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

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.