Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

Abstract

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Figure 1. Developing small molecules that specifically inhibit human copper-trafficking proteins and an overview of the screening process

a, Copper trafficking in eukaryotes and the selective inhibition of human copper trafficking by a small molecule (DC_AC50). Green represents the copper chaperone proteins and lilac represents proteins that receive copper from the chaperones. b, The sequence alignment illustrates a significant similarity between human Atox1 and domain I of CCS. Structural comparison of human Atox1 (green; Protein Data Bank (PDB) 1TL4) and domain I of human CCS (hCCS, purple; PDB 2CRL). c, A hierarchical docking strategy was adopted: DOCK4.0 was used to screen the Specs database, which contains more than 200,000 compounds. Out of this screen, and based on their structural features, physical chemistry properties and drug-like characteristics, 237 compounds were selected for further bioactivity testing with six hits discovered. d, The structures of the six representative hits.

Figure 2. Docking model and binding of DC_AC50 to Atox1 and full-length CCS by FRET measurements (from Tyr/Trp to DC_AC50)

a,b, FRET between Atox1 (Tyr), full-length CCS (Trp) and DC_AC50. Atox1 or full-length CCS (1 μM) displayed the maximum fluorescence emission at 335 or 350 nm, respectively, in the absence of DC_AC50. With the addition of DC_AC50 (1-100 μM and less than 5 μl DMSO in 200 μl buffer), the peak at 335 or 350 nm, which corresponds to the emission of Tyr or Trp, respectively, was reduced, whereas the emission of DC_AC50 at 494 nm was increased. FRET changes are shown in coloured lines. c,d, Binding curves of DC_AC50 to Atox1 and CCS. The experiments were performed in 50 mM HEPES, 200 mM NaCl, 1 mM DTT (pH 7.1). e,f, Surface representations that show DC_AC50 (green) and DC_AC2 (purple) binding to Atox1 (e; PDB 1TL4) and CCS (f; PDB 2CRL). Error bars, mean ± s.e.m., n = 3 biological replicates.

Figure 3. DC_AC50 reduces the proliferation of cancer cells and attenuates tumour growth in xenograft nude mice

a,b, Treatment with DC_AC50 inhibits cancer cell proliferation (a) but not normal cell proliferation (b). Cells were grown and treated with DC_AC50 at 0-10 μM for 72 hours. c, Cell proliferation of normal lung BEAS-2B cells showed minimal effects with DC_AC50 (0-10 μM) treatment for 72 hours. d, Reduced cell proliferation on the knockdown (KD) of Atox1 and CCS. Knockdowns of Atox1 and CCS were confirmed via western blotting (Supplementary Fig. 7c). e, Knockdowns of Atox1 and CCS did not significantly reduce cell proliferation in normal HaCaT cells. Knockdowns of Atox1 and CCS were confirmed via western blotting. f, The structures of DC_AC50 and the inactive control compound ZYAT36. g, Treatment with the inactive control compound ZYAT36 at 10 μM showed minimal effects in the H1299 cells for 72 hours. h, Knockdowns of Atox1 and CCS resulted in a diminished further inhibition of cell proliferation by DC_AC50 (0-10 μM). i, Tumour growth and tumour size in xenograft nude mice injected with H1299 cells compared with the group of mice treated with DC_AC50 and the control group treated with vehicle control. PLKO.1 is the name of the lentiviral vector as a control for the stable knockdown assay. Error bars, mean ± s.e.m., n = 3 biological replicates. P values were determined by a two-tailed Student's t test. *P < 0.05, **P < 0.005. n.s., not significant.

Figure 4. DC_AC50 induces copper accumulation, increases ROS level and decreases the NADPH/NADP⁺ ratio

a, The addition of DC_AC50 (10 μM) and the knockdown of Atox1 both led to an increase in the total cellular copper after 12 hours. The knockdown of CCS also led to a slight increase in the cellular total copper content after 12 hours. Knockdowns of Atox1 and CCS were confirmed via western blotting. b,c, Treatment with DC_AC50 or knockdown of Atox1/CCS induced ROS elevation in H1299 cells. d,e, DC_AC50-induced ROS elevation accompanied by a reduced total GSH and increased GSSG level in H1299 cells after 12 hours of treatment. f,g, The ROS levels showed minimal effects in normal HaCaT cells with DC_AC50 treatment (0-10 μM) or in H1299 cells with the inactive control compound ZYAT36 (10 μM) treatment for 12 hours. h,i, The NADPH/NADP⁺ ratio decreased with the DC_AC50 treatment (10 μM) or Atox1/CCS knockdown in H1299 cells. j, DC_AC50-induced ROS can be rescued by NAC in H1299 cells. Cells were pretreated with 1 mM or 3 mM NAC for one hour, followed by 5 μM DC_AC50 for 12 hours. Error bars, mean ± s.e.m., n = 3 biological replicates. *P < 0.05, **P < 0.005, ***P < 0.0005. RLU, relative luminescence units. shRNA, short hairpin RNA; siRNA, small interfering RNA.

Figure 5. Treatment with DC_AC50 or Atox1/CCS knockdown decreases the cellular ATP level, COX activities and the rate of oxygen consumption in cancer cells

a, Atox1/CCS knockdown or DC_AC50 (10 μM)-induced reduction of ATP levels in H1299 cells after 12 hours. b,c, DC_AC50 (10 μM) or Atox1/CCS knockdown significantly lowers the COX activity in H1299 cells after 12 hours. d, The reduced COX activity by DC_AC50 (10 μM) treatment could be rescued by Atox1/CCS overexpression. e,f, The expression levels of COX subunit 1 (COX1) and subunit 2 (COX2) decreased with different concentrations of DC_AC50 (0-10 μM) or Atox1/CCS knockdown after 12 hours. g,h, Oxygen consumption decreased with DC_AC50 (10 μM) or Atox1/CCS knockdown in H1299 cells after 12 hours. i–l, Treatment with the inactive control compound ZYAT36 (10 μM) did not affect the ATP level (i), COX activity (j), oxygen consumption (k) or the expression level of COX1,2 (l) in H1299 cells. Error bars, mean ± s.e.m., n = 3 biological replicates. *P < 0.05, **P < 0.005. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6. DC_AC50-induced mitochondria defects and decreased lipid biosynthesis through AMPK activation

a, Decreased lipid biosynthesis with DC_AC50 (10 μM) treatment in H1299 cells. b, DC_AC50-induced lipid biosynthesis elevation can be rescued by 10 μM compound C (AMPK inhibitor). c, AMPK activation and ACC1 phosphorylation by a variety of DC_AC50 (0–10 μM) treatments for 12 hours. d,e, Knockdown of Atox1 and CCS induced the reduction of lipid biosynthesis (d), AMPK activation and ACC1 phosphorylation (e) in H1299 cells after 12 hours. f, Knockdown of Atox1 and CCS in normal HaCaT cells did not significantly reduce lipid biosynthesis after 12 hours. g,h, Treatment with the inactive control compound ZYAT36 (10 μM) did not induce a reduction of lipid biosynthesis (g), AMPK activation or ACC1 phosphorylation (h) in H1299 cells after 12 hours. i, AMPK activation and ACC1 phosphorylation by DC_AC50 (10 μM) and rescue assays with compound C and NAC in H1299 cells after 12 hours. j, Cell-proliferation inhibition by DC_AC50 (10 μM) could be partially rescued by NAC or compound C. Treatment with both NAC and compound C almost completely rescued cell-proliferation inhibition by DC_AC50 (10 μM) in H1299 cells. Error bars, mean ± s.e.m., n = 3 biological replicates. *P < 0.05, **P < 0.005.