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

Lysyl Oxidase Regulates Breast Cancer Cell Migration and Adhesion

through a Hydrogen Peroxide–Mediated Mechanism

Stacey L. Payne,1,2Ben Fogelgren,

3Angela R. Hess,

1Elisabeth A. Seftor,

1Elizabeth L. Wiley,

1

Sheri F.T. Fong,3Katalin Csiszar,

3Mary J.C. Hendrix,

1and Dawn A. Kirschmann

1

1Children’s Memorial Research Center, Cancer Biology and Epigenomics Program, Robert H. Lurie Comprehensive Cancer Center,Feinberg School of Medicine at Northwestern University, Chicago, Illinois; 2Department of Anatomy and Cell Biology,The Roy J. and Lucille A. Carver College of Medicine at The University of Iowa, Iowa City, Iowa; and 3CardiovascularResearch Center, John A. Burns School of Medicine, The University of Hawaii, Honolulu, Hawaii

Abstract

We have previously shown that lysyl oxidase (LOX) mRNA isup-regulated in invasive breast cancer cells and that cataly-tically active LOX facilitates in vitro cell invasion. Here wevalidate our in vitro studies by showing that LOX expressionis up-regulated in distant metastatic breast cancer tissuescompared with primary cancer tissues. To elucidate themechanism by which LOX facilitates cell invasion, we showthat catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment ofthe invasive breast cancer cell lines, Hs578T and MDA-MB-231,with B-aminopropionitrile (BAPN), an irreversible inhibitorof LOX catalytic activity, leads to a significant decrease in cellmotility/migration and adhesion formation. Conversely,poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that wasreversible with the addition of BAPN. Moreover, a decrease inactivated focal adhesion kinase (FAK) and Src kinase, keyproteins involved in adhesion complex turnover, was ob-served when invasive breast cancer cells were treated withBAPN. Additionally, FAK and Src activation was increased inMCF-7/LOX32-His cells, which was reversible on BAPN treat-ment. Hydrogen peroxide was produced as a by-product ofLOX activity and the removal of hydrogen peroxide bycatalase treatment in invasive breast cancer cells led to adose-dependent loss in Src activation. These results suggestthat LOX facilitates migration and cell-matrix adhesionformation in invasive breast cancer cells through a hydro-gen peroxide–mediated mechanism involving the FAK/Srcsignaling pathway. These data show the need to target LOXfor treatment of aggressive breast cancer. (Cancer Res 2005;65(24): 11429-36)

Introduction

Lysyl oxidase (LOX) was initially reported as a copper-dependentamine oxidase responsible for the catalysis of collagen and elastincross-linking within the extracellular matrix (reviewed in refs. 1–3).In this process, LOX catalyzes the exchange of an amine to analdehyde group on a peptidyl lysine, producing hydrogen peroxideand ammonia as by-products of catalytic activity. However, recentwork has shown that LOX may have intracellular functionsincluding 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 cancercell lines Hs578T and MDA-MB-231 compared with the poorlyinvasive cell lines MCF-7 and T-47D (9). Inhibition of LOX withh-aminopropionitrile (hAPN; an irreversible inhibitor of LOXcatalytic activity) or LOX-specific antisense oligonucleotides ledto a significant decrease in the invasive potential of Hs578T andMDA-MB-231 cells. Conversely, exogenous expression of LOX in thepoorly invasive MCF-7 cell line resulted in an increase in invasiveactivity that was reversible on treatment with hAPN. These resultsshowed that LOX was essential for breast cancer cell invasion butthe 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 extracel-lular matrix, and finally active migration away from the primarytumor (10). Therefore, the ability of a cell to move on its ownaccord is a critical factor in tumor progression from a non-metastatic to metastatic state. A key process in basic cell migrationis the ability of a cell to form a stable adhesion to the extracellularmatrix (11). This process is regulated by two key proteins withincells: Src and focal adhesion kinase (FAK; refs. 12, 13). Inactivationof either of these proteins leads to a dramatic loss in the abilityof a cell to form adhesion complexes. Src and FAK are activatedthrough a series of phosphorylation events. Previous work hasshown that Src is phosphorylated and activated by variousstimuli including integrins, phosphatases, and hydrogen peroxide(12, 14–16). FAK becomes catalytically active through an initialautophosphorylation at Tyr397. On phosphorylation and activationof Src, autophosphorylated FAK is then phosphorylated by Src inthe kinase domain of FAK (17). The activation of the FAK/Srcsignaling pathway leads to the activation and regulation of varioussignaling 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 activeLOX is required for breast cancer cell migration and adhesionformation. Additionally, LOX regulates adhesion formation via theFAK/Src signaling pathway through a hydrogen peroxide–mediatedmechanism. Understanding the molecular mechanisms of breastcancer cell migration may lead to novel therapeutic strategies inthe treatment of metastatic breast cancer.

Materials and Methods

Cells and culture conditions. Breast cancer cell lines were obtained

and maintained as previously described (9). MCF-7 cells were stablytransfected with 1 Ag 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

Requests for reprints: Dawn A. Kirschmann, Children’s Memorial ResearchCenter, 2300 Children’s Plaza, Box 222, Chicago, IL 60614. Phone: 773-755-6558; Fax:773-755-6594; E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1274

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selected with 400 Ag/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 Ag/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 f80% confluent.

LOX32-His and LOX50-His constructs. PCR primers were designed to

introduce EcoRI and XhoI restriction sites at the 5Vand 3V termini of aLOX 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 byDNA 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 weredeparaffinized, rehydrated in distilled water, and antigen retrieval was

done in citrate buffer (pH 6.0) at 97jC. 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 (3,3V-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 microscopeand 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 hAPN (9, 22, 23). Each variable was assayed in triplicate and

100 cells per treatment group were evaluated for motility. Experiments wererepeated 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 hAPN 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 wasmeasured 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 Amol/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 37jC. Thefluorescent 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 formationwas measured as previously described (25). Where indicated, Hs578T, MDA-

MB-231, and MCF-7/LOX32-His cells were treated with hAPN 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 hAPN 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; BDPharMingen, 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 lysatecollection, immunoblot analysis, and adhesion assays were done as

described above.

Results

Intracellular expression of lysyl oxidase in in vivo metastaticbreast cancer cells. We previously showed that LOX mRNA wasincreased in invasive breast cancer cells compared with poorlyinvasive cells. To validate these observations in clinically relevantbreast cancer tissues, we examined LOX protein expression innormal mammary tissue compared with primary and recurrentbreast cancer tissues. Approximately half of these recurrent breastcancer tissues were taken from distant metastatic sites. Normalmammary tissue had very low levels of LOX expression and thisexpression was localized to the stroma and the luminal layer ofepithelial cells compared with negative controls (Fig. 1A and B).In contrast, breast cancer tumor tissues showed increased LOXexpression, which was observed in the cytoplasm and nuclei of cells(Fig. 1C-H). Of the tumor tissues examined for LOX expression, 77were primary breast cancer tissues. Of these, 52% had little to noLOX expression whereas 48% were positive for LOX. Additionally,39 recurrent breast cancer tissues were examined. Only 23% of thesetissues had little to no LOX expression whereas the remaining 77%had high LOX expression. These data show that there is an increasein LOX expression in recurrent metastatic tissues compared withprimary tumors. Furthermore, we were able to match 16 primarybreast cancer tumors with their recurrent/metastatic tumorsfrom the same patient. Half of these matched samples showed anincrease in LOX expression from primary tumor to recurrence(Fig. 1C and D). The remaining 50% maintained similar LOXexpression between primary tumor and recurrence (Fig. 1E and F).Finally, LOXwas highly expressed in breast cancer metastases to thelung and omentum (Fig. 1G and H). Taken together, these datasuggest that LOX is localized intracellularly within breast cancertumor tissues and LOX expression increases in recurrent metastatictissue compared with primary tumors.Lysyl oxidase regulates breast cancer cell motility/migration.

We previously showed that hAPN inhibited the ability of Hs578Tand MDA-MB-231 cells to invade a collagen IV/gelatin/lamininmatrix (9). The in vitro invasion assay in the previous studymeasuredthe end point and the culmination of a number of biologicalactivities required for invasion—primarily cell-extracellular matrixattachment, extracellular matrix degradation, and migration.

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Cancer Res 2005; 65: (24). December 15, 2005 11430 www.aacrjournals.org



Because LOX has previously been shown to promote motility innontransformed cells (5–7), we used two different in vitro motility/migration assays to determine whether the mechanism of actionby which LOX promotes cellular invasion is through facilitation ofa motogenic phenotype. Using the cell motility HitKit, haptokinesisof cells (motility tracks) on a collagen I matrix by displacementof fluorescent beads is evident (Fig. 2A). Subsequent treatment ofthe invasive Hs578T and MDA-MB-231 breast cancer cell lineswith hAPN led to a significant decrease in cell motility (Fig. 2B).A similar decrease in motility was observed when hAPN-treatedHs578Tand MDA-MB-231 cells were plated onto fibronectin-coatedplates (data not shown), suggesting that the type of extracellularmatrix does not affect LOX-facilitated haptokineticmotility in breastcancer cells. Conversely, when the poorly invasive breast cancer cellline MCF-7 was stably transfected with the 32 kDa active form ofLOX (MCF-7/LOX32-His), cell motility was significantly increased

compared with mock-transfected and untransfected cells, as well asMCF-7 cells transfected with the 50-kDa inactive LOX proenzyme(MCF-7/LOX50-His; Fig. 2C). Moreover, this increase in cell motilitywas reversible on hAPN treatment (Fig. 2C). Multiple stablytransfected MCF-7/LOX32-His and MCF-7/LOX50-His clones weretested and similar results were obtained (data not shown).We also measured the ability of cells to migrate through

polycarbonate filters (10 Am pore size) soaked with 0.01% gelatinwithin 5 hours. Treatment of the invasive breast cancer cell linesHs578T and MDA-MB-231 with hAPN significantly decreasedmigration (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 wasreversible on hAPN treatment (Fig. 2E). Intracellular LOX activitywas measured in all cell lines and showed that treatment with

Figure 1. Intracellular expression of LOX in in vivo breastcancer cells. Immunohistochemistry of LOX expression innormal mammary tissue (A) compared with a rabbit IgGnegative control (B ). C, an example of a primary tumortissue with low LOX expression compared with an increasein expression in its matched recurrent breast cancer tissue(D ). E, an example of a primary tumor tissue with similarLOX expression to its matched recurrent breast cancertissue (F ). G, an unmatched breast cancer metastasis tothe lung and omentum (H ). Magnification, all �40 exceptomentum (�20).

Lysyl Oxidase Regulates Cancer Cell Migration




hAPN led to a significant decrease in intracellular activitycompared with untreated controls (Fig. 2F and G). We havepreviously determined that the inhibitory affect of hAPN onbreast cancer invasion was not attributed to cytotoxicity at theconcentrations tested (9). Additionally, MCF-7/LOX32-His cellshad 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 measuredin transfected cells using real-time PCR and was shown to beexpressed at levels similar to endogenous LOX expression in MDA-MB-231 cells (data not shown). Taken together, catalytically activeLOX enhances invasive breast cancer cell motogenic activity.

Lysyl oxidase regulates cell-matrix adhesion in breastcancer cells. To clearly define which aspect of cell motility wasaffected by LOX catalytic activity, we measured cell-matrixadhesion in invasive breast cancer cells treated with hAPN. Asshown in Fig. 3A , hAPN treatment of the invasive breast cancer celllines MDA-MB-231 and Hs578T led to a significant decrease in celladhesion to a fibronectin matrix. These results were recapitulatedon a collagen I matrix (data not shown). Cell-matrix adhesion inMCF-7/LOX32-His cells was significantly higher than untrans-fected, mock-transfected, and MCF-7/LOX50-His cells (Fig. 3B).Furthermore, treatment with hAPN inhibited this increase in cell-matrix adhesion, showing that LOX expression and catalytic

Figure 2. LOX catalytic activity regulatesbreast cancer cell motility/migration. A, motilitytrack of a migratory MDA-MB-231 cell (arrow )and nonmotile MDA-MB-231 cell (arrowhead )as visualized using the cell motility HitKit.Haptotactic motility of untreated andhAPN-treated Hs578T and MDA-MB-231breast cancer cell lines (B ) and MCF-7/LOX32-His–transfected cells (C ) as analyzedusing the cell motility HitKit. Cell motility wascalculated as the total number of migratorytracks / total number of cells counted � 100and normalized to untreated controls (100%).Migratory potential of untreated andhAPN-treated Hs578T and MDA-MB-231breast cancer cell lines (D ) and poorly invasiveMCF-7 cells compared with dsRed-transfected(Mock), MCF-7/LOX50-His, and MCF-7/LOX32-His cells treated with hAPN (E).Migration was calculated as the total numberof cells that migrated through a 0.01%gelatin-coated polycarbonate filter (10 Am poresize) within 5 hours and normalized to theuntreated control (100%). Intracellular LOXcatalytic activity measured in whole-celllysates of untreated and hAPN-treatedHs578T and MDA-MB-231 breast cancercell lines (F ) and poorly invasive MCF-7cells, dsRed-transfected (Mock), MCF-7/LOX50-His, MCF-7/LOX32-His cells, andMCF-7/LOX32-His cells treated with hAPN(G ). *, P < 0.05, compared with untreatedcontrols (ANOVA). **, P < 0.05, compared withMCF-7/LOX32-His untreated controls(ANOVA).

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activity regulates cell-matrix adhesion in breast cancer cell lines(Fig. 3B). MCF-7/LOX32-His cells were able to adhere to fibronectinand collagen I at the same rate as MDA-MB-231 and Hs578T cells(data not shown). These data show that the stable transfection ofcatalytically active LOX has allowed the poorly invasive MCF-7 cellsto function in a manner similar to invasive breast cancer cell lines.Collectively, LOX expression and activation facilitates cell-matrixadhesion in breast cancer cells.Lysyl oxidase activity facilitates focal adhesion kinase and

Src activity in breast cancer cells. Because cell-matrixadhesion was altered by LOX catalytic activity in breast cancercell lines, we examined FAK expression and activation as acandidate in the signaling pathway regulated by LOX. FAK hasbeen previously shown to play an important role in adhesionformation and turnover (13). We observed that treatment of theinvasive breast cancer cells Hs578T and MDA-MB-231 with hAPNdid not lead to a change in FAK expression or in phosphory-lation 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 wasinhibited (Fig. 3C), which shows a decrease in FAK catalyticactivity. Additionally, MCF-7/LOX32-His cells showed an increasein phosphorylation of Tyr576 compared with untransfected and

MCF-7/LOX50-His controls. Moreover, this phosphorylation wasinhibited with hAPN treatment (Fig. 3D). A slight change in FAKexpression was observed in MCF-7/LOX32-His cells; however, thisdid 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 notshown).Because phosphorylation of FAK at Tyr576 was affected by LOX

activity, we examined the regulation of Src expression and activityas an additional signaling pathway candidate. Active Src isrecruited to autophosphorylated FAK and leads to the phosphor-ylation of FAK within the kinase domain and subsequent catalyticactivation (17). Therefore, we measured Tyr418 phosphorylationof Src in invasive breast cancer cells treated with hAPN as anindicator of Src activity. We observed that Src activation isinhibited in MDA-MB-231 and Hs578T cells treated with hAPNcompared with untreated controls (Fig. 3E). Additionally, Srcprotein expression was not affected by hAPN treatment (Fig. 3E).Conversely, MCF-7/LOX32-His cells showed an increase in Srcphosphorylation/activation compared with untransfected andMCF-7/LOX50-His controls, which was reversed on hAPN treat-ment (Fig. 3F). No change in Src protein expression was observed(Fig. 3F). Collectively, these data show that LOX activity facilitates

Figure 3. LOX catalytic activity regulates breastcancer cell-matrix adhesion and activates FAK andSrc kinases. Cell adhesion of untreated andhAPN-treated Hs578T and MDA-MB-231 breastcancer cell lines (A) and poorly invasiveMCF-7 cells, dsRed-transfected (Mock), MCF-7/LOX50-His, and MCF-7/LOX32-His cells treatedwith hAPN (B ). Adhesion was calculated asthe number of cells that adhered to afibronectin-coated plate after 45 minutes andnormalized to the untreated control (100%).Immunoblot analysis of FAK expression andactivation of untreated and hAPN-treated Hs578Tand MDA-MB-231 invasive breast cancer cell lines(C ) and MCF-7, MCF-7/LOX32-His treated withhAPN, and MCF-7/LOX50-His cells (D ). Srcexpression and activation of untreated andhAPN-treated Hs578T and MDA-MB-231 invasivebreast cancer cell lines (E ) and MCF-7, untreated,and hAPN-treated MCF-7/LOX32-His cells andMCF-7/LOX50-His cells (F ). Cells were culturedon fibronectin and treated with hAPN (whereindicated) for 24 hours. Whole-cell lysates werecollected and electrophoresed on a 7.5% (FAK) or10% (Src) SDS-polyacrylamide gel and probedwith phosphospecific antibodies (P-FAK Y397,P-FAK Y576, or P-Src Y418), stripped and reprobedwith anti-FAK or Src antibodies (FAK, Src), andrestripped and reprobed with an anti-actin antibodyto control for equal loading. *, P < 0.05, comparedwith untreated controls (ANOVA). **, P < 0.05,compared with MCF-7/LOX32-His untreatedcontrols (ANOVA).





Src activation, which in turn leads to FAK activation in breastcancer cells.Lysyl oxidase regulates Src activation through a hydrogen

peroxide–mediated mechanism. Because LOX activity did notaffect FAK autophosphorylation, but rather its catalytic activationby Src, we hypothesized that LOX regulated FAK/Src signalingthrough an interaction with Src. LOX produces hydrogen peroxideon regeneration of catalytic activity (2) and it has recently beenshown that the presence of hydrogen peroxide can facilitate Srcactivation (15). Therefore, we examined the ability of hydrogenperoxide 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 (whichcatalyzes the decomposition of hydrogen peroxide into molecularoxygen and water) led to a dose-dependent decrease in Srcactivation (Fig. 4A). Conversely, untreated poorly invasive MCF-7cells and MCF-7/LOX50-His cells did not show any Src activationand 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 ofSrc phosphorylation (Fig. 4A). Conversely, in MCF-7/LOX32-Hiscells expressing high amounts of LOX (clone 170), an increasedamount of catalase (100 units/mL) was required to decrease Srcphosphorylation (Fig. 4A). We verified that LOX activity isincreased in the high LOX-expressing cells compared with thelower-expressing cells (data not shown). These data suggest thatthe requirement for increased amounts of catalase to inhibit Srcphosphorylation in high LOX-expressing cells is due to theincreased production of hydrogen peroxide through increasedLOX catalytic activity. Taken together, we show that LOX regulatesSrc 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-matrixadhesion. Figure 4B shows that treatment of Hs578T, MDA-MB-231, and MCF-7/LOX32-His cells with catalase led to a significantinhibition of cell adhesion. Taken together, these data show thatthe production of hydrogen peroxide by catalytically active LOXregulates Src activation and leads to an increase in cellularadhesion.

Discussion

Breast cancer is the second leading cause of cancer death inwomen; however, mortality rates have begun to decline since 1995(27). This decrease in breast cancer mortality rates is presumed tobe due to earlier detection and improved treatment with aremarkable 97% 5-year survival rate for patients with localizedbreast cancer. Regardless of improved treatment regimens, the5-year survival rate for breast cancer patients with regional tumorinvasion decreases to 78%, and for women with distant metastasesthe survival rate decreases to 23%. These decreased survival ratesunderscore our lack of understanding of breast cancer biology anddisease progression and accentuate the need for directed anti-invasive/metastatic therapeutic modalities. Therefore, it is impor-tant to have a better understanding of the molecular mechanismsunderlying breast cancer metastasis for the design of noveltherapeutics. Toward this goal, our study shows that LOXexpression is increased in breast cancer tumor tissues comparedwith normal tissues. Additionally, LOX is expressed intracellularlywithin breast cancer cells and facilitates cell migration through theregulation of cell-matrix adhesion formation. Furthermore, thechanges in cell-matrix adhesion are regulated by the FAK/Srcsignaling 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 tissuescompared with a dispersed localization in tumor tissues. Addi-tionally, LOX expression was observed to a much higher extent inrecurrent breast cancer tissues compared with primary tumortissues. Furthermore, LOX staining was present in all primarytumor tissues that eventually progressed to a recurrent cancer,thus showing the possibility that LOX may eventually be used as aprognostic factor in the treatment of breast cancer. However, thisfinding must be further investigated with a larger cohort ofmatched primary and recurrent breast cancer tissues.We further showed that LOX regulates cell motility/migration by

inhibiting LOX activity with hAPN and showing a correspondingdecrease in cell motility and migration. Additionally, transfection ofthe active 32-kDa LOX enzyme, but not of the inactive 50-kDaproenzyme, led to a significant increase in motility and migrationcompared with controls.

Figure 4. LOX regulates Src activation through a hydrogen peroxide–mediated mechanism. A, immunoblot analysis of Src activation and expression of Hs578T,MDA-MB-231, and two clones of MCF-7/LOX32-His cells treated with increasing concentrations of catalase to remove hydrogen peroxide. B, cell-matrix adhesion ofHs578T, MDA-MB-231, and MCF-7/LOX32-His cells treated with catalase. Cells were treated with 100 units/mL catalase and allowed to adhere for 45 minutes.Adhesion was calculated as the number of cells that adhered to a fibronectin-coated plate and normalized to the untreated control (100%). *, P < 0.05, compared withuntreated controls (ANOVA).

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Our study also addressed the role of LOX in cell migration byfocusing on cell-matrix adhesion. We observed that inhibition ofLOX activity led to a significant decrease in cell adhesion to acollagen I or fibronectin matrix. Additionally, transfection of LOXinto poorly invasive breast cancer cells led to a significant increasein adhesion. Furthermore, we show that LOX modulates thesechanges in cell-matrix adhesion through the regulation of FAK andSrc kinases. FAK and Src kinases are two key proteins involved incell adhesion formation and turnover, and here we show that LOXactivity 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 breastcancer cells led to a dose-dependent decrease in Src phosphor-ylation and activation. Additionally, treatment of these same cellswith catalase led to a significant inhibition in cell adhesion. Takentogether, the data show that LOX regulates cell adhesion througha hydrogen peroxide–mediated mechanism that activates Srckinase leading to downstream changes in cell adhesion andmigration.Our hypothetical model of the mechanism by which LOX

regulates cell migration and adhesion is depicted in Fig. 5. In thismodel, the 32-kDa active LOX enzyme is generated through thecleavage of the 50-kDa proenzyme by bone morphogenetic protein-1 in the extracellular matrix. Previous data have shown that activeLOX can subsequently be translocated from the extracellularmatrix into the cytoplasm and nucleus of cells (23). Full catalytic

activation of LOX requires the binding of a copper ion in its copperbinding domain, as well as the binding of a lysine residue in its LTQdomain. This lysine residue eventually becomes oxidized, changingfrom an amine to an aldehyde, leading to the production ofhydrogen peroxide and ammonia as by-products (3). Our modelshows that intracellular LOX interacts with a currently unknownprotein(s), which leads to the increased production of hydrogenperoxide within the cell. This excess peroxide facilitates thephosphorylation and activation of Src. Consequently, active Srcphosphorylates and activates FAK, leading to the induction ofvarious signaling pathways which regulate cell adhesion andmigration.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 varioussignaling pathways involved in these processes (29). Specifically,hydrogen peroxide functions by activating various protein tyrosinekinases within cells. Indeed, both Src and FAK have been shownto be regulated by hydrogen peroxide (15, 16, 30). Although thespecific mechanism by which hydrogen peroxide activates theseproteins is not understood, it is known that hydrogen peroxideincreases phosphorylation of various protein tyrosine kinases, thusleading to their activation.The data presented here show that LOX regulates Src activation

through the production of hydrogen peroxide. However, thisinformation leads to another question: what intracellular protein(s)is LOX interacting with in these cells to produce hydrogenperoxide? Previous work has shown that LOX can interact with avariety of basic, globular proteins that include histones H1 and H2(8). Additionally, LOX has been shown to oxidize basic fibroblastgrowth factor and to interact with cellular fibronectin (22, 31). It isalso possible that LOX may be generating hydrogen peroxideoutside of the cell and this peroxide may enter the cell, causing thefunctional changes observed here. However, the increases inintracellular LOX activity observed in our cells, as well as previousdata showing that LOX can interact with intracellular proteins,suggest that LOX is producing this peroxide intracellularly and thatthis excess leads to the changes observed in Src activation andadhesion formation.It is also possible that LOX may regulate Src through additional

mechanisms, such as an interaction with integrins and thestimulation of outside-in signaling cascades. Integrins are a familyof transmembrane proteins that serve as a link between the cellularcytoskeleton and the extracellular matrix and are central toregulating adhesion and cell migration (reviewed in ref. 32). One ofthe major proteins that integrins recruit on cell adhesion is FAK.Therefore, LOX-integrin relationships are an important area thatremains to be further studied and developed. Taken together, thesedata show the significance of identifying the proteins that LOX mayfunctionally interact with to elucidate the multiple roles that LOXmay have both intracellularly and extracellularly.A growing number of researchers have documented the role of

copper in cancer metastasis. Copper is a highly regulated traceelement in most organisms. Additionally, several proteins, includ-ing LOX, require copper binding for full catalytic activity (3).Therefore, copper balance can regulate protein activity. One of thefirst clinical signs of copper deficiency involves connective tissuedefects. This may be explained by the loss of LOX activity (as wellas other proteins) which regulates collagen and elastin cross-linking. Copper has been shown to stimulate proliferation andmigration in endothelial cells (33, 34). However, it is currently

Figure 5. Putative model of LOX regulation of cell migration. LOX is cleavedfrom a 50-kDa inactive proenzyme to its active 32-kDa enzyme by bonemorphogenetic protein-1 (BMP-1). LOX translocates into the cell where it canenzymatically interact with a target substrate, producing hydrogen peroxide as aby-product. Hydrogen peroxide can then facilitate the phosphorylation andactivation of Src, which can subsequently phosphorylate and activate FAK. Srcand FAK are key proteins known to regulate cell adhesion, and changes inadhesive ability lead to subsequent changes in cell migration.





unknown how copper directly regulates these processes. The datapresented here show a possible mechanism by which copper canregulate LOX activity which will lead to changes in cell migration.Copper has also been shown to be a potent inducer ofangiogenesis, a key process leading to a more aggressive cancerphenotype (35, 36). In fact, copper deficiency therapies are nowbeing tested in clinical trials to inhibit cancer progression (37).Previous animal models have shown that copper depletionsuppresses tumor growth and may combine with other cytotoxictherapies with additive effects (38–40). Phase I and II trials haverecently been completed in the study of copper deficiency as ananticancer therapy (41, 42). Both studies have shown thatanticopper therapy leads to a stable disease. However, no patientshave shown a partial or complete response. Therefore, copperdepletion therapies may have a cytostatic, rather than cytotoxic,effect. These data correspond to observations made in thelaboratory in which inhibition of LOX activity by hAPN does notseem to have any cytotoxic or growth-altering effect (9). Takentogether, these data show that inhibition of copper leads to a lossof LOX activity, which may explain some of the clinical findingsobserved in patients, such as changes in cell migration.

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

Acknowledgments

Received 4/12/2005; revised 9/1/2005; accepted 9/22/2005.Grant support: DAMD17-99-1-9225 and Eisenberg Scholar Research Award (D.A.

Kirschmann), NIH grants AR47713 and G12RR03961 (K. Csiszar), the American HeartAssociation grant 0315258Z (B. Fogelgren), Specialized Program of ResearchExcellence 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 pagecharges. This article must therefore be hereby marked advertisement in accordancewith 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.

Cancer Research


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2005;65:11429-11436. Cancer Res Stacey L. Payne, Ben Fogelgren, Angela R. Hess, et al. Mechanism

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

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