Acquisition of the Metastatic Phenotype Is Accompanied by ... · engages DOC180, a guanine nucleotide exchange factor, to activate Rac-1 and consequential actin rearrangement. Reactive

Signal Transduction

Acquisition of the Metastatic Phenotype Is Accompanied byH2O2-Dependent Activation of the p130Cas SignalingComplex

Nadine Hempel1, Toni R. Bartling1, Badar Mian2, and J. Andres Melendez1

AbstractReactive oxygen species (ROS) have emerged as cellular signaling molecules and are implicated in metastatic

disease by their ability to drive invasion andmigration.Here, we define the signaling adaptor protein p130Cas (Crk-associated substrate) as a key redox-responsivemolecular trigger that is engaged in highly invasivemetastatic bladdertumor cell lines. Endogenous shifts in steady-state hydrogen peroxide (H2O2) that accompany the metastaticphenotype increase p130Cas phosphorylation, membrane recruitment and association with the scaffolding proteinCrk, and subsequent Rac1 activation and actin reorganization. Both enzymatic and nonenzymatic scavenging ofH2

O2 abrogates p130Cas-dependent signaling and themigratory and invasive activity of themetastatic bladder tumorcells. Disruption of p130Cas attenuates both invasion andmigration of the metastatic variant (253J-BV). 253J-BVcells displayed an increase in global thiol oxidation and a concomitant decrease in total phosphatase activity,common target proteins of active-site cysteine oxidation. The dependence of phosphatases on regulation ofp130Cas was highlighted when depletion of PTPN12 enhanced p130cas phosphorylation and the migratorybehavior of a noninvasive parental bladder tumor control (253J). These data show that the metastatic phenotypeis accompanied by increases in steady-state H2O2 production that drive promigratory signaling and suggestthat antioxidant-based therapeuticsmay prove useful in limiting bladder tumor invasiveness.MolCancer Res; 11(3);303–12. �2013 AACR.

IntroductionThe ability of a tumor cell to invade and migrate through

the basement membrane and extracellular matrix (ECM) is avital first step in tumor metastasis. Tumor cell invasion isaccompanied by protease-driven ECM degradation andreorganization of cellular signaling components at the lead-ing and lagging edges of the cell, ultimately leading torestructuring of the actin cytoskeleton and migration. Animportant signaling cascade involved in this process is thefocal adhesion kinase (FAK) pathway. Initiation of thiscascade involves phosphorylation and engagement of thekinases FAK and Src, leading to recruitment of adaptorproteins such as p130cas (Crk-associated substrate) and Crkto the focal contacts at the leading edge. This complexengages DOC180, a guanine nucleotide exchange factor,to activate Rac-1 and consequential actin rearrangement.

Reactive oxygen species (ROS) are known DNA damag-ing agents involved in carcinogenesis. However, at sublethallevels, ROS are implicated in regulating a multitude ofcellular signaling cascades, primarily due to their ability toreversibly oxidize thiolate anions of cysteine residues (1, 2).Endogenous ROS are often elevated in cancer cells and this isaccompanied by alterations in the cells' antioxidant-scav-enging potential (3–5). Thus, cancer cells have uniquelyacquired the ability to survive and even thrive despite thischronic, sublethal oxidant challenge.Intracellular ROS production has been linked to meta-

static disease progression (6). The loss of mitochondrialgenome integrity is accompanied by an increase in ROSlevels that enhance the metastatic potential of tumor cells(7). ROS of both mitochondrial and nonmitochondrialorigin are associated with regulating cellular signaling path-ways, which are implicated in metastatic disease (2, 8). Wehave shown that enhanced invasion and migration areassociated with intrinsic increases in the intracellular hydro-gen peroxide (H2O2) milieu and changes in antioxidantexpression, using a model of metastatic bladder cancerprogression (9, 10). This represents a pathophysiologicallyrelevant model that does not rely on manipulation ofmitochondrial genome integrity (7), NADPH oxidase stim-ulation, following receptor or integrin engagement (6), normanipulating antioxidant expression (11, 12) to changeintracellular redox status. Two-fold picomolar increases inintracellular H2O2 (18 ! 32 pmol/L) in the metastatic

Authors' Affiliations: 1College of Nanoscale Sciences and Engineering,University at Albany, SUNY; and 2Stratton VA Medical Center and AlbanyMedical Center, Albany, New York

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Authors: J. Andres Melendez, College of NanoscaleSciences and Engineering, University at Albany, SUNY, 257 Fuller Rd.,NFE-4313, Albany, NY 12203. Phone: 518-956-7360; Fax: 518-437-8687;E-mail: [email protected]; andNadineHempel, [email protected]

doi: 10.1158/1541-7786.MCR-12-0478

�2013 American Association for Cancer Research.

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bladder cancer cell variant 253J-BV are associated withenhanced matrix metalloproteinase (MMP)-9 and VEGFexpression and increased clonogenicity compared with therelated parental cells 253J (9, 10). Here, we set out to test thehypothesis thatmetastatic cancer cells have uniquely adaptedto thrive with an enhanced intracellular ROS milieu andevolved to use oxidation as a novel mechanism to drivepromigratory signaling events by H2O2-dependent activa-tion of the p130Cas signaling complex.

Materials and MethodsCell culture, treatments, and transfection253J and 253J-BV cells were created and cultured as

described previously (9, 10). Cells were treated with H2O2 (Sigma-Aldrich) in serum-free Dulbecco's modifiedEagle's medium (DMEM). Cells were pretreated in DMEMþ10% FBS with either 500 U/mL bovine catalase (CAT;Sigma-Aldrich) or 2 mmol/L N-acetyl-L-cysteine (NAC;Sigma-Aldrich) for 18 to 24 hours, followed by the sametreatments in serum-free media for the duration of theexperiments, as indicated. Alternatively, CAT or controlb-galactosidase (Lac-Z; MOI 100) were expressed usingadenoviral gene delivery (10). CAT activity was determinedas described previously (10).The following siRNA constructs were transfected into

200,000 cells using RNAi Max Lipofectamine reagent (Invi-trogen Life Technologies): 200 pmol PTPN12 Silencer SelectsiRNA (Ambion), 100 pmol p130Cas siRNA (Santa CruzBiotechnology), or equal molar concentration of Nontarget-ing siRNA (control #1, Dharmacon-Thermo Scientific).

Wound healing and Matrigel invasion assaysWound healing and Matrigel invasion assays were essen-

tially carried out as previously described (12). For Matrigeltranswell (BD Biosciences) experiments, 25,000 253J-BVcells were applied to the top of the chamber, with indicatedtreatments, and allowed to invade (24 hours) toward 10%serumDMEM as the chemoattractant. Cells invading to theunderside of the filter were fixed, stained (0.8% NaCl, 50%ethanol, 5% formaldehyde, and 0.2% crystal violet), andquantified. Images of wounds and Matrigel filters wereobtained using a Zeiss Axio Observer.Z1 with an ECPlan-Neofluar 5�/0.15 M27 objective and AxiocamMR3.No appreciable invasion throughMatrigel could be observedwith 253J cells with the same assay conditions.

Antibodies, immunoblotting, and immunoprecipitationThe following antibodies were used in the study: Crk,

p130Cas, anti-fluorescein isothiocyanate (FITC; BD Bios-ciences), phospho-p130Cas Y165, Fak Y397, Src, Src Y416(Cell Signaling), PTP-PEST, EGF-R (Santa Cruz Biotech-nology), PTPN12, CAT, PY20 phospho tyrosine (Abcam),glyceraldehyde-3-phosphate dehydrogenase (GAPDH;Ambion Life Technologies). Cells were lyzed using radio-immunoprecipitation assay (RIPA) buffer [150 mmol/LNaCl, 50 mmol/L Tris pH 7.5, 1% NP-40, 0.5% deox-ycholate, protease inhibitor cocktail (Roche), 1 mmol/Lsodium orthovanadate]. Cleared lysates were quantified for

protein concentration (BCA, Thermo Scientific) and loadedon either 4% to 12%NuPAGE (Life Technologies) or 10%SDS-PAGE. For immunoprecipitation, protein (1 mg) wasincubated with anti-Crk antibody (1 mg) and protein Gsepharose, and eluded in 2� reducing SDS-PAGE buffer.Following electrophoresis, transfer to nitrocellulose mem-brane (iBlot, Life Technologies) and blocking, primaryantibody incubation was carried out in 5% bovine serunalbumin (BSA) TBS-0.1% Tween (1:1,000). Secondaryhorseradish peroxidase–conjugated mouse or rabbit antibo-dies (GE Healthcare) at 1:10,000 dilutions were addedfollowed by chemiluminescence detection (Pico & FemtoECL reagents, Thermo Scientific).

Membrane/cytoskeletal fractionationThemembrane/cytoskeletal fractionswere obtained using a

protocol adapted from Zhao and colleagues (13). Cells werelyzed in cytoskeleton (CSK) buffer [0.5% Triton X-100, 10mmol/LTris–HCl (pH 6.8), 50mmol/LNaCl, 300mmol/Lsucrose, 3mmol/LMgCl2, 1mmol/L sodium orthovanadate,and protease inhibitors] at 4�C. Following centrifugation(13,600 � g, 30 minutes, 4�C), the supernatant, Triton Xsoluble cytosolic fraction was removed and analyzed forprotein content. The Triton X insoluble pellet (membrane/cytoskeletal fraction) was washed, resuspended in SDS buffer[1%SDS,10mmol/LTris–HCl (pH7.5), 2mmol/LEDTA,1 mmol/L sodium orthovanadate, and protease inhibitors]and boiled. The supernatant Triton X insoluble fraction wascleared from cell debris by centrifugation (13,600 � g, 30minutes 4�C). Equivalent amounts of Triton X insolublefraction in reference to soluble fraction protein concentrationwere electrophoresed, followed by immunoblotting.

Immunofluorescence stainingCells grown on glass coverslips were fixed with 4%

paraformaldehyde/PBS, permeabilized (0.1% Triton X100/PBS) and blocked (3% FBS/PBS). Incubation withphospho-p130Cas Y165 antibody (1:100) was followed byanti-rabbit Alexa-Fluor 488–conjugated antibody (LifeTechnologies, 1:1,000). F-actin was stained with PhaloidinTexas-Red (Life Technologies, 1:50) and nuclear DNAwith 40,6-diamidino-2-phenylindole (DAPI). Cells weremounted (Prolong Gold Antifade, Life Technologies) andimages taken on a Zeiss Axio Observer.Z1FluorescenceMicroscope (HBO100 illuminator), using an EC Plan-Neofluar 63� 1.25 NA, oil immersion objective and Axio-Cam MR3 with Apotome attachment. Image analysis wasconducted using Axiovision software (Zeiss). Alternatively,images were acquired using an Olympus Fluoview 1000Confocal Microscope with a UPLSAPO 60�/1.2 NA waterimmersion objective and analyzed using Fluoview software(Olympus). Subsequent processing was undertaken usingImage J or Adobe Photoshop to uniformly change size,brightness and contrast of the entire image.

Rac1 activation assayActive GTP-bound Rac1 was precipitated from lysates

using the p21-binding domain (PBD) of p21-activated

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protein kinase (PAK1) bound to agarose beads. The Rac1/Cdc42 Activation Assay was purchased from Millipore andcarried out as per manufacturer's instructions.

Iodoacetamide labeling of disulfides and reducedcysteinesIn situ reverse 5-iodoacetamine fluorosceine (5-IAF) label-

ing was adapted from Yang and colleagues (14). FollowingH2O2 treatment, cells were fixed in methanol and permea-bilized (Triton X 100). Free/reduced cysteines were blockedwith 200 mmol/L iodoacetic acid (IAA; Sigma-Aldrich) in100 mmol/L Tris (pH8.3), 5 mmol/L EDTA (37�C, 1hour). Following washes (TBS/EDTA), oxidized thiols werereduced with 1 mmol/L dithiothreitol (DTT), 100 mmol/LTris (pH8.3), 5 mmol/L EDTA (30 minutes, room tem-perature), with IAA alkylated residues being protected fromthis reduction step. Re-reduced thiols were subsequentlylabeled with 1 mmol/L 5-IAF (Life Technologies) in 100mmol/L Tris (pH8.3), 5mmol/L EDTA (30minutes, roomtemperature) and cells mounted (Prolong). Images weretaken as described earlier, background corrected and fluo-rescence intensity quantified using Fluoview software.

Protein phosphatase activity assayTotal phosphatase activity of cellular lysates was assessed

using cholorimetric analysis of dephosphorylation of para-nitrophenol phosphate (pNPP, Thermo Scientific) accord-ing to Streit and colleagues (15).

Statistical analysisAll figures are representative of at least 3 replicate experi-

ments. Data are presented as mean � SEM. The t tests andone-way ANOVA with Tukey multiple comparison postt-ests were conducted using Prism 5.01 (GraphPad Software).

ResultsIntracellular increases in ROS contribute to enhancedmigration and invasion of metastatic bladder cancer cellsThe highlymetastatic 253J-BV cell linewas derived from a

poorly metastatic parental human bladder carcinoma cellline, 253J, following 5 successive bladder xenografts (9).We have previously shown that 253J-BV cells display anearly 2-fold increase in intracellular steady-state H2O2compared with 253J cells (18 ! 32 pmol/L; ref. 10).253J-BV cells also exhibit an altered antioxidant profile,with increased manganese superoxide dismutase (Sod2)expression, decreased CAT expression, and a decrease inreduced to oxidized glutathione ratio (GSH/GSSG; ref. 10).We next asked whether the metastatic 253J-BV cell linedisplays increases in its in vitro migratory and invasivebehavior. Using a classical scratch-wound assay to measurebasic cell migration parameters, the metastatic 253J-BVvariant exhibited enhanced migration in vitro when com-pared with the parental 253J line (Fig. 1A). Similarly, usingMatrigel-coated transwell assays to assess invasion, only the253J-BV cells were able to invade through the Matrigelmatrix. Addition of the H2O2-detoxifying enzyme CAT orthe antioxidant NAC significantly attenuated the migratory

capacity of 253J-BV cells (Fig. 1B). 253J-BV cell invasionwas also impaired by CAT and NAC treatments (Fig. 1C).Treatment of both cells with low dose H2O2 (5–50 mmol/L)stimulated migration (Fig. 1D). This low dose H2O2 treat-ment did not result in cytotoxicity to either cell line.Interestingly, the basal migration rate of 253J cells was notsignificantly altered by CAT or NAC treatment (Supple-mentary Fig. S1). These data implicate ROS as participantsin regulating the migratory and invasive behavior of themetastatic 253J-BV cells.

Redox-dependent p130Cas phosphorylation regulatesfocal adhesion kinase signalingBecause of the important contribution of ROS in cellular

signaling, we monitored whether shifts in steady-state H2O2augment prometastatic signaling networks within 253J-BVcells. We first evaluated the phosphorylation state of FAK, asit plays an important role in cancer cell migration and isredox-responsive (16–19). We found that both total FAKand its (Y397) phosphorylation were moderately elevated in253J-BV cells and this was attenuated by CAT treatment(Fig. 2A). FAKY397 creates a binding site for Src kinase whose(Y416) phosphorylation state remained constant betweenthe 2 cell lines. Interestingly, total Src levels were diminishedin 253J-BV lysates relative to the 253J parental cells, andmay reflect a depletion of its cytosolic pools. This findingmay suggest that SrcY416 predominates in the metastaticvariant, which in turn facilitates FAK-Src signaling. Inter-estingly, Src phosphorylation remained unchanged follow-ing CAT treatment (Fig. 2A).Active FAK-Src facilitates p130Cas binding and phos-

phorylation. The adaptor protein p130Cas links FAK-Src toDOC180, enabling this guanine nucleotide exchange factorto activate Rac-1. Phosphorylation of p130Cas (Y165) wasrobustly enhanced in 253J-BV cells (4.2 � 0.7-fold com-pared with 253J) and this increase was attenuated by 68%following treatment with exogenous CAT (Fig. 2A) or byadenoviral-mediated CAT expression (Fig. 2B), indicatingthat phospho-p130Cas status is H2O2-dependent. Con-versely, p130Cas phosphorylation was increased in nonme-tastatic 253J cells following treatment with low dose H2O2(Fig. 2C). The effect of exogenous H2O2 treatment was lessevident in 253J-BV cells, presumably due to the highendogenous phospho-status of p130Cas in the metastaticcells.

Intracellular redox status regulates membranelocalization of p130CasPhosphorylation of p130Cas is necessary for itsmembrane

recruitment to focal contacts in migrating cells (20, 21).Cellular fractionation revealed that total and phosphory-lated p130CasY165 levels were significantly higher inmembrane fractions from 253J-BV cells under controlconditions compared with the parental line (Fig. 3A and3B). Membrane association of both p130Cas andp130CasY165 were significantly enhanced in non-meta-static 253J cells following treatment with increasing con-centrations of H2O2 (Fig. 3A and B; see Supplementary

Prometastatic Redox Signaling via p130Cas

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Fig. S2 for fractionation controls). Phosphorylationoccurred rapidly and was observed as early as 10 minutespost-H2O2 treatment. Furthermore, 253J-BV cells dis-played enhanced p130CasY165 localization to membraneprotrusions, as assessed by immunofluorescence. Equiva-lent analysis of the nonmetastatic 253J cells revealed adistinct uniform cellular distribution of p130CasY165 (Fig.3C). In addition, 253J-BV cells exhibited differential actincytoskeletal distribution, with enhanced accumulation ofactin fibers at the cell periphery.The localization p130Cas was also strongly enhanced in

filopodia-like protrusions of metastatic cells. In thescratch-wound assay, the frequency of p130CasY165-pos-itive protrusions at the leading edge of 253J-BV cells wasquantified and shown to be significantly higher than in253J cells (Fig. 3D, F, and H). Treatment of 253J cells

with 50 mmol/L H2O2 significantly increased the numberof positive protrusions for p130CasY165 (Fig. 3D, E, andH). Conversely, CAT treatment drastically reducedp130CasY165-positive protrusions in 253J-BV cells (Fig.3F, G, and H). These data suggest that the increases inp130CasY165 in 253J-BV cells, relative to 253J cells, are inlarge part redox-dependent and likely contribute to theredistribution of F-actin in membrane protrusions ofmetastatic cells.

Cellular redox state influences p130Cas-mediateddownstream signalingFollowing phosphorylation and membrane localization,

p130Cas associates with Crk to initiate GTPase recruit-ment and downstream signaling. Analysis of Crk-associ-ated p130Cas using coimmunoprecipitation revealed a

Figure 1. Intracellular redox statusregulates migration and invasion ofmetastatic bladder cancer cells. A,metastatic 253J-BV cells migrateat a faster rate than 253J cells in awound healing assay. Wound edgeat time 0 is marked on the imageand percentage distance migratedby leading edge after 16 hoursquantified (n ¼ 8; mean � SEM;t test; ��, P < 0.0001). B,antioxidants CAT (500 U/mL)and NAC (2 mmol/L) inhibitmigration of 253J-BV cells. Cellswere pretreated for 18 hours incomplete media. Wound healingwas carried out in 0% serumDMEM containing the sameantioxidant concentrations for 24hours (n ¼ 5; mean � SEM; one-way ANOVA; �, P < 0.05; ��,P < 0.001). C, CAT (500 U/mL) andNAC (2 mmol/L) inhibit 253J-BVinvasion. Cells were pretreated for18 hours with antioxidants incomplete media and assayed forinvasion through Matrigel-coatedtranswell chambers toward thechemoattractant (10% serum).Cells on the bottom surface ofthe chamber were quantified(n ¼ 3; mean � SEM; one-wayANOVA; �, P < 0.05). D, sublethallevels of H2O2 enhance bladdercancer cell migration. 253J and253J-BV cells were allowed tomigrate for 21 hours (n¼ 8;mean�SEM; one-way ANOVA; �, P < 0.05;��, P < 0.001, compared with0 mmol/L H2O2).

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marked increase in p130Cas-bound Crk in the metastatic253J-BV cells that was attenuated by CAT treatment (Fig.4A). Furthermore, the amount of active GTP-bound Rac-1 was decreased in the 253J-BV cells, following bothadenoviral CAT expression and treatment with exogenousCAT (Fig. 4B). These findings indicate that p130Casassociation with Crk is under redox-control and likelyparticipates in the robust migratory signaling activity ofthe metastatic 253J-BV cells. To directly assess the role ofp130Cas in migratory signaling, we silenced its expressionin both the 253J and 253J-BV cells and observed asignificant decrease in the migratory behavior of both celllines as compared with control siRNA-treated cells (Fig.4C). The impact of p130Cas suppression on 253J-BVinvasion was more pronounced, displaying a near com-plete inhibition of invasion (Fig. 4D). The nonmetastatic253J cells display no invasive activity, thus the impact onp130Cas suppression using this assay was not assessed.These findings clearly establish the important contribu-tion of p130Cas to the invasive activity of the metastatic253J-BV bladder cell line.

Redox status of metastatic bladder cancer cells influencesglobal protein thiol disulfide formation and proteintyrosine phosphatase inactivationROS play an important role as second messengers in

cellular signaling through reversible, oxidative inactivationof regulatory phosphatases (22). Protein tyrosine phos-phatases (PTP) contain active site cysteines, which aresusceptible to thiol oxidation due to their low pKa. Thisoxidation can lead to a number of reversible modifications,

such as sulfenic acid and disulfide formation. Thus,oxidative phosphatase inactivation is commonly linkedto increases in kinase activity. We hypothesized thatincreases in steady state H2O2 in the 253J-BV cells mightresult in a global shift in thiol oxidation and a concomitantdecrease in phosphatase activity. To monitor global thioloxidation we used an in situ reverse labeling assay.Reduced thiols were first alkylated with IAA, whereasoxidized residues are protected from this alkylation. Cellsare then treated with a strong reductant to reduce alloxidized thiols. The newly reduced nonalkylated thiols aresubsequently labeled with fluorescently tagged 5-IAF.253J-BV cells display an increase in 5-IAF labeling relativeto the 253J cells, indicating a general increase in thioloxidation formation (Fig. 5A) which was further enhancedby H2O2 treatment. Increased thiol oxidation was alsoaccompanied by a global decrease in phosphatase activityof the 253J-BV cells, which was exacerbated by sublethalH2O2 treatment (Fig. 5B). These findings suggest thatoxidative inactivation of phosphatases may contribute tothe enhanced migratory signaling activity of metastatic253J-BV cells.The primary phosphatase responsible for regulation of

p130Cas phosphorylation is PTPN12 (23). The importanceof PTPN12 in regulating p130Cas phosphorylation andmigration was shown when expression of PTPN12 wasdecreased in the nonmetastatic 253J cells. An approximately25% knockdown of PTPN12 dramatically increased thelevel of p130Cas phosphorylation in 253J cells (Fig. 5D).Thismoderate knockdown resulted in statistically significantincreases in 253J cell migration (Fig. 5D). These data

Figure 2. Redox regulation ofprometastatic signaling viap130Cas. A, cells were pretreated for24 hours with recombinant CAT(500 U/mL), followed by 18-hourserum deprivation containing thesame CAT treatment before cell lysisand immunoblotting with phospho-specific antibodies against Y397FAK, Y416 Src, and Y165 p130Cas(B) phosphorylation of Y165 p130casin 253J-BV cells is decreasedfollowing expression of CAT byadenoviral gene delivery(lac-Z ¼ control). CAT activity wassignificantly enhanced followingadenoviral genedelivery (n¼3,mean� SEM; t test; ��, P < 0.01). C, Y165p130Cas phosphorylation increasesfollowing exogenous sublethal H2O2

treatment. Cells were treated withindicated doses for 16 hours inserum-free media.






Figure 3. Intracellular redox status regulates p130Cas cellular localization. A, p130Cas is more abundant in the membrane/cytoskeletal fraction of 253J-BVcells than 253J cells. H2O2 treatment (10 minutes) enhances p130Cas membrane recruitment as assessed by membrane/cytosol fractionation. B,quantification of p130Cas immunoreactive protein in membrane fractions normalized to GAPDH levels. C, immunofluorescent staining revealsenhanced membrane distribution of phospho-p130Cas in metastatic 253-BV compared with 253J cells. Cells were fixed and stained for phospho-p130CasY165 (Alexa-Fluor 488, green), nucleus (DAPI, blue), and F-actin (Phalloidin Texas Red). Following mounting, images were acquired using ZeissAxioObserver.Z1 with AxioCamM3, Apotome (63x/1.25NA oil objective; scale bar, 10 mm). D to G, phospho-p130Cas localization at the leading edge isdependent on redox status of cells. 253J or 253J-BV cells were plated on coverslips, allowed to reach a confluent monolayer and monitored for migrationfollowing scratchwounding in serum-freemedia, with orwithout 50mmol/LH2O2 (253J, D andE) or 500U/mLCAT (253J-BV, F andG). After 6 hours, cells werefixed and immunofluorescently labeled for phospho-p130Cas Y165 (Alexa-Fluor 488, green), nucleus (DAPI, blue), and F-actin (Phalloidin Texas Red).White arrows point to examples of phospho-p130Cas–positive protrusions. Images were taken of cells at the wound edge migrating into the direction of theopposing leading edge of cells (indicated by red arrows), using Zeiss AxioObserver.Z1 with AxioCamM3, Apotome (63x/1.25NA oil objective; scale bar,10 mm). H, quantification of the number of phospho-p130Cas–positive protrusions (6–10 images per treatment quantified, with a range of 5–20 protrusionsper image; one-way ANOVA; ��, P < 0.01; ��, P < 0.001).

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indicate that decreases in PTPN12 activity either by oxida-tion or direct suppression can dramatically influence migra-tory signaling by maintaining p130CasY165.

DiscussionIn the present study, we show that subtle increases in

steady-state H2O2 levels within metastatic bladder cancercells are sufficient and necessary to drive promigratorysignaling via regulation of p130Cas. These changes are allreliant on subtle picomolar increases in endogenous H2O2levels within the metastatic variant cells (i.e., 253J: 18 pmol/L; 253J-BV: 32 pmol/L) without prior treatment or stim-ulation of cells (10). We also show that intracellular shifts insteady state H2O2 production result in detectable changes inpromigratory signaling and further shed light on a redox-

signaling network that may play an important role inmetastatic disease progression.The role of intracellularH2O2was validated in all cell lines

when coexpression or treatment with recombinant CATeffectively abrogated these prometastatic phenotypes andsignaling cascades (12, 24). These data also suggest thatantioxidant enzyme expression can have profound effects onpromigratory cellular signaling pathways. Of note aremarkedly lower basal CAT expression levels previouslyreported in 253J-BV cells when compared with their paren-tal control (10), which likely explains some of the robust H2O2-dependent signaling observed in these cells. It is inter-esting to note that changes in antioxidant expression profilecorrelate with metastatic disease. Increased Sod2 levels arecommonly associated with more invasive disease and poor

Figure 4. p130Cas downstream signaling is redox dependent. A, intracellular redox status regulates Crk-p130Cas interaction. Cells were lysed using RIPAbuffer, followed by immunoprecipitation (IP) with antibody against Crk and immunoblotting (IB) with indicated antibodies. CAT treatment (as in Fig. 2)abrogates association of Crk with p130Cas. B, Rac1 activation in 253J-BV cells is decreased following CAT adenoviral delivery or exogenous CATtreatment. GTP-bound active Rac1was isolated from cell lysates using PAK-1–binding domain–conjugated agarose, as described inMaterials andMethods.Total Rac1, phospho-p130Cas, and total p130Cas levels were assessed by IB of input. C, p130Cas knockdown significantly abrogates 253J-BVmigration in a wound healing assay. Cells were mock transfected (�) with siRNA construct against p130Cas or scramble control before wound healing assay(n ¼ 7; mean � SEM; t test; �, P < 0.05; ��, P < 0.0001, compared with scramble control). Immunoblot for p130Cas shows efficiency of p130Casprotein knockdown. D, siRNA p130Cas knockdown decreases invasive properties of 253J-BV cells through Matrigel. Cells invaded through to the bottomsurface of the transwell chamber were quantified (n ¼ 3; mean � SEM; t test; ��, P < 0.01, compared with scramble control).






patient outcome in a number of cancer types and areconstitutively high in 253J-BV cells (5, 10, 25–27). ElevatedSod2 levels can increase steady state H2O2 levels. In addi-tion, recombinant Sod2 expression in 253J cells can signif-icantly increase their invasive potential throughMatrigel, aneffect that is abrogated following CAT coexpression (12).Furthermore, analysis of microarray data suggests thatincreasing stage and grade of human bladder cancer isassociated with enhanced Sod2 and decreased CAT expres-

sion (5, 10). The factors responsible for driving this differ-ential expression of Sod2 and CAT in these 2 cell lines areunknown. Nuclear factor erythroid 2-related factor (Nrf-2)is a known regulator of both Sod2 and CAT gene transcrip-tion, and NF-kB is a critical player in Sod2 expression. It isinteresting to note that in studies usingNrf-2 null mice, Nrf-2 has been shown to be necessary for induction of CATexpression (28), whereas there is evidence that it negativelyregulates Sod2 transcription (29, 30). Shifts in steady-stateH2O2 could alter the activation of these important redox-sensitive regulators, and help explain the changes in antiox-idant enzyme expression that accompany the metastaticphenotype.H2O2 is believed to be the ROS that best fits the

requirements for a second messenger, based on attributes,such as its relative stability and ability to traverse membranesthrough diffusion or channels (1, 31). Previous studies haveimplicatedH2O2 as a regulator of cellularmigration (32–34)and showed that FAK phosphorylation can be regulated byH2O2 in a dose-dependent manner (16–19). Interestingly,the effects on FAK phosphorylation were relatively modest,and we observed that endogenous H2O2 shifts regulatemigratory phenotype via redox-dependent changes inp130Cas phosphorylation. This scaffolding molecule of theFAK/Src signaling cascade has been implicated in regulatingcell transformation and migration (35, 36), with significantincreases in expression observed in breast, prostate, andchronic myeloid leukemia (37–40). p130Cas has also beenfound to regulate filopodia formation (41) and 3-dimendio-nal migration in a collagen matrix (42). In that context, weobserved a more enhanced inhibitory effect on invasionthough Matrigel compared with migration over a 2-dimen-sional surface when inhibiting p130Cas expression in 253J-BV cells (Fig. 4). While redox-dependent shifts in both FAKand Src phosphorylation were observed, the most strikingdifference was observed at the level of p130CasY165. Inter-estingly, Fonseca and colleagues reported that p130Casphosphorylation was difficult to detect unless cells werepretreated with vanadate, highlighting the importance ofphosphatases in regulating p130Cas phosphorylation (21).It was previously shown that phosphorylation stabilizesp130Cas and results in decreased protein degradation(43). This may explain the higher total levels of this proteinobserved in some of our experiments from cell lines display-ing enhanced intracellular ROS levels. We are activelyinvestigating whether this ROS-dependent change inp130Cas phosphorylation enhances protein stability andare also not ruling out a potential redox component to thetranscriptional regulation of p130Cas.Oxidation of PTPs is increasingly being recognized as a

molecular mechanism by which ROS act as second messen-gers. It is therefore not surprising that an enhanced redoxenvironment, as observed in metastatic 253J-BV cells, issufficient in mediating oxidation of PTPs involved in reg-ulating promigratory signaling. We show that endogenousshifts in steady state H2O2 that accompanies acquisition ofthe metastatic phenotype enhance thiol oxidation anddecreases total PTP activity. PTPN12, the primary PTP

Figure 5. Enhanced intracellular redox status leads to increase in PTPN12oxidation. A, total protein disulfide status of cells with or without 50 mmol/L H2O2 (10 minutes) was assessed using reverse in situ 5-IAF labeling asoutlined in Materials and Methods. All reduced thiol residues werealkylated using nonlabeled IAA. The remaining disulfides were reducedwith DTT and acetylated using 5-IAF. Significant increases in in situ 5-IAFlabeling were observed in 253J-BV cells compared with 253J cells usingan Olympus Fluoview1000 Confocal Microscope. Average intensity unitsper cell weremeasured following background correction (n¼ 30;mean�SEM; t test; �, P < 0.05; ��, P < 0.001). B, total phosphatase activity isdecreased in 253J-BV cells. Cells were treated with or without50 mmol/L H2O2 for 10 minutes in serum-free media and the ability of celllysates to dephosphorylate p-nitrophenol-phosphate was assessedcolorimetrically. C, knockdown of PTPN12 in 253J cells increasesp130Cas Y165 phosphorylation. Cells were mock transfected (�), withsiRNA construct against PTPN12 or scramble control, and cell lysatessubjected to immunoblotting with the indicated antibodies. D, PTPN12knockdown significantly enhances migration of 253J cells in woundhealing assays.Cellswere transfected as inD,monitored formigration for17 hours and percentage distance migrated by leading edge quantified(n ¼ 4; mean � SEM; t test; �, P < 0.05; ��, P < 0.01, compared withscramble control).

Hempel et al.





involved in p130Cas dephosphorylation is important inregulating cellular migration and focal adhesion assembly,specifically by regulating the activation of Rac-1, and haspreviously been shown to be under regulation by oxidation(23, 44–47). Furthermore, subtle decreases in PTPN12levels had a profound impact tumor cell migration andp130Cas signaling (Fig. 5C).The ability of cancer cells to migrate from their primary to

a secondary sited involves coordinating events that inducedetachment, alter integrin engagement and allow migrationthrough ECM. ROS through H2O2 regulate a multitude ofcellular signaling cascades primarily due to their ability toreversibly oxidize thiolate anions of cysteine residues (1, 8).Many of the redox-sensitive signals control distinct aspects ofthe migratory and invasive phenotype. Lee and colleaguesfirst identified the dual lipid phosphatase PTEN as reversiblysensitive to H2O2-dependent inactivation (48). PTEN playsa critical role in modulating phosphoinositide distributionduring directed migration (49) and its oxidation by mito-chondrial-derived H2O2 drives both the angiogenic andmigratory phenotype (24). H2O2 generation has beenshown to be targeted at focal complexes in lamellipodia andmembrane ruffles through tetheringNADPHoxidase familymembers via scaffolding proteins. This compartmentalizedproduction of ROS is directed at tyrosine phosphatases thatare enriched in these compartments (50, 51). Our priorwork indicates that lamellipodia are also enriched inmitochondria and provide an additional source formigratory ROS production. Indeed, recent work usinga mitochondrial-targeted CAT construct developed in ourlaboratory (52) indicates that mitochondrial ROS gener-ation contributes to VEGF-dependent endothelial cellmigration (53). We have also established that many ofthe key migratory signaling molecules that are engagedduring endothelial cell migration also drive the invasive-ness of metastatic bladder cancer cells. Thus, the increasein steady state H2O2 associated with acquisition of themetastatic phenotype likely drives phosphatase inactiva-tion leading to activation of FAK, Rho-related smallGTPase Rac1 and mitogen-activated protein kinase(MAPK) signaling. However, a key difference is that theseredox-driven signals are constitutively active in metastatic

bladder cancer. It is our hypothesis that oxidants convergeto drive the invasiveness of bladder tumors, thus opening atherapeutic window to limit metastatic disease progressionusing targeted-antioxidant based therapeutics.These studies indicate that the ability to endure chronic

oxidative stress is a feature acquired by metastatic cancercells. Current neoadjuvant therapies for metastatic bladderdisease display similar therapeutic efficacy and despiteinitial high response rates these treatments are not cura-tive. Our preliminary work has identified imbalances inthe redox-milieu as contributing to the metastatic phe-notype. It is likely that oxidizing species impinge on anumber of molecular targets that as a whole serve toengage metastatic disease progression. Thus, antioxidantsmay prove beneficial in limiting disease relapse whenappropriately administered.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N. Hempel, B. Mian, J.A. MelendezDevelopment of methodology: N. Hempel, B. Mian, J.A. MelendezAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): N. Hempel, B. Mian, J.A. MelendezAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): N. Hempel, J.A. MelendezWriting, review, and/or revision of the manuscript:N.Hempel, T.R. Bartling, J.A.MelendezAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J.A. MelendezStudy supervision: J.A. Melendez

AcknowledgmentsThe authors thankMs. Erin Moore, Mr. Ryan Clark, Ms. Usawadee Dier, and Dr.

Hanqing Ye for their technical assistance. The authors also thank Dr. Colin P.N.Dinney (M.D. Anderson Cancer Center, Houston, TX) for providing 253J and 253J-BV cell lines.

Grant SupportThis work was supported by NIH award numbers F32CA134165 and K99/

R00CA143229 (to N. Hempel) and R01AG031067 (to J.A. Melendez).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received August 7, 2012; revised October 22, 2012; accepted January 4, 2013;published OnlineFirst January 23, 2013.

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2013;11:303-312. Published OnlineFirst January 23, 2013.Mol Cancer Res Nadine Hempel, Toni R. Bartling, Badar Mian, et al. -Dependent Activation of the p130Cas Signaling Complex

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