Lysyl Oxidase Regulates Breast Cancer Cell Migration and Adhesion through a Hydrogen Peroxide–Mediated Mechanism

Lysyl oxidase (LOX) was initially reported as a copper-dependent amine oxidase responsible for the catalysis of collagen and elastin cross-linking within the extracellular matrix (reviewed in refs. 1–3). In this process, LOX catalyzes the exchange of an amine to an aldehyde group on a peptidyl lysine, producing hydrogen peroxide and ammonia as by-products of catalytic activity. However, recent work has shown that LOX may have intracellular functions including the regulation of cell differentiation, motility/migration, and gene transcription (4–8). We have previously identified an up-regulation of LOX mRNA expression in the invasive breast cancer cell lines Hs578T and MDA-MB-231 compared with the poorly invasive cell lines MCF-7 and T-47D (9). Inhibition of LOX with β-aminopropionitrile (βAPN; an irreversible inhibitor of LOX catalytic activity) or LOX-specific antisense oligonucleotides led to a significant decrease in the invasive potential of Hs578T and MDA-MB-231 cells. Conversely, exogenous expression of LOX in the poorly invasive MCF-7 cell line resulted in an increase in invasive activity that was reversible on treatment with βAPN. These results showed that LOX was essential for breast cancer cell invasion but the molecular mechanism(s) underlying this finding was unclear.

Tumor cell invasion is a complex process that involves attachment to, degradation of, and detachment from an extracellular matrix, and finally active migration away from the primary tumor (10). Therefore, the ability of a cell to move on its own accord is a critical factor in tumor progression from a nonmetastatic to metastatic state. A key process in basic cell migration is the ability of a cell to form a stable adhesion to the extracellular matrix (11). This process is regulated by two key proteins within cells: Src and focal adhesion kinase (FAK; refs. 12, 13). Inactivation of either of these proteins leads to a dramatic loss in the ability of a cell to form adhesion complexes. Src and FAK are activated through a series of phosphorylation events. Previous work has shown that Src is phosphorylated and activated by various stimuli including integrins, phosphatases, and hydrogen peroxide (12, 14–16). FAK becomes catalytically active through an initial autophosphorylation at Tyr³⁹⁷. On phosphorylation and activation of Src, autophosphorylated FAK is then phosphorylated by Src in the kinase domain of FAK (17). The activation of the FAK/Src signaling pathway leads to the activation and regulation of various signaling molecules involved in cell migration and adhesion (18).

In the present study, we tested the hypothesis that LOX facilitates breast cancer migration through the regulation of cell-matrix adhesion formation. The data show that catalytically active LOX is required for breast cancer cell migration and adhesion formation. Additionally, LOX regulates adhesion formation via the FAK/Src signaling pathway through a hydrogen peroxide–mediated mechanism. Understanding the molecular mechanisms of breast cancer cell migration may lead to novel therapeutic strategies in the treatment of metastatic breast cancer.

Cells and culture conditions. Breast cancer cell lines were obtained and maintained as previously described (9). MCF-7 cells were stably transfected with 1 μg of LOX32-His DNA, LOX50-His DNA, or the pDsRed2-N1 vector (Clontech, Palo Alto, CA) using Effectene per specifications of the manufacturer (Qiagen, Valencia, CA). LOX-expressing MCF-7 cells were selected with 400 μg/mL G418, cloned by limiting dilution, and LOX expression was verified by real-time PCR (Hs00184700 LOX primer/probe set, Applied Biosystems, Foster City, CA). DsRed2-expressing MCF-7 cells (mock-transfected) were selected with 400 μg/mL G418 and sorted using fluorescence-activated cell sorting (two sequential sorts, FACSAria, Becton Dickinson, San Jose, CA). At least two clones from each transfection were used for each variable analyzed. Cell cultures were determined to be free of Mycoplasma contamination using the Mycoplasma PCR ELISA kit (Roche Diagnostics, Indianapolis, IN). All cells were harvested when cultures were ∼80% confluent.

LOX32-His and LOX50-His constructs. PCR primers were designed to introduce EcoRI and XhoI restriction sites at the 5′ and 3′ termini of a LOX cDNA template, respectively (19), and cloned into the pcDNA3.1/His A expression vector downstream and in frame with the 6× histidine (His) epitope tag (Invitrogen, Carlsbad, CA). The catalytically active form of LOX (amino acids 169-417) was designed to start at the bone morphogenetic protein-1 cleavage site (20). The noncatalytically active form of LOX (amino acids 1-417) was designed to encode the full-length proenzyme. The pcDNA3.1/LOX32-His and pcDNA3.1/LOX50-His constructs were verified by DNA sequencing.

Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue microarrays of normal human mammary tissue, primary tumor tissues, and recurrent tissues (containing local and distant metastases) from breast cancer patients were obtained from the Pathology Core Facility (Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL). Human metastatic breast tissue was also obtained from Dr. Ruth Lininger (University of North Carolina, Chapel Hill, NC). Sections were deparaffinized, rehydrated in distilled water, and antigen retrieval was done in citrate buffer (pH 6.0) at 97°C. Tissue was blocked sequentially with hydrogen peroxide, avidin, biotin, and serum-free protein block for 15 minutes each. Tissues were incubated with anti-LOX (1:50) or anti-rabbit immunoglobulin G (IgG; DAKO, Carpinteria, CA) antibody, biotinylated linking antibody, and streptavidin peroxidase. Sections were stained with chromogen (3,3′-diaminobenzidine, Richard Allen Scientific, Kalamazoo, MI) and counterstained with hematoxylin. Antibody was generated to the LOX peptide KYSDDNPYYNYYDTYERPRPGG (amino acids 176-197; Zymed Laboratories, Inc., San Francisco, CA) and specificity was evaluated in rat brain tissues (21). Tissues were imaged using an Axioscope 2 microscope and Spot 2 camera using the Zeiss Axiovision 2.0.5 software (Thornwood, NY). Tissues were scored on a +/− scale based on the extent of LOX staining compared with negative controls. All samples were scored blinded.

Motility analysis. Analysis and quantitation of haptokinetic motility of breast cancer cells was done using the Cell Motility HitKit (Cellomics, Inc., Pittsburgh, PA) as per specifications of the manufacturer. Where indicated, Hs578T, MDA-MB-231, and MCF-7/LOX32-His cells were pretreated for 24 hours with βAPN (9, 22, 23). Each variable was assayed in triplicate and 100 cells per treatment group were evaluated for motility. Experiments were repeated thrice. Statistical significance was evaluated by ANOVA using Microsoft Excel 2000 spreadsheets.

Migration assay. The in vitro cellular migration assay is a modification of the Membrane Invasion Culture System assay previously described (24). Where indicated, breast cancer cells were pretreated with βAPN for 24 hours before and during the migration assay. Experiments were repeated thrice. Statistical significance was evaluated by ANOVA using Microsoft Excel 2000 spreadsheets.

Lysyl oxidase activity assay. LOX activity of whole-cell lysates was measured using the Amplex Red fluorescence assay (Molecular Probes, Eugene, OR). The assay reaction mixture consisted of 50 mmol/L sodium borate (pH 8.2), 1.2 mol/L urea, 50 μmol/L Amplex Red, 0.1 units/mL horseradish peroxidase, and 10 mmol/L 1,5-diaminopentane substrate. Protein samples were added to the reaction mix and incubated at 37°C. The fluorescent product was excited at 560 nm and the emission was read at 590 nm every 5 minutes for 2 hours using a BMG FLUOstar Optima (Durham, NC). LOX activity was measured as fluorescent units and normalized to untreated controls. Experiments were repeated thrice. Statistical significance was evaluated by ANOVA using Microsoft Excel 2000 spreadsheets.

In vitro cell-matrix adhesion assay. Cell-matrix adhesion formation was measured as previously described (25). Where indicated, Hs578T, MDA-MB-231, and MCF-7/LOX32-His cells were treated with βAPN for 24 hours before the assay. Percentage of adherent cells was determined by calculating the number of adherent cells and normalizing to untreated controls (100%). Experiments were repeated thrice. Statistical significance was evaluated by ANOVA using Microsoft Excel 2000 spreadsheets.

Electrophoresis and immunoblotting. Cells were plated on fibronectin-coated flasks and allowed to grow for 24 hours. Where indicated, Hs578T, MDA-MB-231, and MCF-7/LOX32-His cells were treated with βAPN during this time. Whole-cell lysate collection and immunoblot analysis were done as previously described (25). Blots were incubated with anti-FAK (pTyr⁵⁷⁶ or pTyr³⁹⁷, 1:1,000; Biosource, Camarillo, CA), anti-FAK (1:500; BD PharMingen, San Diego, CA), anti-Src (pTyr⁴¹⁸, 1:1,000; Biosource), anti-Src (1:250; Upstate, Lake Placid, NY), or anti-actin (1:10,000; Chemicon Intl., Temecula, CA).

Catalase treatment. Cells were plated on fibronectin-coated dishes and, where indicated, treated once with increasing concentrations of catalase (26). Cells were then allowed to grow for 24 hours for protein lysate collection or adhere for 45 minutes for adhesion assay. Whole-cell lysate collection, immunoblot analysis, and adhesion assays were done as described above.

Intracellular expression of lysyl oxidase in in vivo metastatic breast cancer cells. We previously showed that LOX mRNA was increased in invasive breast cancer cells compared with poorly invasive cells. To validate these observations in clinically relevant breast cancer tissues, we examined LOX protein expression in normal mammary tissue compared with primary and recurrent breast cancer tissues. Approximately half of these recurrent breast cancer tissues were taken from distant metastatic sites. Normal mammary tissue had very low levels of LOX expression and this expression was localized to the stroma and the luminal layer of epithelial cells compared with negative controls (Fig. 1A and B). In contrast, breast cancer tumor tissues showed increased LOX expression, which was observed in the cytoplasm and nuclei of cells (Fig. 1C-H). Of the tumor tissues examined for LOX expression, 77 were primary breast cancer tissues. Of these, 52% had little to no LOX expression whereas 48% were positive for LOX. Additionally, 39 recurrent breast cancer tissues were examined. Only 23% of these tissues had little to no LOX expression whereas the remaining 77% had high LOX expression. These data show that there is an increase in LOX expression in recurrent metastatic tissues compared with primary tumors. Furthermore, we were able to match 16 primary breast cancer tumors with their recurrent/metastatic tumors from the same patient. Half of these matched samples showed an increase in LOX expression from primary tumor to recurrence (Fig. 1C and D). The remaining 50% maintained similar LOX expression between primary tumor and recurrence (Fig. 1E and F). Finally, LOX was highly expressed in breast cancer metastases to the lung and omentum (Fig. 1G and H). Taken together, these data suggest that LOX is localized intracellularly within breast cancer tumor tissues and LOX expression increases in recurrent metastatic tissue compared with primary tumors.

Lysyl oxidase regulates breast cancer cell motility/migration. We previously showed that βAPN inhibited the ability of Hs578T and MDA-MB-231 cells to invade a collagen IV/gelatin/laminin matrix (9). The in vitro invasion assay in the previous study measured the end point and the culmination of a number of biological activities required for invasion—primarily cell-extracellular matrix attachment, extracellular matrix degradation, and migration. Because LOX has previously been shown to promote motility in nontransformed cells (5–7), we used two different in vitro motility/migration assays to determine whether the mechanism of action by which LOX promotes cellular invasion is through facilitation of a motogenic phenotype. Using the cell motility HitKit, haptokinesis of cells (motility tracks) on a collagen I matrix by displacement of fluorescent beads is evident (Fig. 2A). Subsequent treatment of the invasive Hs578T and MDA-MB-231 breast cancer cell lines with βAPN led to a significant decrease in cell motility (Fig. 2B). A similar decrease in motility was observed when βAPN-treated Hs578T and MDA-MB-231 cells were plated onto fibronectin-coated plates (data not shown), suggesting that the type of extracellular matrix does not affect LOX-facilitated haptokinetic motility in breast cancer cells. Conversely, when the poorly invasive breast cancer cell line MCF-7 was stably transfected with the 32 kDa active form of LOX (MCF-7/LOX32-His), cell motility was significantly increased compared with mock-transfected and untransfected cells, as well as MCF-7 cells transfected with the 50-kDa inactive LOX proenzyme (MCF-7/LOX50-His; Fig. 2C). Moreover, this increase in cell motility was reversible on βAPN treatment (Fig. 2C). Multiple stably transfected MCF-7/LOX32-His and MCF-7/LOX50-His clones were tested and similar results were obtained (data not shown).

We also measured the ability of cells to migrate through polycarbonate filters (10 μm pore size) soaked with 0.01% gelatin within 5 hours. Treatment of the invasive breast cancer cell lines Hs578T and MDA-MB-231 with βAPN significantly decreased migration (Fig. 2D). Furthermore, the migration of MCF-7/LOX32-His cells was significantly increased compared with mock-transfected and untransfected controls, as well as MCF-7/LOX50-His cells. Subsequently, this increase in cell migration was reversible on βAPN treatment (Fig. 2E). Intracellular LOX activity was measured in all cell lines and showed that treatment with βAPN led to a significant decrease in intracellular activity compared with untreated controls (Fig. 2F and G). We have previously determined that the inhibitory affect of βAPN on breast cancer invasion was not attributed to cytotoxicity at the concentrations tested (9). Additionally, MCF-7/LOX32-His cells had a 2-fold increase in LOX activity compared with mock-transfected and untransfected controls, as well as MCF-7/LOX50-His cells (Fig. 2G). Exogenous LOX expression was also measured in transfected cells using real-time PCR and was shown to be expressed at levels similar to endogenous LOX expression in MDA-MB-231 cells (data not shown). Taken together, catalytically active LOX enhances invasive breast cancer cell motogenic activity.

Lysyl oxidase regulates cell-matrix adhesion in breast cancer cells. To clearly define which aspect of cell motility was affected by LOX catalytic activity, we measured cell-matrix adhesion in invasive breast cancer cells treated with βAPN. As shown in Fig. 3A, βAPN treatment of the invasive breast cancer cell lines MDA-MB-231 and Hs578T led to a significant decrease in cell adhesion to a fibronectin matrix. These results were recapitulated on a collagen I matrix (data not shown). Cell-matrix adhesion in MCF-7/LOX32-His cells was significantly higher than untransfected, mock-transfected, and MCF-7/LOX50-His cells (Fig. 3B). Furthermore, treatment with βAPN inhibited this increase in cell-matrix adhesion, showing that LOX expression and catalytic activity regulates cell-matrix adhesion in breast cancer cell lines (Fig. 3B). MCF-7/LOX32-His cells were able to adhere to fibronectin and collagen I at the same rate as MDA-MB-231 and Hs578T cells (data not shown). These data show that the stable transfection of catalytically active LOX has allowed the poorly invasive MCF-7 cells to function in a manner similar to invasive breast cancer cell lines. Collectively, LOX expression and activation facilitates cell-matrix adhesion in breast cancer cells.

Lysyl oxidase activity facilitates focal adhesion kinase and Src activity in breast cancer cells. Because cell-matrix adhesion was altered by LOX catalytic activity in breast cancer cell lines, we examined FAK expression and activation as a candidate in the signaling pathway regulated by LOX. FAK has been previously shown to play an important role in adhesion formation and turnover (13). We observed that treatment of the invasive breast cancer cells Hs578T and MDA-MB-231 with βAPN did not lead to a change in FAK expression or in phosphorylation of Tyr³⁹⁷, its major autophosphorylation site (Fig. 3C). However, there was an inhibition in phosphorylation of Tyr⁵⁷⁶, located in the kinase domain of FAK, when LOX activity was inhibited (Fig. 3C), which shows a decrease in FAK catalytic activity. Additionally, MCF-7/LOX32-His cells showed an increase in phosphorylation of Tyr⁵⁷⁶ compared with untransfected and MCF-7/LOX50-His controls. Moreover, this phosphorylation was inhibited with βAPN treatment (Fig. 3D). A slight change in FAK expression was observed in MCF-7/LOX32-His cells; however, this did not lead to any changes in the phosphorylation of Tyr³⁹⁷ (Fig. 3D). Additionally, on longer exposure time of immunoblots, FAK was shown to be expressed in all cell lines (data not shown).

Because phosphorylation of FAK at Tyr⁵⁷⁶ was affected by LOX activity, we examined the regulation of Src expression and activity as an additional signaling pathway candidate. Active Src is recruited to autophosphorylated FAK and leads to the phosphorylation of FAK within the kinase domain and subsequent catalytic activation (17). Therefore, we measured Tyr⁴¹⁸ phosphorylation of Src in invasive breast cancer cells treated with βAPN as an indicator of Src activity. We observed that Src activation is inhibited in MDA-MB-231 and Hs578T cells treated with βAPN compared with untreated controls (Fig. 3E). Additionally, Src protein expression was not affected by βAPN treatment (Fig. 3E). Conversely, MCF-7/LOX32-His cells showed an increase in Src phosphorylation/activation compared with untransfected and MCF-7/LOX50-His controls, which was reversed on βAPN treatment (Fig. 3F). No change in Src protein expression was observed (Fig. 3F). Collectively, these data show that LOX activity facilitates Src activation, which in turn leads to FAK activation in breast cancer cells.

Lysyl oxidase regulates Src activation through a hydrogen peroxide–mediated mechanism. Because LOX activity did not affect FAK autophosphorylation, but rather its catalytic activation by Src, we hypothesized that LOX regulated FAK/Src signaling through an interaction with Src. LOX produces hydrogen peroxide on regeneration of catalytic activity (2) and it has recently been shown that the presence of hydrogen peroxide can facilitate Src activation (15). Therefore, we examined the ability of hydrogen peroxide to regulate Src activation in our cell lines.

Treatment of the Hs578T, MDA-MB-231, and MCF-7/LOX32-His cell lines with increasing concentrations of catalase (which catalyzes the decomposition of hydrogen peroxide into molecular oxygen and water) led to a dose-dependent decrease in Src activation (Fig. 4A). Conversely, untreated poorly invasive MCF-7 cells and MCF-7/LOX50-His cells did not show any Src activation and were not affected by catalase treatments (data not shown). Moreover, in MCF-7/LOX32-His cells expressing low levels of LOX (clone 7), a low dose (1 unit/mL) of catalase led to the loss of Src phosphorylation (Fig. 4A). Conversely, in MCF-7/LOX32-His cells expressing high amounts of LOX (clone 170), an increased amount of catalase (100 units/mL) was required to decrease Src phosphorylation (Fig. 4A). We verified that LOX activity is increased in the high LOX-expressing cells compared with the lower-expressing cells (data not shown). These data suggest that the requirement for increased amounts of catalase to inhibit Src phosphorylation in high LOX-expressing cells is due to the increased production of hydrogen peroxide through increased LOX catalytic activity. Taken together, we show that LOX regulates Src activation through a hydrogen peroxide–mediated mechanism.

To further elucidate the mechanism by which LOX mediates cell adhesion, we examined the effect of catalase on cell-matrix adhesion. Figure 4B shows that treatment of Hs578T, MDA-MB-231, and MCF-7/LOX32-His cells with catalase led to a significant inhibition of cell adhesion. Taken together, these data show that the production of hydrogen peroxide by catalytically active LOX regulates Src activation and leads to an increase in cellular adhesion.

Breast cancer is the second leading cause of cancer death in women; however, mortality rates have begun to decline since 1995 (27). This decrease in breast cancer mortality rates is presumed to be due to earlier detection and improved treatment with a remarkable 97% 5-year survival rate for patients with localized breast cancer. Regardless of improved treatment regimens, the 5-year survival rate for breast cancer patients with regional tumor invasion decreases to 78%, and for women with distant metastases the survival rate decreases to 23%. These decreased survival rates underscore our lack of understanding of breast cancer biology and disease progression and accentuate the need for directed anti-invasive/metastatic therapeutic modalities. Therefore, it is important to have a better understanding of the molecular mechanisms underlying breast cancer metastasis for the design of novel therapeutics. Toward this goal, our study shows that LOX expression is increased in breast cancer tumor tissues compared with normal tissues. Additionally, LOX is expressed intracellularly within breast cancer cells and facilitates cell migration through the regulation of cell-matrix adhesion formation. Furthermore, the changes in cell-matrix adhesion are regulated by the FAK/Src signaling pathway via a hydrogen peroxide–mediated mechanism.

Histologic data presented in Fig. 1 showed that LOX expression was specific to the luminal layer of normal breast tissues compared with a dispersed localization in tumor tissues. Additionally, LOX expression was observed to a much higher extent in recurrent breast cancer tissues compared with primary tumor tissues. Furthermore, LOX staining was present in all primary tumor tissues that eventually progressed to a recurrent cancer, thus showing the possibility that LOX may eventually be used as a prognostic factor in the treatment of breast cancer. However, this finding must be further investigated with a larger cohort of matched primary and recurrent breast cancer tissues.

We further showed that LOX regulates cell motility/migration by inhibiting LOX activity with βAPN and showing a corresponding decrease in cell motility and migration. Additionally, transfection of the active 32-kDa LOX enzyme, but not of the inactive 50-kDa proenzyme, led to a significant increase in motility and migration compared with controls.

Our study also addressed the role of LOX in cell migration by focusing on cell-matrix adhesion. We observed that inhibition of LOX activity led to a significant decrease in cell adhesion to a collagen I or fibronectin matrix. Additionally, transfection of LOX into poorly invasive breast cancer cells led to a significant increase in adhesion. Furthermore, we show that LOX modulates these changes in cell-matrix adhesion through the regulation of FAK and Src kinases. FAK and Src kinases are two key proteins involved in cell adhesion formation and turnover, and here we show that LOX activity facilitates FAK and Src phosphorylation and activation.

Finally, we show that LOX regulates Src phosphorylation and adhesion through a hydrogen peroxide–mediated mechanism. Removal of hydrogen peroxide by catalase in the invasive breast cancer cells led to a dose-dependent decrease in Src phosphorylation and activation. Additionally, treatment of these same cells with catalase led to a significant inhibition in cell adhesion. Taken together, the data show that LOX regulates cell adhesion through a hydrogen peroxide–mediated mechanism that activates Src kinase leading to downstream changes in cell adhesion and migration.

Our hypothetical model of the mechanism by which LOX regulates cell migration and adhesion is depicted in Fig. 5. In this model, the 32-kDa active LOX enzyme is generated through the cleavage of the 50-kDa proenzyme by bone morphogenetic protein-1 in the extracellular matrix. Previous data have shown that active LOX can subsequently be translocated from the extracellular matrix into the cytoplasm and nucleus of cells (23). Full catalytic activation of LOX requires the binding of a copper ion in its copper binding domain, as well as the binding of a lysine residue in its LTQ domain. This lysine residue eventually becomes oxidized, changing from an amine to an aldehyde, leading to the production of hydrogen peroxide and ammonia as by-products (3). Our model shows that intracellular LOX interacts with a currently unknown protein(s), which leads to the increased production of hydrogen peroxide within the cell. This excess peroxide facilitates the phosphorylation and activation of Src. Consequently, active Src phosphorylates and activates FAK, leading to the induction of various signaling pathways which regulate cell adhesion and migration.

Reactive oxygen species, such as hydrogen peroxide, have long been known to cause changes in cell adhesion and migration (26, 28). These changes occur through the regulation of various signaling pathways involved in these processes (29). Specifically, hydrogen peroxide functions by activating various protein tyrosine kinases within cells. Indeed, both Src and FAK have been shown to be regulated by hydrogen peroxide (15, 16, 30). Although the specific mechanism by which hydrogen peroxide activates these proteins is not understood, it is known that hydrogen peroxide increases phosphorylation of various protein tyrosine kinases, thus leading to their activation.

The data presented here show that LOX regulates Src activation through the production of hydrogen peroxide. However, this information leads to another question: what intracellular protein(s) is LOX interacting with in these cells to produce hydrogen peroxide? Previous work has shown that LOX can interact with a variety of basic, globular proteins that include histones H1 and H2 (8). Additionally, LOX has been shown to oxidize basic fibroblast growth factor and to interact with cellular fibronectin (22, 31). It is also possible that LOX may be generating hydrogen peroxide outside of the cell and this peroxide may enter the cell, causing the functional changes observed here. However, the increases in intracellular LOX activity observed in our cells, as well as previous data showing that LOX can interact with intracellular proteins, suggest that LOX is producing this peroxide intracellularly and that this excess leads to the changes observed in Src activation and adhesion formation.

It is also possible that LOX may regulate Src through additional mechanisms, such as an interaction with integrins and the stimulation of outside-in signaling cascades. Integrins are a family of transmembrane proteins that serve as a link between the cellular cytoskeleton and the extracellular matrix and are central to regulating adhesion and cell migration (reviewed in ref. 32). One of the major proteins that integrins recruit on cell adhesion is FAK. Therefore, LOX-integrin relationships are an important area that remains to be further studied and developed. Taken together, these data show the significance of identifying the proteins that LOX may functionally interact with to elucidate the multiple roles that LOX may have both intracellularly and extracellularly.

A growing number of researchers have documented the role of copper in cancer metastasis. Copper is a highly regulated trace element in most organisms. Additionally, several proteins, including LOX, require copper binding for full catalytic activity (3). Therefore, copper balance can regulate protein activity. One of the first clinical signs of copper deficiency involves connective tissue defects. This may be explained by the loss of LOX activity (as well as other proteins) which regulates collagen and elastin cross-linking. Copper has been shown to stimulate proliferation and migration in endothelial cells (33, 34). However, it is currently unknown how copper directly regulates these processes. The data presented here show a possible mechanism by which copper can regulate LOX activity which will lead to changes in cell migration. Copper has also been shown to be a potent inducer of angiogenesis, a key process leading to a more aggressive cancer phenotype (35, 36). In fact, copper deficiency therapies are now being tested in clinical trials to inhibit cancer progression (37). Previous animal models have shown that copper depletion suppresses tumor growth and may combine with other cytotoxic therapies with additive effects (38–40). Phase I and II trials have recently been completed in the study of copper deficiency as an anticancer therapy (41, 42). Both studies have shown that anticopper therapy leads to a stable disease. However, no patients have shown a partial or complete response. Therefore, copper depletion therapies may have a cytostatic, rather than cytotoxic, effect. These data correspond to observations made in the laboratory in which inhibition of LOX activity by βAPN does not seem to have any cytotoxic or growth-altering effect (9). Taken together, these data show that inhibition of copper leads to a loss of LOX activity, which may explain some of the clinical findings observed in patients, such as changes in cell migration.

The results shown here are consistent with our hypothesis that LOX is up-regulated in breast cancer cells with metastatic ability and that LOX facilitates breast cancer cell migration and adhesion through the hydrogen peroxide–mediated regulation of the FAK/Src signaling pathway. Elucidation of the mechanism(s) by which LOX facilitates breast cancer cell migration is essential for our understanding of tumor cell progression and raises the possibility of targeting LOX expression in tumor cells either as a predictive/prognostic indicator of metastasis or for development of a novel antimetastatic therapy.

Grant support: DAMD17-99-1-9225 and Eisenberg Scholar Research Award (D.A. Kirschmann), NIH grants AR47713 and G12RR03961 (K. Csiszar), the American Heart Association grant 0315258Z (B. Fogelgren), Specialized Program of Research Excellence in Breast Cancer National Cancer Institute grant 5-P50-CA089018-05 (E.L. Wiley), and the Michael Sweig Foundation (M.J.C. Hendrix).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Kenneth L. van Golen, Lynne-Marie Postovit, Naira V. Margaryan, Lisa M.J. Lee, and Keith S.K. Fong for helpful scientific discussions.