CLT1 Targets Bladder Cancer through Integrin 5 1 and CLIC3 · Signal Transduction CLT1 Targets Bladder Cancer through Integrin a5b1 and CLIC3 Lynn M. Knowles1, James Zewe1, Gunjan

Signal Transduction

CLT1 Targets Bladder Cancer through Integrin a5b1 andCLIC3

Lynn M. Knowles1, James Zewe1, Gunjan Malik1, Anil V. Parwani2,3, Jeffrey R. Gingrich1,3, and Jan Pilch1,3

AbstractHigh-grade non–muscle-invasive bladder cancer is commonly treated with Bacillus Calmette-Gu�erin, an

immunotherapeutic that depends on fibronectin and tumor cell integrin a5b1 for internalization into bladdercancer cells. We previously showed that the anti-angiogenic peptide CLT1 forms cytotoxic complexes withfibronectin that are cooperatively internalized into proliferating endothelium through ligation of integrins andchloride intracellular channel 1.While CLT1 has no effect onmature, differentiated cells, we show here that CLT1is highly cytotoxic for a panel of bladder tumor cell lines as well as a variety of cell lines derived from kidney, lung,breast, and prostate cancer. Paralleling our previous results, we found CLT1-induced tumor cell death to beincreased in the presence of fibronectin, which mediated CLT1 internalization and subsequent autophagic celldeath in amechanism that depends on tumor cell integrina5b1 and chloride intracellular channel 3 (CLIC3). Thismechanistic link was further supported by our results showing upregulation of a5b1 and CLIC3 in CLT1-responsive tumor cell lines and colocalization with CLT1 in tumor tissues. Incubating tumor tissue from patientswith bladder cancerwithfluorescein-conjugatedCLT1 resulted in a strong and specific fluorescencewhereas normalbladder tissue remained negative. On the basis of its affinity for bladder tumor tissue and strong antitumor effects,we propose thatCLT1 could be useful for targeting bladder cancer.MolCancer Res; 11(2); 194–203.�2012AACR.

IntroductionBladder cancer is the second most common and the third

deadliest malignancy of the genitourinary tract, with amortality of approximately 20% (1). The majority ofpatients (75%–85%) have non–muscle-invasive bladdercancer (NMIBC) at the time of diagnosis, which is typicallytreated with transurethral resection (TURBT; ref. 2). WhileTURBT is effective in patients with low-risk bladder cancer(i.e., small single tumors, noninvasive, and low-grade), therate of recurrence and progression in patients with interme-diate- or high-risk disease is considerable (3, 4). To reducerecurrence, TURBT is usually accompanied by an adjuvantintravesical treatment with chemo- or immunotherapy(5, 6). However, despite these therapies, the rate of recur-rence and progression is up to 70% and 30%, respectively(5). Other strategies to control bladder cancer includehyperthermia, photodynamic therapy, and diverse imaging

strategies that are intended to improve tumor visualizationduring cystoscopy and TURBT (2, 7, 8). While some ofthese approaches (e.g., hyperthermia) have shown benefit inthe short term, their efficacy of controlling bladder cancerover a long period of time is not clear (7). Together, thesedata underscore the continued need to develop efficient andwell-tolerated adjuvant treatments for high-grade NMIBC.The adjuvant treatment of choice for high-risk NMIBC is

the intravesical application of live bacteria, Bacillus Calm-ette-Gu�erin (BCG), which is superior in reducing the risk ofrecurrence compared with intravesical chemotherapy withmitomycin (9–11). BCG has also been shown to reduce therisk of progression, but it is not clear whether the treatmentyields a significant survival advantage (12, 13). The 2 majordrawbacks of BCG are nonresponse and a high rate of sideeffects (�90% of cases) ranging from irritative voiding,hematuria, and cystitis to BCG-induced sepsis (13, 14).While the majority of BCG-induced side effects are con-sidered as nonsevere, it has been estimated that they causenearly 30% of the patients to discontinue therapy (14). Themechanism of action for BCG is to induce an immunereaction that orchestrates the eradication of bladder tumorcells and treatment complications are a direct result of thisresponse (15). As such, side effects correlate positively withthe efficacy of BCG, whereas the lack of a sufficient immuneresponse is predictive of treatment failure.Using phage display, we previously identified a tumor-

homing peptide, CLT1 that associates with clotted plasmain tumor interstitial spaces (16). More recently, we foundthat CLT1 has strong anti-angiogenic activity in vivo and

Authors' Affiliations: Departments of 1Urology and 2Pathology, Universityof PittsburghSchool ofMedicine,ShadysideMedicalCenter, and 3Prostateand Urological Cancers Program, University of Pittsburgh Cancer Institute,Pittsburgh, Pennsylvania

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

Corresponding Author: Jan Pilch, University of Pittsburgh School ofMedicine, Shadyside Medical Center, Suite G33, 5200 Centre Avenue,Pittsburgh, PA 15232. Phone: 412-623-3917; Fax: 412-623-3907; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-12-0300

�2012 American Association for Cancer Research.

MolecularCancer

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attributed this function to the capacity of CLT1 to inducean unfolded protein response and autophagic cell death inproliferating endothelial cells (17). Cytotoxicity towardendothelial cells was supported by fibronectin, whichforms co-aggregates with CLT1 and, as such, mediatesbinding to endothelial integrins and subsequent internal-ization through a novel CLT1 receptor, chloride intracel-lular channel 1 (CLIC1; ref. 17). This mechanism isreminiscent of the role of fibronectin for BCG opsoniza-tion, which contributes to the integrin-mediated internal-ization of fibronectin-bound BCG into bladder tumorcells as a prerequisite of BCG antitumor activity (18, 19).On the basis of the role of fibronectin for BCG antitumorefficacy, we sought to determine whether CLT1 hasantitumor properties and if this function is pronouncedin bladder cancer.

Materials and MethodsPeptidesAll peptides were purchased from Primm Biotech. A

lysine/alanine scan of CLT1 yielded CLT1 variant peptidesGA (CALIIQKNEC), LK (CGKIIQKNEC), IK1(CGLKIQKNEC), IK2 (CGLIKQKNEC), QA (CGLIIA-KNEC), KA (CGLIIQANEC), NA (CGLIIQKAEC), andEA (CGLIIQKNAC). CLT1 peptides were cyclized byexposure to air, which leads to the formation of intramo-lecular disulfide bonds between C- and N-terminalcysteines. Linear CLT1 (LCLT1, AGLIIQKNEA) wasgenerated by replacing the cysteines with alanine. Carboxy-fluorescein was conjugated to the peptides via a 2-ami-noethoxy-2-ethoxyacetic acid (AEEA) linker for fluorescentstudies.

Cell lines and treatmentsJ82, T24, UMUC3, TCCSUP, 5637 (bladder),

DU145, PC-3 (prostate), RCC4, 786-0 (renal), MDA-MB-231, MCF-7 (breast), A549 (lung), PANC1 (pan-creas), and RD (muscle) tumor cell lines were purchasedfrom American Type Culture Collection and cultured permanufacturer's specifications. Human bladder epithelialcells were from Lifeline Cell Technology and culturedaccording to manufacturer recommendations in serum-free Prostalife Basal Medium supplemented with L-gluta-mine, extract P, epinephrine, rh TGF-a, hydrocortisonehemisuccinate, rh insulin, apo-transferrin, and calciumchloride. Cells were cultured at 37�C under a humidified5% CO2 atmosphere. All cells were treated at a platingdensity of about 50% to ensure a linear growth rate.Peptides were diluted in H2O to 2 mg/mL and addedto cells at concentrations ranging from 7.5 to 150 mg/mLin the presence of 2% FBS. Human plasma fibronectin(Sigma; 3–100 mg/mL), fibrinogen (Enzyme ResearchLaboratories; 30 mg/mL), nocodazole (Sigma; 10 mmol/L),GRGDSP (RGD) or GRADSP (RAD) peptides (each300 mmol/L; EMD Chemicals) were added at the timeof peptide treatment. Bafilomycin A1 (EMD Chemicals;40–400 nmol/L) was added 1 hour before CLT1 addition.Where indicated, FBS was depleted of fibronectin by

passing the serum through a gelatin agarose column. Forexperiments using serum-free media, cells were serumstarved for 8 hours before study onset.

Cytotoxicity assayCell death was assessed after 24 hours by measuring

lactate dehydrogenase (LDH) release using the Cytotox-icity Detection Kit (Roche Applied Science). Resultsare normalized for background cell death observed incontrol cells treated in absence of CLT1 to the specifiedmedium conditions. Maximal cell death was achieved bysimultaneous treatment with camptothecin (1.4 mmol/L;Sigma-Aldrich) and staurosporine (100 nmol/L; AlexisBiochemicals).

MicroscopyTo analyze internalization of CLT1 in tumor cell lines,

cultured cells grown on coverslips coated with 10 mg/mLvitronectin (BD Biosciences) overnight at 4�C were incu-bated with 25 mg/mL fluorescein-conjugated peptides for 24hours in the presence of 30 mg/mL fibronectin or fibrinogenwhere indicated, fixed in 4% paraformaldehyde (PFA) andstained with 40,6-diamidino-2-phenylindole (DAPI)-con-taining mounting media (Vectashield; Vector Laboratories).Cellular localization was analyzed at �40 magnificationusing a fluorescence microscope (Zeiss Axioplan 2) withimage processing unit. Lysosomes were labeled with 100nmol/L Lysotracker Red DND-99 (Invitrogen) for 1 hourbefore fixation. For fibronectin and CLIC3 staining, PFA-fixed cells were permeabilized with 0.5% Triton-X100 andincubated with anti-fibronectin (Millipore), anti-CLIC3(Abcam), or isotype control, followed by incubation withAlexa Fluor 546–conjugated secondary antibody (Invitro-gen) and analyzed using a confocal microscope (LeicaTCSSL). Cell morphology was imaged by reflection confocalmicroscopy. To visualize nuclei, cells were stained withDraq5 (eBioscience).De-identified tissues from clinical bladder cancer as well as

normal adjacent bladder tissue were inspected by an ana-tomic pathologist following resection and then immediatelydistributed through the tissue bank of the University ofPittsburgh Cancer Institute (Pittsburgh, PA). Upon receiptin our laboratory, tissue was placed in keratinocyte growthmedia containing bovine pituitary extract, hEGF, insulin,hydrocortisone, and antibiotics with or without 25 mg/mLfluorescein-conjugated CLT1 or control peptides (IK1,LCLT1). Fibronectin and fibrinogen were added whereindicated. After overnight incubation at 37�C and 5%CO2, tissue samples were washed in PBS and either placedon a microscopy slide for en-face confocal microscopy orfrozen in optimum cutting temperature (OCT). Frozentissues were sectioned, fixed in acetone, and analyzed forpeptide uptake by fluorescence microscopy. For immuno-histochemistry, acetone-fixed sections were incubated withanti-CLIC3 or anti-a5 integrin (BD Bioscience) and AlexaFluor 546–conjugated secondary antibody before confocalmicroscopy. Digitized images were processed with AdobePhotoshop.

CLT1 Targets Bladder Cancer

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Western blot analysisCells were lysed using the Subcellular Protein Fraction-

ationKit (Thermo Fisher Scientific) or by the addition of�2SDS sample buffer. Proteins were separated by SDS-PAGE,transferred onto polyvinylidene difluoride (PVDF), andstained with 0.05% Ponceau S (Sigma) to ensure equivalentprotein loading. Immunoblots were blockedwith 5%bovineserum albumin and probed overnight at 4�C with anti-Cathepsin D, anti-LC3B (Cell Signaling Technology) oranti-CLIC3. Immunoreactivity was detected using peroxi-dase-conjugated anti-rabbit or anti-mouse IgG antibody andvisualized by enhanced chemiluminescence.

siRNA-mediated gene silencingIntegrin a5 (L-008003-00), integrin b3 (L-004124-00),

CLIC1 (L-009530-00), CLIC3 (L-011805-00), CLIC4(L-013553-00), CLIC5 (L-020570-00), and nontargetingcontrol (D-001810-10) On-TARGETplus SMARTpoolsiRNAs were purchased from Dharmacon. Cells were trans-fected with 10 nmol/L siRNA in Opti-MEM medium(Invitrogen) using Lipofectamine 2000 reagent (Invitrogen)for 5 hours and then placed in normal culture medium andgrown for an additional 43 hours before treatment withCLT1. Target knockdown was confirmed by reverse tran-scription polymerase chain reaction (RT-PCR) andWesternblot analysis (Supplementary Fig. S1).

Flow cytometry assayCells were suspended in media supplemented with 2%

FBS and incubated with integrin a5 or isotype controlantibody (BD Biosciences) for 30 minutes at 4�C, washedwith ice-cold media, and incubated for 30 minutes on icewith Alexa Fluor 488 anti-mouse F(ab0)2 (Invitrogen). Tomeasure CLIC3 expression, suspended cells were incubatedon a rotor for 45 minutes at room temperature beforeincubation with CLIC3 antibody for 30 minutes at 4�C.Cell viability was monitored by staining cells with 5 mg/mLpropidium iodide (PI; Roche Applied Science). Fluorescencewas examined on 10,000 viable cells per sample using atabletop cytometer (Accuri C6).

Statistical analysisData were analyzed using unpaired 2-tailed Student t test

or one-way ANOVA followed by the post hocTukeymultiplecomparisons test (GraphPad Prism 5). Treatment differ-ences with a 2-sided P < 0.05 were considered significantlydifferent. Error bars show mean � SEM.

ResultsCLT1 induces tumor cell death in cooperation withfibronectinWe previously showed that CLT1 has significant anti-

angiogenic activity, which correlates with the ability of CLT1to generate cytotoxic complexes with the adhesion proteinfibronectin (17). Here, we show that CLT1 is cytotoxic fora diverse panel of human tumor cell lines derived fromthe bladder, prostate, kidney, breast, and lung (Fig. 1A). Thecytotoxic activity of CLT1 was strongly augmented in the

presence of fibronectin, which alone had no effect on thetumor cell fate (Fig. 1A and B; Supplementary Fig. S2A).Cytotoxicity in proliferating tumor cells occurred within 24hours of treatment at a fibronectin concentration of 10 mg/mL and reached a plateau at a CLT1 concentration of 75 mg/mL (Fig. 1C and D). A second plasma adhesion protein,fibrinogen, had the opposite effect and effectively preventedCLT1-induced cytotoxicity (Fig. 1A). Among the differenttumor types tested, CLT1 activity was pronounced in bladdercancer cells, which responded consistently with extensive celldeath even in the presence of urine (Fig. 1A; SupplementaryFig. S2B). CLT1 was significantly more effective in prolif-erating than in serum-starved bladder tumor cells and largelyinactive in primary bladder epithelial cells (Fig. 1A and B). Tofurther dissect the role of fibronectin for bladder tumor celldeath, we treated TCCSUP, J82, and UMUC3 cells withfibronectin-depleted serum, which in comparison to plasmafibronectin–supplemented serum was significantly less effec-tive in mediating CLT1-induced cell death (Fig. 1E and F).Tumor cell death in response toCLT1was also reducedwhenwe transfected bladder tumor cells with siRNA againstfibronectin; however, this was only the case when tumorcells were treated in absence of exogenous fibronectin, sug-gesting that CLT1 interacts with cellular fibronectin as well asplasma fibronectin (Fig. 1F). Disabling structural features ofCLT1 that are relevant for fibronectin complex formation,such as the LIIQK motif or the C- and N-terminal cysteinesthat mediate cyclization (17), rendered CLT1 inactive (Fig.1G). Together, our results show that CLT1 has substantialantitumor activity,which is pronounced in bladder tumor celllines, and that this activity depends on an intact LIIQKmotifas well as the presence of fibronectin.

Tumor cells undergo autophagic cell death in response toCLT1To further delineate the mechanism of CLT1 cytotoxicity,

we incubated tumor cells with fluorescein-conjugated CLT1andanalyzed its subcellular localizationusingfluorescence andconfocal microscopy. These experiments showed that CLT1uptake was significantly higher in J82 and UMUC3 bladdercancer cells that are sensitive for CLT1-induced cell deaththan in 786-0 kidney cancer cells that are largely resistanttoward CLT1 (Fig. 2A; Supplementary Fig. S3). Conversely,CLT1 cytotoxicity was significantly reduced when we inhib-ited CLT1 uptake with nocodazole (Fig. 2B and C). Costain-ing with lysotracker revealed that CLT1 was transported intolysosomes, which appeared enlarged and dysformed aftertreatment with CLT1 compared with untreated cells (Fig.2D and E). To determine whether CLT1 induces lysosomedysfunction, we analyzed the cytoplasmic fraction of CLT1-treated tumor cells and found increased concentrations of thelysosomal protease cathepsin D as an indicator for lysosomeleakage (Fig. 3A). In addition, we found that CLT1 causesLC3 conversion, indicating that sorting of CLT1 into lyso-somes is mediated in the context of autophagy (Fig. 3B).Moreover, inhibiting fusion of autophagosomes and lyso-somes with the autophagy inhibitor bafilomycin A1 signifi-cantly reduced CLT1 cytotoxicity in a panel of bladder and

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prostate tumor cells (Fig. 3C). Together, our results show thattumor cells internalize CLT1 and that this process leads tolysosome dysfunction and autophagic cell death.

CLT1-induced cell death correlates with fibronectin-mediated internalization of CLT1An important function of fibronectin is to form complexes

with CLT1 that promote CLT1 uptake into the angiogenic

endothelium (17). Paralleling these results, we show thatfibronectin, which colocalized with CLT1 inside the tumorcells, supports CLT1 uptake into bladder and prostate tumorcells whereas fibrinogen largely prevents CLT1 internaliza-tion (Fig. 4A and B). Moreover, CLT1 internalization wassignificantly reduced in the presence of a peptide GRGDSPthat blocks the RGD recognition motif of fibronectin-binding integrins but not in the presence of an inactive

Figure 1. CLT1 is cytotoxic for tumor cells. A, a panel of humanbladder (J82, T24,UMUC3, TCCSUP), prostate (DU145), kidney (RCC4), breast (MDA-MB-231),and lung (A549) tumor cell lines and primary human bladder epithelial cells (HBEC) were treated for 24 hours with CLT1 (75 mg/mL) in the presence of 2%serum supplemented with 30 mg/mL fibronectin (2% þ FN), fibrinogen (2% þ FG), or media þ 2% serum alone (2%). Cell death is shown as percentageof LDH release of cells treatedwith camptothecin/staurosporine. B, LDH released bybladder tumor cells treatedwithCLT1 (75mg/mL) in the presence of 2%þFN, serum-free media supplemented with FN (SF þ FN) or serum-free media alone (SF). LDH release is normalized to absorbance values of vehicle controlcells treated in absence of CLT1. LDH released in response to CLT1 in the presence of 2% þ FN is set to 1. �, P < 0.05; ��, P < 0.001 versus 2% þ FN.C, LDH released by DU145 tumor cells cultured in FN-depleted serum and treated with CLT1 (75 mg/mL) and increasing concentrations of FN (3–100 mg/mL).D, LDH released by DU145 cells treated with increasing concentrations of CLT1 (7.5–150 mg/mL) in the presence of 2% þ FN. E, LDH released bybladder tumor cells treated with CLT1 (75 mg/mL) in the presence of 2%þ FN, fibronectin-depleted serum (FNminus), and FN-depleted serum supplementedwith FN (FN minus þ FN). LDH released in response to CLT1 by cells grown in the presence of 2% þ FN is set to 1. ��, P < 0.01; ��, P < 0.001 versus2%þ FN or FNminusþ FN. F, LDH released by UMUC3 cells transfected with FN or control siRNA and then treated with CLT1 (75 mg/mL) in the presence of2% þ FN, FN minus, or FN minus þ FN media. LDH released in response to CLT1 by control siRNA cells grown in the presence of 2% þ FN is set to 1.��,P < 0.01; NS, nonsignificant versus control siRNA.G, a lysine/alanine scan of CLT1. LDH releasewas assessed after treatingDU145 for 24 hours withCLT1and the variant CLT1 peptides (each 150 mg/mL). ��, P < 0.001 versus CLT1.






control peptide (GRADSP; Fig. 4C). Blocking integrinfunction with the RGD peptide or knocking down integrina5, which binds to fibronectin in an RGD-dependentmanner, also inhibited the cytotoxic function of CLT1,whereas knockdown of another fibronectin-binding integ-rin,avb3, had no effect onCLT1-mediated tumor cell death(Fig. 4D and E). Flow cytometry showed that CLT1-sensitive tumor cell lines generally expressed higher levelsof integrin a5b1 on their cell surface than tumor cell linesthat were resistant toward CLT1 treatment (Fig. 4F, Sup-plementary Fig. S3A). Together, these results indicate thatCLT1 internalization and cytotoxicity is largely determinedby interactions of CLT1–fibronectin complexes with tumorcell integrin a5b1.

CLT1 cytotoxicity is mediated by CLIC3We previously showed that CLT1–fibronectin complexes

are taken up into angiogenic endothelium through interac-tion with integrinavb3 and CLIC1 and that this interactioninduces autophagic cell death (17). To determine whetherCLIC proteins cooperate with fibronectin-binding integrins

in mediating CLT1 cytotoxicity, we transfected tumor cellswith siRNA against various CLIC family members beforetreatmentwithCLT1.While knocking downCLIC1, 4, and5 had no effect, we found that siRNA against CLIC3significantly reduced CLT1-mediated cell death in a panelof bladder cancer cell lines as well as DU145 cells (Fig. 5Aand B). Subcellular fractionation and flow cytometryrevealed that CLIC3 is expressed in the tumor cell mem-brane and extends from there to the cell surface (Fig. 5C andD). Importantly, CLIC3 colocalized with CLT1 insidetumor cells, suggesting a mechanistic link between CLIC3and CLT1 internalization (Fig. 5E). A connection betweenCLIC3 and CLT1 became further apparent by analyzingtotal CLIC3 protein expression levels, which were overallincreased in CLT1-responsive when compared with nonre-sponsive cell lines (Fig. 5F). Together, these results suggestthat CLIC3 plays an important role for mediating thecytotoxic activity of CLT1.

CLT1 binds to tumor tissue from bladder cancer patientsTo determine whether CLT1 interacts with clinical blad-

der cancer, we incubated tumor tissue from 7 patients withbladder cancer with fluorescein-conjugated CLT1 overnightand subsequently analyzed the samples by fluorescence andconfocal microscopy. Corresponding hematoxylin and eosin(H&E)-stained sections were analyzed by a clinical patho-logist for morphologic confirmation of tumor (Supplemen-tary Fig. S4). A summary of patient characteristics is

Figure 2. CLT1 internalization correlates with cytotoxicity. A, uptake offluorescein–CLT1 (25 mg/mL) was analyzed in the presence of 2% serumþ FN after 24 hours in UMUC3, J82, and 786-0 cells by fluorescencemicroscopy. Data are shown as percentage of CLT1-positive cells peroptical field (�40). ��, P < 0.001 versus UMUC3 and J82 cells. B, DU145cells were treated with fluorescein–CLT1 alone or in the presence ofnocodazole (10 mmol/L) for 24 hours and analyzed for peptideinternalization as percentage of CLT1-positive cells per optical field(�40). ��, P < 0.001 versus dimethyl sulfoxide (DMSO). C, LDH releasedby DU145 cells after 24 hours of CLT1 treatment (75 mg/mL) with orwithout nocodazole. ��, P < 0.001 versus DMSO. D, UMUC3 (top) andJ82 cells (middle and bottom) were treated with fluorescein-conjugatedCLT1 (green) for 24 hours and lysotracker (Lyso, red; top and middle) orDMSO (bottom) for 1 hour before confocal microscopy. Merged imagesare shown in yellow. Reflection depicts cell morphology. Scale bar,10 mm. E, DU145 cells were treated with fluorescein–CLT1 (green) orvehicle (Con) for 24 hours, stained with lysotracker (Lyso; red), andanalyzed by fluorescence microscopy. Nuclei were stained with DAPI(blue). Merged images are shown in yellow. Scale bar, 10 mm.

Figure 3. CLT1 internalization induces lysosome dysfunction andautophagic cell death. A, cytosolic fractions from DU145, UMUC3, andJ82 tumor cells were treatedwith vehicle, CLT1, the inactive CLT1 variantIK1 (each 75 mg/mL), FN (30 mg/mL), or CLT1 þ FN for 24 hours andimmunoblotted for cathepsin D (CTSD). B, lysates isolated from UMUC3and J82 cells cultured in 2% serum þ FN were immunoblotted for theautophagymarker LC3 after 24 hours of treatment with CLT1 (25 mg/mL).A and B, Ponceau S (PS) staining shows equal protein loading. C, LDHrelease in DU145, UMUC3, and J82 cells treated with 40 to 400 nmol/Lbafilomycin A1 (BAF) and CLT1 (25 mg/mL) þFN for 24 hours. BAF wasadded 1 hour before CLT1. LDH released in response to CLT1 is set to 1.��, P < 0.001 versus CLT1.

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provided in Supplementary Table S1. Fluorescence micros-copy revealed that CLT1 was strongly taken up in freshlyresected, unfixed tumor tissues from both non-muscle- andmuscle-invasive bladder cancer (Fig. 6A and B). Notably,there was no detectable fluorescence in normal bladder tissuetreated with CLT1 or in tumor tissues treated with aninactive CLT1 variant as control peptide indicating thatthe interaction of CLT1 with bladder cancer is highlyspecific. Additional analysis with confocal microscopyshowed staining of single cells, indicating that CLT1 isinternalized in a manner similar to what we observed incultured tumor cells (Fig. 6C). CLT1 binding was moreefficient when tumor tissues were cultured in the presence ofplasma fibronectin than in fibrinogen (Fig. 7A). The role of

fibronectin for CLT1 binding was further supported by ourfinding that CLT1 colocalized with integrin a5 and CLIC3in bladder tumor tissue sections (Fig. 7B). Together, ourresults show that CLT1binds to clinical bladder tumor tissueand that CLT1 binding is pronounced in areas that are richin integrin a5b1 and CLIC3.

DiscussionWe previously showed that CLT1 forms aggregates with

fibronectin that are cytotoxic for angiogenic endothelium(17). Here, we show that CLT1 is strongly cytotoxic fortumor cells and attribute this activity to interactions betweenCLT1–fibronectin complexes and the fibronectin receptor

Figure 4. CLT1-induced cell death correlates with fibronectin-mediated internalization of CLT1. A, internalization of fluorescein–CLT1 in the presenceof FNor FG (each 30mg/mL)was analyzedby fluorescencemicroscopy and is shown aspercentageofCLT1-positive cells per optical field (�40). ��,P <0.001versus FN. B, DU145 cells treated with fluorescein–CLT1 (green) and FN for 7 hours were stained with anti-fibronectin antibody (FN, red) and analyzedby confocal microscopy. Nuclei were stained with Draq5 (blue). Scale bar, 10 mm. C, DU145 cells were measured by fluorescence microscopy forfluorescein–CLT1 uptake in the presence of either an integrin-blocking RGD peptide or control RAD peptide. Data are shown as percentage of CLT1-positivecells per optical field (�40). ��, P < 0.001 versus RAD. D, LDH release in DU145 cells after treatment with 75 mg/mL CLT1 in the presence of RGD and RADpeptides or following transfection with integrin a5, integrin b3, or control siRNA. ��, P < 0.001 versus vehicle and control siRNA. E, LDH release in CLT1-treated UMUC3, J82, and TCCSUP cells transfected with integrin a5 or control siRNA. LDH released in response to CLT1 by control siRNA cells is set to 1.��,P <0.01; ��,P < 0.001 versus control siRNA. F, cell surface expression of integrina5b1 assessed by flowcytometry (mean fluorescence intensity,MFI) wascompared with CLT1 reactivity (þþ, cell death > 75%; þ, cell death 75%–50%; þ/�, cell death � 25%; �, cell death < 25%).






integrin a5b1, which mediates CLT1 internalization andautophagic cell death in cooperation with CLIC3. Interest-ingly, CLT1 cytotoxicity is particularly pronounced inbladder tumor cell lines that express large amounts ofa5b1 and CLIC3. Paralleling this result, we show thatCLT1 binds strongly and specifically to clinical bladdercancer tissues that express integrin a5b1 and CLIC3.CLT1 (CGLIIQKNEC) contains a unique hydrophobic

peptide sequence, LIIQK, that is, critical for its antitumor

Figure5. CLT1-inducedcell death ismediatedbyCLIC3.A, LDH release inDU145 cells transfected with siRNA against CLIC1, CLIC3, CLIC4, andCLIC5 after treatment with 75 mg/mL CLT1. ��, P < 0.001 versus controlsiRNA. B, LDH release in CLT1-treated UMUC3, J82, and TCCSUP cellstransfected with CLIC3 or control siRNA. LDH released in response toCLT1 by control siRNA cells is set to 1. �, P < 0.05; ��, P < 0.001 versuscontrol siRNA. C, membrane fractions isolated from DU145, J82,TCCSUP, and UMUC3 cells were immunoblotted for CLIC3. DU145comparedwith J82 and TCCSUP (left). DU145 comparedwith UMUC3 at�10 higher protein concentration (right). Ponceau S (PS) staining showsequal protein loading. D, cell surface expression of CLIC3 on DU145 andUMUC3 cells was assessed by flow cytometry. Percentage of cells gatedin M1 was determined after incubation with anti-CLIC3 or controlantibody (IgG) based on fluorescence in FL1. ��, P < 0.01; ��, P < 0.001versus control IgG. E, J82, TCCSUP, and DU145 cells incubated withfluorescein–CLT1 (green) for 24 hours were stained with anti-CLIC3antibody (red) or control IgG (bottom) and analyzed by confocalmicroscopy. Merged images are shown in yellow. Reflection depicts cellmorphology. Scale bar, 10 mm. F, whole-cell lysates from CLT1-reactiveand -nonreactive cellswere probed forCLIC3 expression.CLT1 reactivity(þþ, cell death>75%;þ, cell death 75%–50%;þ/�, cell death�25%;�,cell death < 25%). C and F, PS staining shows protein loading.

Figure 6. CLT1 binds to clinical bladder cancer ex vivo. A, fluorescencemicroscopy of tissues frommuscle-invasive (INV) compared with normaladjacent bladder tissue (NAT) after incubation with fluorescein-conjugated CLT1 (green) or LCLT1 as a control peptide (CP). Numberscorrespond to patients in Supplementary Table S1. Nuclei (blue) werestained with DAPI. B, fluorescence microscopy of non–muscle-invasivebladder cancer (SUP) after incubation with fluorescein-conjugated CLT1or LCLT1 (green). A and B, scale bar, 50 mm. C, en face confocalmicroscopy of freshly resected bladder tumor tissues compared withNAT after incubation with CLT1 (green) or IK1 as a control peptide (CP).Scale bar, 10 mm. C, nuclei (blue) were stained with Draq5 for confocalmicroscopy.

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effects. LIIQK is necessary for the formation of CLT1–fibronectin co-aggregates and, as such, is important forCLT1 internalization, which causes endoplasmic reticulumstress and autophagic cell death (17). As part of the CLT1complex, fibronectin has been shown tomediate interactionswith integrin avb3 on angiogenic endothelium that lead toupregulation of the CLT1 receptor CLIC1 and subsequentinternalization of CLT1–fibronectin complexes (17, 20).The mechanism of CLT1 cytotoxicity is similar in tumorcells, which internalize CLT1 in a fibronectin-dependentmanner; however, instead of using CLIC1 and integrinavb3, uptake of CLT1–fibronectin complexes into tumorcells is mediated through CLIC3 and the fibronectin recep-tor integrin a5b1, both of which are upregulated on CLT1-responsive tumor cells. This internalization mechanism alsoexplains the selectivity of CLT1 for tumor cells over restingepithelial cells, which lack integrin a5b1 and express sig-nificantly less CLIC3 than malignant cells (21–24). How-ever, even in the presence of fibronectin, CLT1 exhibits onlyreduced levels of cytotoxicity in serum-starved bladdertumor cells, suggesting that CLT1–fibronectin complexesare most effective in fast-growing, invasive urothelial cancersthat lack cell-cycle control as a result of mutations in p53,retinoblastoma (RB), or certain receptor tyrosine kinasepathways (25). These mutations are typically associated withsignificant changes to the tumor microenvironment, which

in return lead to the generation of a plethora of factors thatpromote tumor cell proliferation, survival, and invasion (26).Moreover, pro-invasive and anti-apoptotic factors alter theinteraction of integrins with the extracellular matrix byincreasing turnover and recycling of activated integrins thatare subsequently available for binding and internalization ofCLT1–fibronectin complexes (27, 28).The interaction between integrin a5b1 and fibronectin

appears to be a major determinant of CLT1 cytotoxicitybecause eliminating exogenous fibronectin in prostate tumorcells or replacing fibronectin with fibrinogen effectivelyantagonized CLT1 internalization and subsequent celldeath. While there was minimal cell death in prostate tumorcells in the absence of plasma fibronectin, we achieved onlypartial inhibition of cytotoxicity in bladder tumor cellstreated with CLT1 in plasma fibronectin-depleted media.Moreover, transfection with fibronectin siRNA caused addi-tional reduction of CLT1-induced cell death, suggesting thatbladder tumor cells generate endogenous fibronectin tosupport CLT1 internalization. This is in line with previousresults showing that fibronectin is a significant component ofurothelial tumor stroma (29, 30). Notably, for the purposeof CLT1 internalization, the origin of fibronectin appears tobe irrelevant, as both plasma fibronectin and cellular fibro-nectin are able to interact with integrin a5b1 through theirRGD-containing integrin recognition motif located withinthe fibronectin repeat III10 (31). More importantly, theinteraction of CLT1–fibronectin complexes with a5b1 ispreserved in the presence of urine, which is an importantconsideration for the intravesical treatment of bladder cancerin vivo.In addition to fibronectin, we identified CLIC3 as an

important factor for CLT1 cytotoxicity. However, whileinvolved in CLT1 internalization and cytotoxicity, CLIC3was unable to compensate for the loss of fibronectin,indicating that CLIC3 function depends on RGD-depen-dent ligation ofa5b1 with the fibronectin component of theCLT1–fibronectin complexes. This is in agreement with arecent report showing that CLIC3 mediates internalizationand recycling of integrin a5b1 from lysosomes after ligationwith fibronectin (21). Endocytosis of fibronectin is impor-tant for the turnover of focal contacts and recycling ofunligated integrin a5b1 that mediates adhesive interactionswith fibronectin during cell migration and invasion (21, 32).As a consequence, factors that promote recycling of activateda5b1 such as CLIC3 are upregulated in invasive tumor cellphenotypes (21). On the basis of this, we propose a modelwhere amyloidogenic CLT1–fibronectin co-aggregates co-opt a CLIC3-dependent recycling mechanism for integrina5b1 in invasive tumor cells, thereby inducing lysosomedysfunction and autophagic cell death. Considering the highdegree of homology between CLIC3 and the CLT1 receptorCLIC1, this mechanism could also include direct interac-tions of CLT1 with cell surface CLIC3 (33).The mechanism of CLT1 uptake shares similarities with

the integrin a5–dependent internalization of BCG–fibro-nectin complexes into bladder tumor cells, suggesting thatCLT1 could be useful for detection and treatment of clinical

Figure 7. CLT1colocalizeswith integrina5b1andCLIC3 in clinical bladdercancer. A, fluorescence microscopy analysis of bladder tumor sectionsafter incubation with fluorescein-conjugated CLT1 (green) or LCLT1 as acontrol peptide (CP) in the presence of FN or FG (each 30 mg/mL).Scale bar, 50 mm. B, tissue sections from invasive bladder cancer treatedwith fluorescein–CLT1 (green) were stained with anti-a5 integrin,anti-CLIC3 antibody, or control IgG (red) and analyzed by confocalmicroscopy. Merged images are shown in yellow. Scale bar, 10 mm.






bladder cancer (18). This concept is further supported by ourresults showing that CLT1 is particularly effective in induc-ing cell death in bladder tumor cell lines that exhibit highlevels of integrin a5b1. In addition, we show that CLT1binds to human bladder tumor tissue ex vivo in the presenceof integrina5b1, which has been shown to be upregulated inhigh-grade bladder cancer (23). On the basis of these data,we conclude that CLT1 function depends on receptors thatare commonly expressed in bladder cancer. Both BCG andCLT1 induce autophagy (17, 34); however, while internal-ization of BCG has no direct effects on the tumor cell fate(35), we show here that CLT1 causes autophagic cell deathin bladder cancer cells. The antitumor mechanism of CLT1is also different from apoptosis-inducing chemotherapeuticssuch as mitomycin C, suggesting that CLT1 could beeffective as a second line treatment or in combination withBCG,which has been shown to rendermitomycinC inactive(35). Overall, considering the robust antitumor activity ofCLT1 in combination with its specific labeling of bladdertumor tissue, it will be interesting to determine whetherCLT1 can make a significant contribution to the diagnosisand treatment of bladder cancer in vivo.

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

Authors' ContributionsConception and design: L. Knowles, J. PilchDevelopment of methodology: L. Knowles, J. Pilch, A.V. Parwani, J.R. GingrichAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): L. Knowles, G. Malik, A.V. Parwani, J.R. Gingrich, J. PilchAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L. Knowles, J. Zewe, G. Malik, A.V. Parwani, J.R. Gingrich,J. PilchWriting, review, and/or revision of the manuscript: L. Knowles, J. Zewe, G.Malik,A.V. Parwani, J.R. Gingrich, J. PilchAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): L. Knowles, J. ZeweStudy supervision: J. Pilch

AcknowledgmentsThe authors thankMichelle Bisceglia, Patricia Clark, BrandyGreenawalt, andTina

Tomko of the University of Pittsburgh Health Sciences tissue bank for their assistancein obtaining tissues and Drs. Per Basse and Simon Watkins and the UPCI ImagingFacility for help with confocal microscopy.

Grant SupportThis work was supported by NIH grants CA134330 (J. Pilch), P30CA047904

(UPCI CCSG), and Department of Defense grant PC073635-NIA (J. Pilch).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.

ReceivedMay 15, 2012; revised September 13, 2012; accepted November 6, 2012;published OnlineFirst November 30, 2012.

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2013;11:194-203. Published OnlineFirst November 30, 2012.Mol Cancer Res Lynn M. Knowles, James Zewe, Gunjan Malik, et al.

1 and CLIC3β5αCLT1 Targets Bladder Cancer through Integrin

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