Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel · Cell Fate...

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Ovarian Tumor Cell Expression of Claudin-4Reduces Apoptotic Response to PaclitaxelChristopher Breed1,2, Douglas A. Hicks1, Patricia G.Webb1, Carly E. Galimanis1,Benjamin G. Bitler1, Kian Behbakht1,2, and Heidi K. Baumgartner1,2

Abstract

A significant factor contributing to poor survival rates forpatients with ovarian cancer is the insensitivity of tumors tostandard-of-care chemotherapy. In this study, we investigatedthe effect of claudin-4 expression on ovarian tumor cellapoptotic response to cisplatin and paclitaxel. We manipulat-ed claudin-4 gene expression by silencing expression [shorthairpin RNA (shRNA)] in cells with endogenously expressedclaudin-4 or overexpressing claudin-4 in cells that natively donot express claudin-4. In addition, we inhibited claudin-4activity with a claudin mimic peptide (CMP). We monitoredapoptotic response by caspase-3 and Annexin V binding. Weexamined proliferation rate by counting the cell number overtime as well as measuring the number of mitotic cells. Prox-imity ligation assays, immunoprecipitation (IP), and immu-nofluorescence were performed to examine interactions ofclaudin-4. Western blot analysis of tubulin in cell fractions

was used to determine the changes in tubulin polymerizationwith changes in claudin-4 expression. Results show that clau-din-4 expression reduced epithelial ovarian cancer (EOC) cellapoptotic response to paclitaxel. EOCs without claudin-4proliferated more slowly with enhanced mitotic arrest com-pared with the cells expressing claudin-4. Furthermore, ourresults indicate that claudin-4 interacts with tubulin, having aprofound effect on the structure and polymerization of themicrotubule network. In conclusion, we demonstrate thatclaudin-4 reduces the ovarian tumor cell response to micro-tubule-targeting paclitaxel and disrupting claudin-4 with CMPcan restore apoptotic response.

Implications: These results suggest that claudin-4 expressionmay provide a biomarker for paclitaxel response and can be atarget for new therapeutic strategies to improve response.

IntroductionBecause of the lack of adequate early detection screening, the

majority (>85%) of the patients with epithelial ovarian cancer(EOC) are diagnosed at advanced stages of disease. Delayeddetection means that ovarian tumor cells have disseminatedbeyond their site of origin (fallopian tube or ovary) into theperitoneal cavity. The standard of care for these patients is surgicaldebulking of the tumor, followed by adjuvant platinum- andtaxane-based chemotherapies (cisplatin/carboplatin andpaclitax-el/docetaxel; ref. 1). One third of patients with ovarian cancerhave tumors that do not respond to initial chemotherapy and ofthe patients that do respond, approximately 75%of these patientswill have disease recurrence (2). Patients with recurrent EOCsuccumb to the disease following the development ofchemoresistance (3).

There are several proposed mechanisms responsible for thereduced therapeutic response that include both de novo pathways

that inherently make tumor cells resistant and the acquired path-ways that are upregulated in response to repeated chemothera-peutic insult. These pathways include, but are not limited to, drugtransporters (i.e. P-glycoprotein) that either inhibit druguptake orenhance drug efflux (4–6), epigenetic changes (i.e. MLH1 andTap73 methylation, miR-214, and miR-376c upregulation) thatprevent expression of antitumorigenic genes (7–10), expressionor mutations in proapoptotic proteins [i.e., p53, Bcl-2 familyproteins, PTEN (11–14)], and cytoskeletal organization that pre-vents binding of microtubule-targeting agents (15). Despite theincreased understanding of chemoresistance mechanisms, it isunlikely that a single pathway can be targeted to restore tumorsensitivity to all cytotoxic drugs.However, a deeper understandingof patients' genetic and proteomic characteristics, which canpredict response and inform optimal therapeutic strategies, is asignificant step forward.

Claudin-4 is a member of a large family of transmembraneproteins, of which there are 27 different subtypes (16). Clau-dins have been most studied for their canonical role in tightjunctions, where they mediate the specific paracellular barrierproperties of an epithelium. Although claudin-4 has beenshown to alter the permeability of epithelial monolayers incell culture (17–19), it is rarely localized to tight junctions inhuman and mouse tissue and is generally found along baso-lateral membranes and throughout the cytosol (20–22). Thefindings that claudin-4 knockout (KO) mice are phenotypicallynormal with only a subclinical increase in permeability to smallmolecules in lung epithelium, but have a reduced capacity forwound healing, further suggest a noncanonical role for claudin-4 (23). Furthermore, it has been well documented that claudin-4 is significantly upregulated in multiple epithelia-derived

1Department of Obstetrics and Gynecology, Division of Reproductive Sciences,Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado.2Department of Obstetrics and Gynecology, Division of Gynecologic Oncology,Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado.

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

Corresponding Author: Heidi K. Baumgartner, University of Colorado Denver,Mail Stop 8613, 12700 E. 19th Ave. Aurora, CO 80045, Phone: 303-724-3505:Fax: 303-724-3512; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0451

�2019 American Association for Cancer Research.

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cancer cells that lack traditional tight junction structures.High claudin-4 expression has, in fact, been associated witha phenotypically aggressive ovarian cancer cell [chemoresistant,highly mobile, and stem-like (24–26)]. The mechanism bywhich claudin-4 exerts these effects is currently unknown.

To investigate the noncanonical role of claudin-4 in bothnormal and tumor cell biology, we previously designed a smallclaudin mimic peptide (CMP) that interferes with the DFYNPsequence in the second extracellular loop of claudin-4 that isthought to be important in claudin–claudin interactions (27).Wehave recently shown that disrupting claudin-4 activity with CMPincreased tumor cell response to the potent apoptotic inducer,staurosporine, and inhibited cell migration (28). In addition,treatment ofmice bearing EOCcell line–derived xenograft tumorswith CMP significantly reduced the tumor burden. These observa-tions suggest that claudin-4 expression in ovarian cancer cellsplays a functional role in tumor progression.

To determine whether claudin-4 plays a role in the apoptoticresponse of ovarian tumors to standard chemotherapeutics, weexamined whether the presence of the protein altered the apo-ptotic response of EOC cell lines to cisplatin and paclitaxel in thepresence and absence of CMP. We found that claudin-4 expres-sion led to reduced tumor cell sensitivity to paclitaxel-inducedapoptosis. Tumor cell sensitivity to paclitaxel was restored bysilencing claudin-4 expression or co-treatment withCMP.We alsoshow claudin-4 involvement in cell-cycle progression and aninteraction of claudin-4 with microtubules, providing a possiblemechanism by which claudin-4 drives resistance to paclitaxel.

Materials and MethodsCell culture

Human-derived OVCAR3, OVCAR4, OVCAR5, OVCAR8,PEO4, OV429, and DOV13 ovarian tumor cells were obtainedfrom the laboratory of Monique A. Spillman (University ofColorado Denver, Aurora, CO) and cultured in RPMI1640 medi-um (Gibco, Thermo Fisher Scientific) plus 10% heat-inactivatedFBS (Access Biologicals) and 1% penicillin/streptomycin (Gibco,Thermo Fisher Scientific) at 37�C and 5% CO2. Cells were trypsi-nized (0.25% trypsin, EDTA,Mediatech) and plated 1:3 every 3–4days. All cell lines were authenticated at the beginning ofthis study by short tandem repeat profiling, as described previ-ously (29). Cells were passaged approximately 10 times beforethawing out a fresh vial of authenticated cells. Authenticationwas then repeated at the end of the study. Cells were plated at 1�104 cells/well into type I collagen-coated 8-well chamber slides(Lab-Tek, NUNC) for treatment and analysis. Upon 80%–90%confluence, cells were treated with either 400 mmol/L controlpeptide [(NH2-GDGYNPG-OH, D-amino acid conformation,University of Colorado Protein and Peptide ChemistryCore (27)], 400 mmol/L claudin mimic peptide (CMP, NH2-GDFYNPG-OH, D-amino acid conformation, University of Col-orado Protein and Peptide Core), 10 nmol/L paclitaxel (Sigma-Aldrich), and/or 10 mmol/L cisplatin (Sigma-Aldrich) for 24hours. To examine the apoptotic mechanism, cells were alsotreated with 50 mmol/L etoposide (Sigma-Aldrich).

Ovarian tumor samplesDe-identified patient tumor samples were collected from the

University of Colorado Gynecologic Tissue and Fluid Bankunder an Institutional Review Board–approved protocol

(COMIRB 05-1081). Each tumor sample was flash frozen inliquid nitrogen and stored at�80�C until processed for protein.Frozen tissue was placed in lysis buffer [30 mmol/L Tris HClpH7.4, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 2mmol/L EDTA, 0.57 mmol/L phenylmethylsulfonylfluoride(PMSF), 1� cOmplete Protease Inhibitor Cocktail] and homog-enized using a Polytron tissue homogenizer (BrinkmannInstruments, Thermo Fisher Scientific). Lysates were then spunat 13,000 rpm for 10 minutes and the supernatant collected forprotein analysis.

Western blot analysisTo determine levels of claudin-4 protein expression, tumor

cells were scraped from culture plates in the presence of lysisbuffer (30 mmol/L Tris HCl pH 7.4, 150 mmol/L NaCl, 1%Triton X-100, 10% glycerol, 2 mmol/L EDTA, 0.57 mmol/LPMSF, 1� cOmplete Protease Inhibitor Cocktail), placed on ashaker for 10 minutes and spun at 13,000 rpm for 10 minutes.Supernatant was collected and 20 mg of total protein wasdenatured, resolved on 10% SDS-PAGE, and transferred to apolyvinylidene difluoride membrane (Bio-Rad). Membraneswere blocked with 5% nonfat dry milk in Tris-buffered salinewith 0.1% Tween-20 (TBST) for 1 hour at room temperaturebefore treatment with either rabbit anti-human claudin-4(1:500, Invitrogen), mouse anti-human claudin-4 (1:500; Invi-trogen), or rabbit antihuman GAPDH (1:10,000; Sigma) over-night at 4�C. Membranes were then washed four times withTBST for 15 minutes before treatment with horseradish perox-idase (HRP)-conjugated goat anti-rabbit (1:10,000; GE Health-care) or goat anti-mouse (1:10,000; Jackson ImmunoResearchLaboratories) antibodies for 1 hour at room temperature.Membranes were washed with TBST as described above andthen visualized using an ECL Prime Western Blotting DetectionReagent (GE Healthcare) and X-ray film (CL-XPosure Film,Thermo Fisher Scientific).

Caspase activation assayApoptosis was measured by determining the number of cells

exhibiting activated caspase-3. After treatment, cells werewashed with PBS (Gibco, Thermo Fisher Scientific) and fixedwith 10% phosphate-buffered formalin (Thermo Fisher Scien-tific) at room temperature for 15 minutes. Cells were washedtwice with PBS before cell membrane permeablization with0.5% Triton X-100 (IBI Scientific) for 5 minutes and washedagain with PBS. Cells were treated with blocking buffer (2%BSA; Sigma-Aldrich) for 1 hour before application of primaryantibody directed to cleaved caspase-3 (1:400; rabbit anti-human cleaved caspase-3, Cell Signaling Technology) over-night at 4�C. Cells were then washed with PBS five times beforeapplication of secondary antibody conjugated to CY3 (1:100;donkey anti-rabbit, Jackson ImmunoResearch Laboratories)and 5 mg/mL 40,6-diamidino-2-phenylindole (DAPI; Sigma)for 45 minutes at room temperature followed by washing withPBS five times. After removal of PBS, o-phenylenediaminedihydrochloride (20 mg/mL; OPDA) in 1 mol/L Tris (pH 8.5)was added to the slides to preserve fluorescence and cover-slipped. The Olympus FV1000/RICS confocal microscope(University of Colorado AMC Light Microscopy Core) was usedto image fluorescence. The percent of cells positive for activecaspase was determined using SlideBook software (IntelligentImaging Innovations, Inc.).

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shRNA knockdownOVCAR3 and PEO4 cells were plated 3.2 � 104 in a 96-well

plate and incubated at 37�C for 24hours.When cells reached 70%confluence, 10 mL of claudin-4 short hairpin RNA (shRNA; TRC#:TRCN0000116627, TRCN0000116628, or TRCN0000116631)or control shRNA (SHC001, pLK0.1-puro EmptyVector) lentiviralsuspension (Sigma-Aldrich MISSION shRNA, University ofColorado Functional Genomics Facility) was added to the cellsand incubated overnight at 37�C. Fresh medium was added toremove lentivirus and cells were allowed to recover for 24 hoursbefore being treated with 0.5 mg/mL puromycin for selection andexpansion of transduced cells. Western blot analysis (as describedabove) confirmed loss of claudin-4 expression.

Overexpression of claudin-4GFP-tagged claudin-4 and aGFP-only control (pLenti-C-mGFP

vector; OriGene) were transduced into OVCAR8 and OVCAR4cells via lentiviral particles in the presence of 10 mg/mL hexadi-methrine bromide. GFP-positive cells were flow-sorted andexpanded.

Mitotic assaysTo examine the number of cells in mitosis, we examined the

images of DAPI fluorescence in OVCAR3 cells cultured in 8-wellchamber slides. Cells that had clearly condensed chromatin thataligned along a single plane were counted as mitotic cells andcalculated as the percentage of total number of nuclei per field ofview (at least 12 fields of view were analyzed for each condition).In addition, a Mitotic Assay Kit (Active Motif) was used as permanufacturer's instructions. Briefly, cells were cultured in 96-wellplates, fixed, and treated with a phospho-histone H3 (Ser 28)monoclonal primary antibody followed by an HRP-conjugatedsecondary antibody. Absorbance at 450 nm was read after 20-minute incubation with the Developing Solution. Crystal violetwas used to normalize for total cell number.

Proliferation assayA total of 10,000 cells were plated in each well of 12-well

culture plates. Cells were trypsinized (0.25% trypsin, EDTA) and

counted by aCountess AutomatedCell Counter (Invitrogen) after24, 48, and 72 hours in culture.

Cell-cycle analysisCells were plated in 100-mm culture dishes at 5 � 105 cells

per dish. After 24 hours in culture, media were changed toserum-free RPMI1640 for 48 hours. Cells were then exposed tomedia containing serum and harvested 6 hours later. Cells wereplaced in Krishan stain (0.224 g sodium citrate 2H2O, 9.22 mgpropidium iodide, 2.0 mL 1% NP40 in H2O, 2.0 mL 1 mg/mLRNase in 200 mL H2O) overnight at 4�C before being sent tothe University of Colorado Flow Cytometry Core Facility,Aurora, CO). Cells were analyzed by flow cytometry (BeckmanCoulter Gallios, University of Colorado Flow Cytometry CoreFacility, Aurora, CO), measuring fluorescence emission at >575nm (FL3). FlowJo software was used to determine number ofcells in G2–M phase.

Proximity ligation assayClaudin-4 interaction with tubulin was examined using the

DuoLink Proximity Ligation Assay Kit (Sigma), followingthe manufacturer's protocol. Briefly, cells were plated in 8-wellchamber slides and cultured to 70% confluence before beingfixed, permeablized, and blocked as described above forcapase-3 immunofluorescence. Cells were then treated with anti-bodies directed to claudin-4 (mouse anti-human claudin-4;1:200; Invitrogen) and a-tubulin (rabbit anti-human a-tubulin;1:100; Abcam) or b-tubulin (rabbit anti-human b-tubulin; 1:100;Abcam) overnight at 4�C. After washing with PBS, cells weretreated with PLA probes (1:5; anti-mouse PLUS, anti-rabbitMINUS) for 1 hour at 37�C in a preheated humidity chamber.After washing, Ligation Solution was added for 30 minutes at37�C followed by washing and addition of amplification poly-merase solution for 100 minutes at 37�C in a humidity chamber.Slides were washed and dried before adding mounting mediumand placing coverslip. Fluorescence was imaged using an Olym-pus FV1000/RICS confocal microscope (University of ColoradoAMC Light Microscopy Core).

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Figure 1.

Claudin-4 expression in HGSOCcells. A, CLDN4 gene expressionlevels in laser capturemicrodissected specimens ofhuman high-grade serous ovariantumors compared with isolatednormal human ovarian surfaceepithelial cells (36). Western blotanalysis of claudin-4 protein levelsin human patient samples of normalfallopian tube and ovarian cancer(B) as well as high-grade serousovarian tumor cell lines (C), usingGAPDH as a loading control. Mean� SEM, n¼ 10 HOSE, n¼ 10–53HGSOC tumor specimens, n¼ 7other EOC subtypes (� , P < 0.05;�� , P < 0.0001).

Claudin-4 Reduces Response to Paclitaxel

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ImmunoprecipitationClaudin-4 (and interacting proteins) was pulled from cell

lysates with an antibody directed to claudin-4 (mouse anti-human claudin-4, Invitrogen) bound to Protein A/G Dyna-beads Magnetic beads, using the Pierce Crosslink Immunopre-cipitation (IP) Kit (Thermo Fisher Scientific). Beads were incu-bated with lysate (�500 mg protein) for 1 hour at roomtemperature and then collected using a Magnetic SeparationRack (New England BioLabs). Proteins were eluted from beadsper manufacturer's protocol. Eluted proteins were run on 10%SDS-PAGE and Western blot analysis was performed asdescribed above, using antibodies directed to claudin-4 (rabbitanti-human claudin-4; 1:500) and tubulin (rabbit antihumana-tubulin or b-tubulin; 1:1,000).

Visualization of claudin-4 and microtubulesImmunofluorescence was performed, as described above, for

caspase-3 activation as well as to visualize claudin-4 and tubulinin tumor cells. Primary antibodies used in these assays were the

same (including dilutions) as those described above for theproximity ligation assays.

Tubulin polymerization assayThe Microtubules/Tubulin In Vivo Assay Kit (Cytoskeleton

Inc.) was used to determine changes in tubulin polymerizationwith changes in claudin-4 expression. Cells were grown to 80%confluence before being washed with PBS and scraped from100-cm2 plates in tubulin stabilization buffer provided by themanufacturer of the kit and homogenized by pipetting. Cellsuspension was centrifuged at 1,000 � g (37�C) for 5 minutes.Cell pellets and supernatant were collected separately andresolved on 10% SDS-PAGE gel. Primary tubulin antibody andHRP-conjugated secondary antibody provided by the manu-facturer of the kit were used to determine polymerized (pelletfraction) and nonpolymerized (supernatant fraction) tubulinlevels. Densitometry, using ImageJ, was performed to quantifychanges in levels of polymerized to nonpolymerized tubulin ineach cell line.

Figure 2.

Disruption of claudin-4 activity with CMP enhances paclitaxel response. A, Representative images of nuclei (DAPI staining) and activated caspase-3 (fluorescentantibody directed to cleaved caspase-3) from OVCAR3 cells. Quantification of caspase-3 activation in OVCAR3 (B) and OVCAR8 (C) cells treated for 24 hourswith vehicle control (Veh cont), 400 mmol/L inactive control peptide (ContP), 400 mmol/L CMP, 10 mmol/L Cisplatin (Cis), CMPþ Cisplatin, 10 nmol/L paclitaxel(taxol), or CMPþ paclitaxel. Percent of cell population positive for caspase-3 activation was calculated using SlideBook software (3i). Mean� SEM; n¼ 3; NS, notsignificant; �� , P < 0.001 versus control/drug only.

Breed et al.





Statistical analysisData are presented as mean � SEM. An unpaired Student t test

was used for statistical comparison between control and treat-ment groups. A one-way ANOVA was used to determine varianceamong multiple groups, with a Bonferroni Multiple Comparisonposttest to determine significance between individual groups. A Pvalue of

paclitaxel with CMP cotreatment. As a secondary assay ofapoptotic response, we used flow cytometry to measure num-ber of OVCAR3 cells exhibiting Annexin V binding and con-firmed enhanced apoptosis (Annexin V binding) in response topaclitaxel when cotreated with CMP (Supplementary Fig. S3).

Loss of claudin-4 expression improves tumor cell apoptoticresponse to paclitaxel

Using a small hairpin RNA (shRNA) specific to claudin-4,we knocked down claudin-4 expression in OVCAR3 cells("shCLDN4_2" and shCLDN4_3; Fig. 3A). A control, emptyvector, shRNA, and shCLDN4_1 did not impact expression ofthe claudin-4 protein ("shCTRL" and "shCLDN4_1", respective-ly). Similar to parental OVCAR3 cells, shCTRL and shCLDN4_1cells showed increased apoptosis with CMP or paclitaxel aloneand a significantly enhanced apoptotic response to paclitaxel inthe presence of CMP (P < 0.0001; Fig. 3B). CMP did not induce anapoptotic response in OVCAR3 shCLDN4_2 and shCLDN4_3cells (cells that successfully lost claudin-4 expression) and CMPcotreatment did not enhance paclitaxel-induced apoptosis (P ¼0.8890 and 0.8851, respectively, for shCLDN4_2; P¼ 0.5024 and0.7734, respectively, for shCLDN4_3). However, the response topaclitaxel alone was significantly (P < 0.0001) higher in theshCLDN4_2 and shCLDN4_3 cells compared with shCTRL andshCLDN4_1 cells, indicating that disrupting claudin-4 augmentsthe apoptotic response to paclitaxel. Similar results were seen inthe claudin-4–expressing PEO4 cell line (Supplementary Figs. S4and S5).

Overexpression of claudin-4 reduces tumor cell response topaclitaxel

We next evaluated the impact of overexpressing claudin-4 inOVCAR8 andOVCAR4 cells, which endogenously express no/lowclaudin-4 protein (Fig. 1).We transfected both cell lineswithGFP-tagged claudin-4 or a control GFP vector. Western blot analysisshowed strong expression of the claudin-4-GFP (Fig. 4A). GFPcontrol cells didnot respond toCMP treatment; apoptosiswas notinduced with CMP and paclitaxel apoptotic response was notenhanced with CMP cotreatment. However, cells with claudin-4-GFP overexpression had a decreased apoptotic response to pac-litaxel and were responsive to CMP, with induction of apoptosiswith CMP alone and enhanced apoptotic response to paclitaxelin the presence of CMP (Fig. 4B, representative images of nucleiand activated caspase-3 in claudin-4 nonexpressing control andclaudin-4 overexpressing tumor cells are shown in SupplementaryFig. S6). Similar responses were seen in OVCAR4 cells (Supple-mentary Fig. S7).

Claudin-4 expression results in increased mitotic fidelityWe have shown in the experiments above that claudin-4

diminishes the apoptotic response to paclitaxel. We nextwanted to determine a potential mechanism by which clau-din-4 could affect cell biology to influence paclitaxel response.Upon examination of nuclei in the OVCAR3 cell line, weobserved significantly more mitotic figures in shCLDN4_2 cellscompared with the claudin-4–expressing shCTRL cells (P <0.0001; Fig. 5A and B). To confirm this observation, we useda colorimetric mitotic assay kit that utilizes a phospho-histoneH3 (Ser28) mAb with HRP-conjugated secondary antibody todetect the level of mitosis. The loss of claudin-4 expressionsignificantly increased the number of cells in the M-phase of

mitosis (Fig. 5C, P < 0.0001). To determine whether thisincreased mitotic index resulted in an increase in cell prolifer-ation, we measured cell number over time in the shCTRL andshCLDN4_2 cell lines. We found that the claudin-4 KO cellsproliferated significantly more slowly than the control cells(P ¼ 0.0453, Fig. 5D). Cell-cycle analysis, using flow cytometry(Fig. 5E and F), confirmed significantly (P < 0.0001) moretumor cells in the G2–M phase at 6 hours postreleasefrom serum-free conditions in cells that did not express clau-din-4 (shCLDN4_2) compared with the cells that did expressclaudin-4 (shCTRL). These findings suggest that the increase inmitotic figures in the cells lacking claudin-4 expression is notthe result of increased proliferation, but reflects an inhibition ofG2–M progression.

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Figure 4.

Overexpression of claudin-4 reduces paclitaxel response. A,Western blotanalysis of claudin-4 protein in OVCAR8 cells transduced with GFP only orclaudin-4-GFP. GAPDHwas used as a loading control. B,Immunofluorescence analysis of fixed OVCAR8 cells that were treated with400 mmol/L inactive control peptide (ContP), 400 mmol/L CMP, 10 nmol/Lpaclitaxel (taxol), or CMPþ paclitaxel for 24 hours. Cells were treated withfluorescent antibody directed to cleaved caspase-3 and DAPI (nuclei) andpercent of cell population positive for caspase-3 was calculated usingSlideBook software (3i). Mean� SEM; n¼ 3 per treatment group; NS, notsignificant; �� , P < 0.01; �� , P < 0.001 versus control/paclitaxel only.

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Microtubules are known to play an important role in bothpaclitaxel response and cell-cycle progression. Therefore, weexamined whether claudin-4 interacted with microtubules.Proximity ligation assays were performed on OVCAR3 cellswith antibodies directed to claudin-4 and either a-tubulin orb-tubulin. Claudin-4 was found to be within interacting dis-tance of both a-tubulin and b-tubulin (Fig. 6A). IP of claudin-4confirmed the interaction with a-tubulin (Fig. 6B), but wewere unable to confirm the interaction with b-tubulin by thismethod. However, immunofluorescence assays revealed thatclaudin-4 was colocalized with b-tubulin at the mitotic spindlein tumor cells undergoing mitosis (Fig. 6C). In addition,images of microtubules in claudin-4–expressing (shCTRL cells)and nonexpressing (shCLDN4_2) OVCAR3 cells revealed sig-nificant changes in microtubule structure when claudin-4expression was present (Fig. 6D).

Claudin-4 expression alters polymerization of tubulinTo identify a potential role for claudin-4 in altering micro-

tubule stability, we examined the levels of tubulin withinmicrotubules (polymerized tubulin) compared with free tubu-lin (nonpolymerized tubulin) in OVCAR3 cells expressing andnot expressing claudin-4. Figure 7A shows a shift betweenpolymerized (pellet: p) and nonpolymerized (supernatant: s)tubulin levels when claudin-4 expression is silenced. Densi-tometry analysis revealed that there was a significant (P ¼0.0054) decrease in the percentage of total tubulin foundwithin microtubules (polymerized) in the claudin-4 KO

(shCLDN4_2) compared with claudin-4 expressing control(shCTRL) cells (Fig. 7B), suggesting that claudin-4 is enhancingthe polymerization of microtubules.

DiscussionIn this study, we have shown that claudin-4 expression reduces

the apoptotic response of ovarian tumor cells to paclitaxel.Disruption of claudin-4 expression (shRNA knockdown) or activ-ity (CMP treatment) can enhance or restore the paclitaxel apo-ptotic response through activation of apoptosis. In addition, wedemonstrated that loss of claudin-4 delayed progression throughthe cell cycle and reduced proliferation. The effect on bothapoptosis and the proliferative response in tumor cells may bedue the interaction of claudin-4 withmicrotubules and alterationof tubulin polymerization.

Previously, high claudin-4 expression was shown to be asso-ciated with ovarian tumor cells that are resistant to chemother-apeutic agents (24, 25, 31). For example, CD44þ ovarian tumorcells were shown to have significantly higher expression levels ofclaudin-4 compared with CD44� populations isolated from thesame tumor. The CD44þ/high claudin-4–expressing cells weremore resistant to carboplatin and paclitaxel treatment comparedwith the low/no expressing cells (25). In addition, Gao andcolleagues have demonstrated that inhibiting claudin-3 and -4expression with a fragment of the Clostridium perfringens entero-toxin increased the ovarian tumor cell response to both paclitaxeland carboplatin (31). Although our data shows that cells

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Loss of claudin-4 leads to delayed mitotic progression. A, Representative images of DAPI (DNA) staining in fixed monolayers of control knockdown(shCTRL) and claudin-4 knockdown (shCLDN4_2) OVCAR3 cells. Yellow circles highlight mitotic figures. B, Visual counting of mitotic figures fromDAPI images. C, Colorimetric measurement of phosphorylated H2B (mitotic marker) to quantify population of mitotic cells in shCTRL (white bars) andshCLDN4_2 (black bars) OVCAR3 cells. D, Proliferation rates of shCTRL (circles/solid line) and shCLDN4_2 (triangles/dotted line) OVCAR3 cells.E, Representative histograms of propidium iodide staining 6 hours after release from serum starvation synchronization in shCTRL and shCLDN4_2cells. F, Quantification of the percent of the total cell population in the G2–M phase of the cell cycle using FlowJo software. Mean � SEM; n ¼ 3(� , P < 0.05; �� , P < 0.001 versus control/paclitaxel only).






expressing high levels of claudin-4 (OVCAR3) are more resistantto cisplatin compared with the cells that do not express claudin-4(OVCAR8), CMP was unable to enhance the apoptotic responseto cisplatin in the claudin-4–expressing tumor cells. These resultssuggest the possibility that claudin-4 may regulate the apoptoticresponse to cisplatin through a different mechanism than pacli-taxel, potentially through an interaction of claudin-4 that doesnot involve the second extracellular loop (which is targeted byCMP).

Previous studies investigating a role for claudin-4 in prolifer-ation have not been consistent. Agarwal and colleagues demon-strated that overexpressing claudin-4 in normal HOSE cellsenhanced survival, but did not alter proliferation (32). On theother hand,Ma and colleagues demonstrated that loss of claudin-4 expression inhibits proliferation in MCF7 breast cancer cellsin vitro and in vivo (33). The discrepancies could be due todifferences in the regulation (or loss of regulation) of claudin-4in normal versus tumor cells. In addition, our results demonstratethat tumor cells still proliferate in the absence of claudin-4. Thissuggests that claudin-4 is not required for proliferation but doesenhance proliferationwhen it is present. The observation that loss

of claudin-4 leads to increased number of cells inmitosis indicatesthat claudin-4 likely enhances proliferation by facilitating pro-gression through mitosis.

A proteomic analysis of proteins within interacting distanceof claudin-4 in MDCK cells was performed by Fredricksson andcolleagues and b-tubulin was identified as a potential interact-ing partner of claudin-4 (34). Our results confirm an interac-tion of claudin-4 with both a-tubulin and b-tubulin in ovariantumor cells and that this interaction changes during the cellcycle. We were able to capture an interaction of claudin-4 atsites at or near the plasma membrane as well as within themitotic spindle. Further analysis of this interaction will beneeded to fully understand how the interaction of claudin-4with microtubules may be regulating tumor cell survival andgrowth. Our results do suggest that interaction of claudin-4with tubulin may alter the polymerization of tubulin. McGrailand colleagues have shown that ovarian tumor cells that areresistant to paclitaxel-induced death have altered microtubulestability (35). Therefore, it is possible that claudin-4 altersmicrotubule dynamics in a way that makes tumor cells moreresistant to paclitaxel-induced death.

Whole

Claudin-4 / α-tubulin

Claudin-4 / β-tubulin a-Tubulin

Claudin-4

A B

co-localization

Claudin-4β tubulinDAPI

cld4 β tubulin DAPIC

20 µm

20 µm

D shCTRL (+ claudin-4)Tubulin DAPI

Tubulin

10 μm

shCLDN4_2 (- claudin-4)Tubulin DAPI

Tubulin

10 μm

Figure 6.

Claudin-4 interacts with tubulin. A, Proximity ligation assay using antibodies directed at claudin-4 and a-tubulin or b-tubulin. Red fluorescence indicatesprotein–protein interaction. Yellow outlined boxes are higher magnification of cell from image. Arrows point to sites of protein-protein interaction. B, IP ofclaudin-4 (cld-4 IP) and IgG (mouse IgG IP) from OVCAR3 lysates blotted for presence of a-tubulin and claudin-4. C, Immunofluorescence of claudin-4 (green)and b-tubulin (red), with DAPI (blue), in mitotic OVCAR3 cell. Yellow color indicates colocalization of claudin-4 and b-tubulin. D, Immunofluorescence ofmicrotubules (b-tubulin; white/red) and nuclei (DAPI/blue) in cells expressing (shCTRL) and not expressing (shCLDN4_2) claudin-4.

Breed et al.





In conclusion, we have demonstrated that claudin-4 plays animportant role in tumor cell resistance to paclitaxel. Therefore,expression of claudin-4 by tumor cells may be a biomarker forpatient response to paclitaxel. In addition, the development of aclaudin-4 therapeutic (e.g., CMP) could provide a novel way toimprove the patient response to paclitaxel.

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

Authors' ContributionsConception and design:C.A. Breed, B.G. Bitler, K. Behbakht, H.K. BaumgartnerDevelopment ofmethodology:C.A. Breed, D.A. Hicks, B.G. Bitler, K. Behbakht,H.K. BaumgartnerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.A. Breed, D.A. Hicks, C.E. Galimanis, B.G. Bitler,K. Behbakht, H.K. BaumgartnerAnalysis and interpretation of data (e.g., statistical analysis,biostatistics, computational analysis): C.A. Breed, D.A. Hicks, K. Behbakht,H.K. BaumgartnerWriting, review, and/or revision of the manuscript: C.A. Breed, B.G. Bitler,K. Behbakht, H.K. BaumgartnerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.G. Webb, C.E. Galimanis, K. Behbakht,H.K. BaumgartnerStudy supervision: K. Behbakht, H.K. Baumgartner

AcknowledgmentsThe authors wish to acknowledge Dziuleta Cepeniene of the Peptide and

Protein Chemistry Core at the University of Colorado for synthesizing CMP.Wewould also like to acknowledge that imaging was made possible by theUniversity of Colorado Advanced Light Microscopy Core, supported in partby NIH/NCRR CCTSI grant UL1 RR025780. Flow cytometry was performed bythe University of Colorado Cancer Center Flow Cytometry Shared Resource(supported by NCI Cancer Center Support Grant P30CA046934). Funding wasalso provided by the Department of Obstetrics and Gynecology, University ofColorado Denver.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 3, 2018; revised October 11, 2018; accepted December 20,2018; published first January 3, 2019.

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Claudin-4 alters tubulin polymerization. A, RepresentativeWestern blotanalysis of polymerized (P, pellet fraction) and nonpolymerized (S,supernatant fraction) tubulin levels in OVCAR3 cells expressing claudin-4(shCTRL) and cells with silenced claudin-4 expression (shCLDN4_2). B,Quantification of the percent of the total tubulin that is polymerized. Mean�SEM; n¼ 4 (�� , P < 0.01).






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Breed et al.




2019;17:741-750. Published OnlineFirst January 3, 2019.Mol Cancer Res Christopher Breed, Douglas A. Hicks, Patricia G. Webb, et al. Response to PaclitaxelOvarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic

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