ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis

Abstract

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.

Keywords: breast cancer; copper; lung cancer; lysyl oxidase; metastasis.

Fig. 1.

ATP7A is necessary and sufficient for the copper-dependent activity of LOX family members. (A) Immunoblot analysis of ATP7A in 4T1-WT cells and 4T1 clones subjected to CRISPR/Cas9 targeting of ATP7A (C3/ATP7A− and C8/ATP7A− cells). Tubulin was detected as a loading control. (B) Immunofluorescence analysis of ATP7A protein in 4T1-WT, C3/ATP7A− and C8/ATP7A− cells. ATP7A protein (arrows; green) was detected in the perinuclear region of 4T1-WT cells and was absent in C3/ATP7A− and C8/ATP7A− clones. Nuclei were labeled with DAPI (blue). (C) LOX activity in conditioned media of 4T1-WT, C3/ATP7A− and C8/ATP7A− cells (mean ± SEM; **P < 0.01). Activity is expressed as relative fluorescence units (RFU) normalized against 4T1-WT cells. BAPN (0.5 mM), an irreversible inhibitor of LOX, was used to confirm LOX activity in 4T1-WT media. (D) Restoration of LOX activity in the media of C3/ATP7A− cells by stable transfection of a human ATP7A expression plasmid (mean ± SEM; ***P < 0.001). (E) Rescue of LOX activity in C3/ATP7A− cells by copper supplementation. LOX activity was measured in the conditioned media of 4T1-WT and C3/ATP7A− cells supplemented for 24 h with the indicated copper concentrations (mean ± SEM; **P < 0.01, ***P < 0.001); ns, not significant. (F) ATP7A is required for the activity of LOX, LOXL1 and LOXL2. The 4T1-WT and C3/ATP7A− cells were transiently transfected with a plasmid encoding GFP alone (control) or in combination with plasmids encoding LOX, LOXL1 or LOXL2. After a 24 h recovery, LOX activity was assayed in the media and normalized to cellular GFP expression to control for transfection efficiency (mean ± SEM; *P < 0.05); ns, not significant.

Fig. 2.

Deletion of ATP7A suppresses tumor growth and metastasis to the lungs in the orthotopic 4T1 murine breast cancer mouse model. (A) Primary tumors were isolated from mammary glands of BALB/c mice (n = 8 per group) 4 wk after injection with 4T1-WT, C3/ATP7A− or C8/ATP7A− cells (mean ± SEM; ****P < 0.0001). (B) Copper concentrations in primary tumors (mean ± SEM; *P < 0.05, **P < 0.01). (C) Representative images of metastatic nodules in the lungs of tumor-bearing mice. Lungs were fixed in Bouin’s solution to highlight lung tumor nodules. (D) Quantification of lung nodules (mean ± SEM; ****P < 0.0001). (E) Loss of ATP7A in 4T1 tumors reduces recruitment of CD11b+ and CD117+ myeloid cells to the lungs. Representative flow cytometry scatter plot analysis of dissociated lung tissues isolated from BALB/c mice bearing 4T1-WT or C3/ATP7A tumors (n = 4). Cells were stained with antibodies against CD11b conjugated to PerCP, CD117 conjugated to APC and CD45 conjugated to VioBlue. (F) Quantification of CD11b+ and CD117+ cells as a percentage of CD45+ immune cells (mean ± SEM, *P < 0.05).

Fig. 3.

ATP7A is required for cell motility and LOX-mediated phosphorylation of focal adhesion kinase (FAK1). (A) Immunoblot analysis of FAK1 phosphorylation in 4T1-WT, C3/ATP7A−, C8/ATP7A− and shRNA-ATP7A cells. Total FAK1 was detected as a loading control. (B) Immunoblot analysis of C3/ATP7A− cell lysates showing the restoration of FAK1 phosphorylation by the addition of exogenous purified LOX (1 µg/mL) to the culture medium for the indicated times. (C) Immunoblot band intensities were used to determine the ratio of p-FAK1/total FAK1 at the 20 min time point in B. Values are mean ± SEM; *P < 0.05; ns, not significant. (D) Detection of vinculin (green) at focal contacts in 4T1-WT and C3/ATP7A− cells using confocal immunofluorescence microscopy. Nuclei were stained using DAPI (blue). (E) Enumeration of vinculin clusters at focal adhesions in 4T1-WT and C3/ATP7A− cells (mean ± SEM; ***P < 0.001; n = 15 fields per sample). (F) Immunoblot analysis of total vinculin protein in 4T1-WT and C3/ATP7A− cells. Tubulin was detected as a loading control. (G) In vitro scratch assay demonstrating diminished motility of C3/ATP7A− cells compared with 4T1-WT cells. Representative images indicating the extent of closure are shown at 24 h after scratch formation. Dashed lines indicate scratch starting points. (H) Rate of gap closure of scratched 4T1-WT and C3/ATP7A− cell monolayers (mean ± SEM; ***P < 0.001). The LOX inhibitor BAPN (0.5 mM) was added to media of 4T1-WT cells as a positive control.

Fig. 4.

Deletion of ATP7A suppresses tumorigenesis and metastasis in the LLC mouse model. (A) Immunoblot analysis of ATP7A expression in wild-type LLC cells and LLC^7A-1 and LLC^7A-2 cell clones produced by CRISPR/Cas9 targeting of ATP7A. Tubulin was detected as a loading control. (B) LOX activity in culture medium of LLC, LLC^7A-1 and LLC^7A-2 cells (mean ± SEM; ***P < 0.001). (C) Weights of primary tumor isolated from C57BL/6 mice injected s.c. with LLC, LLC^7A-1, or LLC^7A-2 cells (mean ± SEM; ***P < 0.001). (D) LLC, LLC^7A-1, and LLC^7A-2 cells stably expressing mCherry were injected into the tail veins of C57BL/6 mice. After 3 wk, lungs and liver were harvested for RNA isolation and qPCR analysis of mCherry expression. Data are mean ± SEM. **P < 0.01; ***P < 0.001. A higher dCt value denotes a smaller metastatic burden.

Fig. 5.

Kaplan-Meier curve of breast cancer patients stratified by ATP7A expression. (A) Kmplot.com analysis of ATP7A expression levels as a function of distant metastasis-free survival of ER-negative breast cancer patients. (B) Proposed model for ATP7A function in tumor growth and metastasis. ATP7A delivers copper to LOX family members and facilitates copper export to maintain cellular copper homeostasis (Left). Deletion of ATP7A (Right) results in hyperaccumulation of copper, increased ROS, loss of LOX activity and a reduction in LOX-mediated metastatic pathways (e.g., FAK1 phosphorylation and myeloid recruitment at metastatic sites).