Metformin reveals a mitochondrial copper addiction of mesenchymal cancer cells

Abstract

The clinically approved drug metformin has been shown to selectively kill persister cancer cells through mechanisms that are not fully understood. To provide further mechanistic insights, we developed a drug surrogate that phenocopies metformin and can be labeled in situ by means of click chemistry. Firstly, we found this molecule to be more potent than metformin in several cancer cell models. Secondly, this technology enabled us to provide visual evidence of mitochondrial targeting with this class of drugs. A combination of fluorescence microscopy and cyclic voltammetry indicated that metformin targets mitochondrial copper, inducing the production of reactive oxygen species in this organelle, mitochondrial dysfunction and apoptosis. Importantly, this study revealed that mitochondrial copper is required for the maintenance of a mesenchymal state of human cancer cells, and that metformin can block the epithelial-to-mesenchymal transition, a biological process that normally accounts for the genesis of persister cancer cells, through direct copper targeting.

Fig 1. Metformin and more potent surrogate Met preferentially alter the proliferation of cancer cells in a mesenchymal state.

(A) Molecular structures of metformin, phenformin and synthesis of Met. (B) Dose response viability curves of cancer cells treated as indicated for 72 h. (C) Dose response viability curves of cells in epithelial (dark) and mesenchymal (green) states of MDA-MB-468 cells treated as indicated for 72 h. (D) Dose response viability curves of HMLER cells in epithelial (dark) and mesenchymal (green) states treated as indicated for 72 h. Data points and error bars, mean values and SD of three independent biological replicates. Cells were treated at the IC₅₀ concentration at 72 h of Metformin or Metforminyn respectively, unless stated otherwise. See also S1, S2, S3, S4 and S5 Figs.

Fig 2. Biguanides directly target mitochondria and promote copper oxidation.

(A) Schematic illustration of the labeling of Met in cells using click chemistry. (B) Fluorescence microscopy images of labeled Met (green). Cells were treated with Met and subjected to click-labeling as described in Methods. Mitochondria were detected using cytochrome c immunostaining (red) and 4',6-diamidino-2-phenylindole (DAPI) stains nuclear DNA (blue). Scale bar, 10 μm. (C) Fluorescence microscopy detection of Cu²⁺ in cancer cells treated as indicated for 48 h. Scale bar, 10 μm. (D) Flow cytometry analysis of mitochondrial Cu²⁺ in cancer cells treated as indicated for 48 h. Bar chart represents an average of three independent experiments. (E) Flow cytometry analysis of mitochondrial Cu⁺ in cancer cells treated as indicated for 48 h. Bar chart represents an average of three independent experiments. (F) Cyclic voltammetry measurements towards oxidation potentials (purple arrow) of a Cu⁺ solution. Data recorded in the absence (black) and presence of 2 mol equiv. metformin (blue) or 2 mol equiv. Met (red). Redox peak potentials are marked with dashed lines. (G) Flow cytometry analysis of Fe²⁺ in cancer cells treated as indicated for 48 h. MDA-MB-468 cells were used in Fig 2C, 2D, 2E and 2G and were treated as described in Methods. Cells were treated at the IC₅₀ concentration at 72 h of Metformin or Metforminyn respectively, unless stated otherwise. See also S6, S7, S8, S9, S10 and S11 Figs. Bar chart represents an average of three independent experiments.

Fig 3. Copper is required to maintain a mesenchymal state of cancer cells.

(A) Western blot analysis of levels of a mesenchymal marker (Vimentin), Ctr1 and copper-containing mitochondrial proteins (SOD1, Cox4) in cell treated with EGF for 72 h. (B) Flow cytometry analysis of Ctr1 protein level in cells treated with EGF for 72 h. (C) ICP-MS analysis of Cu content of cells treated with EGF for 72 h. Error bars represent the standard deviation of four independent analyses. (D) Flow cytometry analysis of Cu²⁺ in cells treated with EGF for 72 h. Bar chart represents an average of three independent experiments. (E) Western blot analysis of mesenchymal markers (Vimentin, Fibronectin) in cells treated as indicated for 72 h. (F) Fluorescence microscopy images of E-cadherin (red) and Phalloidin (green) in cells treated as indicated for 72 h. DAPI stains nuclear DNA (blue). Scale bar, 10 μm. (G) Western blot analysis of mesenchymal markers and EMT-TF in cancer cells treated as indicated for 72 h. (H) Flow cytometry analysis of cells surface markers of cancer cells treated as indicated for 72 h and corresponding quantification. Bars and error bars, mean values and SD of three independent biological replicates. MDA-MB-468 cells were used throughout the figure. Cells were treated at the IC₅₀ concentration at 72 h of Metformin or Metforminyn respectively, unless stated otherwise. See also S12 and S13 Figs.