulting in increased oncogenic roles in breast cancer. Importantly, activation of AMPK with its agonist metformin markedly enhances CTR1 levels, and leads to the combinational usage of AMPK agonists and copper chelators for breast cancer treatment.

Conclusions

Our findings not only reveal the crosstalk between energy response and copper uptake via AMPK-mediated CTR1 phosphorylation and stability but also highlight the strategy to combat breast cancer by a combination of AMPK agonists and copper chelators.

Significance

The connection between energy response and copper homoeostasis is linked by AMPK phosphorylating and stabilising CTR1, which provides a promising strategy to combat breast cancer by combining AMPK agonists and copper chelators.

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Fig. 1: AMPK enhances CTR1 protein levels.
Fig. 2: AMPK activation facilitates CTR1 membrane location.
Fig. 3: AMPK directly phosphorylates CTR1.
Fig. 4: AMPK-mediated CTR1 phosphorylation affects its protein level and localisation.
Fig. 5: AMPK activation blocks Nedd4l-mediated CTR1 degradation.
Fig. 6: Metformin synergises with copper chelator TTM in breast cancer cells.
Fig. 7: Metformin synergises with copper chelator TTM in vivo.

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Data availability

Data are available on request to the authors.

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Acknowledgements

We thank members of the Guo laboratory for critical reading and kind suggestions for the manuscript. We thank Xiaolin Tian and Dr Haiteng Deng in Center of Protein Analysis Technology, Tsinghua University, for MS analysis. This work was supported by National Natural Science Foundation of China to JG (32070767, 31871410), China Postdoctoral Science Foundation (XZ, 2020M672954).

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  1. These authors contributed equally: Xiaomei Zhang, Qiwei Jiang.

Authors and Affiliations

  1. Department of Breast and Thyroid Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510275, China

    Xiaomei Zhang, Zicheng Sun & Ying Lin

  2. Institute of Precision Medicine, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510275, China

    Xiaomei Zhang, Qiwei Jiang, Yaqing Su, Lang Bu, Zicheng Sun, Xueji Wu, Bing Gao, Lei Wang, Wei Xie & Jianping Guo

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Contributions

This study was conceived and designed by JG and XZ. Development of methodology: XZ and QJ. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): XZ, QJ, YS, LB. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): XZ, QJ, YS, LB. Writing, review, and/or revision of the manuscript: JG, XZ, QJ, YL. Administrative, technical, or material support (i.e., reporting or organising data, constructing databases): WX, XW, BG, ZS, LW. Study supervision: JG, WX, YL. Approved manuscript: all authors.

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Correspondence to Ying Lin, Wei Xie or Jianping Guo.

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All procedures followed are in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

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Zhang, X., Jiang, Q., Su, Y. et al. AMPK phosphorylates and stabilises copper transporter 1 to synergise metformin and copper chelator for breast cancer therapy. Br J Cancer 128, 1452–1465 (2023). https://doi.org/10.1038/s41416-022-02127-4

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