The discovery that copper concentrations in serum and tumors are elevated in cancer patients and are correlated with disease severity has prompted speculation that copper delivery to oncogenic enzymes may be rate-limiting for tumor growth and metastasis (9C11). in animal models of malignancy (12, 13). Clinical studies show that copper chelation prolongs the survival of patients with late-stage breast malignancy (14) and kidney malignancy (15). These studies raise the possibility that blocking specific actions in the delivery of copper to oncogenic metalloenzymes may provide an approach to treating malignancy. LOX is usually a Mouse monoclonal to CRTC2 secreted copper-dependent amine oxidase that plays critical functions in the development of connective tissue and remodeling of the extracellular matrix by catalyzing the cross-linking of collagen and elastin (16). In addition to LOX, several LOX-like enzymes (LOXL1C4) have been identified that share a conserved catalytic copper-binding domain name. To date, functional functions CDDO-EA for LOX or LOXL enzymes have been documented in breast, colorectal, prostate, gastric, hepatic, pancreatic, and head and neck cancers, as well as in cancers of the skin, including melanoma (6, 17C19). LOX has been shown to promote tumor cell migration and adhesion via the activation of focal adhesion kinase (FAK1) (20C22). In addition, LOX and LOXL2 activity is required to facilitate the recruitment of myeloid cells to metastatic sites, creating a favorable environment for the subsequent invasion and growth of tumor cells (18, 19, 23). The importance of LOX and LOXL proteins in malignancy has led to rigorous efforts to develop inhibitors of these enzymes as potential anticancer therapies. Since all LOX family members share a functional requirement for copper, we surmised that inhibiting copper incorporation into these enzymes may be an effective strategy to inhibit LOX-dependent metastasis. However, the CDDO-EA mechanism of copper incorporation into LOX and LOXL proteins is usually poorly comprehended. The copper-binding catalytic domain name of each LOX family member is lumenally oriented (extracytoplasmic), suggesting that copper insertion occurs via CDDO-EA a conserved mechanism during the biosynthesis of these enzymes en route through the secretory pathway. Previous studies have shown that this Golgi-localized copper transporter ATP7A delivers copper to tyrosinase, a metalloenzyme with an extracytoplasmic copper-binding catalytic domain name (24). In addition, LOX activity is usually reduced in Menkes disease fibroblasts, which contain mutations in the gene (25). However, the extent to which ATP7A is required for the activity of different users of the LOX family of enzymes and, by extension, the importance of ATP7A in LOX-mediated pathologies remain unknown. Here we demonstrate that ATP7A is required for the activity of multiple users of the LOX family, and that silencing ATP7A in two different malignancy cell lines suppresses tumorigenesis and metastasis in mice. Importantly, elevated ATP7A expression was found to be significantly correlated with reduced survival in breast malignancy patients, suggesting a role for ATP7A in human cancer. These studies identify ATP7A as a target for inhibiting LOX-dependent malignancies. Results ATP7A Is Necessary and Sufficient for Copper-Dependent Activity of LOX Family Members. The 4T1 cells were chosen as a model of metastatic human breast cancer CDDO-EA because when orthotopically implanted into the mammary glands of mice, these cells readily metastasize to the lung in a LOX-dependent manner (7, 23, 26). We found that all known LOX family members were expressed in 4T1 cells under standard culture conditions (mRNAs relative to the adjacent stromal tissue (gene expression in murine 4T1 breast carcinoma cells using a CRISPR-Cas9 approach. Immunoblot analysis confirmed the absence of ATP7A protein in two impartial CRISPR clones, C3/ATP7A? and C8/ATP7A? (Fig. 1 0.01). Activity is usually expressed as relative fluorescence models (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. (expression plasmid (mean SEM; *** 0.001). ( 0.01, *** 0.001); ns, not significant. ( 0.05); CDDO-EA ns, not significant. These observations were not due to off-target effects of CRISPR-Cas9, because reduced LOX activity was also observed in 4T1 cells in which ATP7A was silenced using an RNAi approach (and expression plasmid (Fig. 1and = 8 per group) 4 wk after injection with 4T1-WT, C3/ATP7A? or C8/ATP7A? cells (mean SEM; **** 0.0001). ( 0.05, ** 0.01). ( 0.0001). (= 4). Cells were stained.
