IKKbRegulatesVEGFExpressionandIsaPotential...

Small Molecule Therapeutics

IKKbRegulatesVEGFExpressionand IsaPotentialTherapeutic Target for Ovarian Cancer as anAntiangiogenic TreatmentYasuto Kinose1, Kenjiro Sawada1, Hiroshi Makino2, Tomonori Ogura2, Tomoko Mizuno2,Noriko Suzuki2, Tomoyuki Fujikawa3, Eiichi Morii4, Koji Nakamura1, Ikuko Sawada1,Aska Toda1, Kae Hashimoto1, Aki Isobe1, Seiji Mabuchi1, Tsuyoshi Ohta5, Akiko Itai3,Ken-ichirou Morishige2, Hirohisa Kurachi5, and Tadashi Kimura1

Abstract

The prolongation of progression-free survival (PFS) inpatients with advanced ovarian cancer by antiangiogenic ther-apy has been shown in several clinical trials. However,although an anti-VEGF antibody (bevacizumab) is the onlyoption currently available, its efficacy is limited and it is notcost effective for use in all patients. Therefore, the developmentof a novel antiangiogenic drug, especially composed of small-molecule compounds, could be a powerful armament forovarian cancer treatment. As NF-kB signaling has the potentialto regulate VEGF expression, we determined to identify wheth-er VEGF expression is associated with NF-kB activation and toinvestigate the possibility of a novel IKKb inhibitor, IMD-0354(IMMD Inc.), as an antiangiogenic drug. Tissue microarraysfrom 94 ovarian cancer tissues were constructed and immu-nohistochemical analyses performed. We revealed that IKK

phosphorylation is an independent prognostic factor (PFS:26.1 vs. 49.8 months, P ¼ 0.011), and is positively correlatedwith high VEGF expression. In in vitro analyses, IMD-0354robustly inhibited adhesive and invasive activities of ovariancancer cells without impairing cell viabilities. IMD-0354 sig-nificantly suppressed VEGF production from cancer cells,which led to the inhibition of angiogenesis. In a xenograftmodel, the treatment of IMD-0354 significantly inhibitedperitoneal dissemination with a marked reduction of intratu-moral blood vessel formation followed by the inhibition ofVEGF expression from cancer cells. IMD-0354 is a stable small-molecule drug and has already been administered safely tohumans in other trials. Antiangiogenic therapy targeting IKKbis a potential future option to treat ovarian cancer. Mol CancerTher; 14(4); 1–11. �2015 AACR.

IntroductionThe prognoses of patients with ovarian cancer have remained

much the same since the late 1990s, and, for this reason, a largenumber of novel molecular targeted agents and innovative ther-apeutic associations of chemotherapy have been attempted andremain under investigation (1, 2). Among the new drugs studiedfor ovarian cancer, bevacizumab, an anti-VEGF antibody, hasshown promising activities in combination with standard che-motherapy in several phase III trials, such as the ICON7 andGOG218 studies (3, 4).

Angiogenesis has been shown to be an important contributorto ovarian carcinogenesis and progression. Among various angio-genic factors, VEGF, themost endothelial cell–specific angiogenicfactor characterized to date, induces endothelial cell proliferation,promotes cell migration, and inhibits apoptosis. VEGF is firmlybelieved to be a key regulator of physiologic and pathologicangiogenesis (5). Thus, it is likely that bevacizumab would bea powerful tool for ovarian cancer treatment. It has shownrevolutionary efficacies not only as a first-line therapy combinedwith paclitaxel plus carboplatin but also as a treatment forrelapsed cases. However, several concerns remain unresolved. Inphase III trials (ICON7 and GOG218), although maintenancetreatment with bevacizumab extended the length of PFS, thisadvantage stopped 10months after the cessation of treatment andonly ICON7 showed a trend toward improved overall survival(OS) in a subgroup of patients with high-risk disease (3, 4). Inplatinum-sensitive relapse, although bevacizumab has also beenshown to improve PFS (OCEANS; ref. 6), no OS benefit wasfound. In platinum-resistant relapse, a recent phase III trial(AURELIA) reported an increase in response rate and a doublingof PFS (6.7 vs. 3.4 months) in those who received single-agentchemotherapy plus bevacizumab compared with chemotherapyalone, while the effect on OS has not been reported (2, 7). Theseclinical data indicate that, in order to improve OS, long-termcontinuous antiangiogenic maintenance therapy would be indis-pensable until patients show progressive disease. However, the

1Department of Obstetrics and Gynecology, Osaka University Gradu-ate School of Medicine, Osaka, Japan. 2Department of Obstetrics andGynecology,GifuUniversityGraduate School ofMedicine,Gifu, Japan.3IMMD Inc.,Tokyo, Japan. 4Departmentof Pathology,OsakaUniversityGraduate School of Medicine, Osaka, Japan. 5Department of Obstet-rics and Gynecology, Yamagata University Faculty of Medicine,Yamagata-shi, Japan.

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

Corresponding Author: Kenjiro Sawada, Department of Obstetrics and Gyne-cology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka Suita,Osaka 5650871, Japan. Phone: 816-6879-3351; Fax: 816-6879-3359; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-0696

�2015 American Association for Cancer Research.

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Published OnlineFirst January 30, 2015; DOI: 10.1158/1535-7163.MCT-14-0696

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pharmacoeconomic issue is obviously a major obstacle for clin-ical long-term use of bevacizumab in every patient. Hensley andcolleagues commented that bevacizumab costs $78.3 million for3.8 progression-free months for 600 women (8). Hence, webelieve that it would be meaningful to find better compoundsthat specifically inhibit angiogenesis with safety profiles, giventhat antiangiogenic therapy is to be used in combination withcytotoxic chemotherapies. Indeed, antiangiogenicmolecules havebeen developed and many clinical trials are underway (9).

NF-kB is an inducible transcription factor that regulates awide variety of gene expressions. Constitutive NF-kB signalinghas been identified in tumors of epithelial origin, includingbreast, colon, lung, and ovarian carcinomas (10), and numer-ous in vitro and in vivo studies have suggested that NF-kB playsan important role in cancer progression, including angiogenesis(11). Although various studies have suggested that NF-kBsignaling correlates with angiogenesis, the importance of NF-kB in terms of VEGF expression remains controversial (12–14).In addition, the prognostic value of high NF-kB activity onovarian carcinomas is also controversial. Kleinberg and col-leagues reported that the nuclear localization of NF-kB/RelA(p65) is frequently expressed in advanced stage serous ovariancarcinomas, and is associated with poor PFS (15), whileAnnunziata and colleagues wrote that high expression of cyto-plasmic NF-kB1 (p50) was associated with poor survival inpatients with advanced-stage ovarian cancer (16). However,more recently, Yang and colleagues showed that the nuclearaccumulation of p65 in epithelial ovarian carcinomas is sig-nificantly associated with a good response to chemotherapyand can predict longer OS of patients (17). For these reasons,we were encouraged to analyze whether NF-kB signaling reg-ulates VEGF expression in ovarian cancer tissues as well aswhether targeting this signaling can be an alternative option forthe inhibition of VEGF expression, leading to a new antiangio-genic therapy.

The NF-kB/Rel family includes NF-kB1 (p50), NF-kB2 (p52),RelA (p65), RelB, and c-Rel. They can all form homo- andheterodimers, with the most abundant form being p50/p65(18). These dimers are present in the cytosol in an inactive form,complexed with proteins of the IkB family. Upon stimulation,phosphorylation of IkB by IkB kinase (IKK) leads to degradationof IkB and renders NF-kB free to translocate into the nucleus. Thenuclear NF-kB binds to promoters and enhancers, leading to genetranscription (11). Two IkB kinases, IKKa and IKKb, have beenwell described. It is known that they have nonequivalent func-tions and that activation of IKK-b, rather than IKK-a, participatesin the primary pathway, by which proinflammatory stimuliactivate NF-kB (18). Given that the indispensable roles playedby NF-kB in many biologic processes raise the concern that acomplete shutdown of this pathway would have significant det-rimental effects on normal cellular function, drugs that selectivelytarget only IKK-b activity could be of greater therapeutic value(19). IMD-0354 is a newly synthesized low molecular weightcompound that selectively inhibits IKKb in the IKK complex.IMD-1041 is a prodrug of IMD-0354 for oral administration andon its way to clinical application at present. A phase I study wascompleted and up to 12 weeks of IMD-1041 administrationproved to be sufficiently safe with very few adverse events rankedas mild (20). Therefore, we determined to elucidate the effect ofthis drug on ovarian cancer cells, focusing especially on VEGFexpression.

With these goals in mind, we first constructed tissue micro-arrays (TMA) from ovarian cancer samples and immunostainedthem with phosphorylated-IKK and VEGF antibody. We revealedthat IKK phosphorylation is an independent prognostic factorand that its expression was positively correlated with high VEGFexpression, suggesting that NF-kB activation regulates VEGFexpression in ovarian cancer tissues. Furthermore, we assessedthe potential of a small molecular weight IKK inhibitor, IMD-0354, as a novel therapeutic option for antiangiogenic treatmentagainst ovarian cancer.

Materials and MethodsMaterials

IMD-0354, a synthetic IKK-b inhibitor, was supplied by IMMDInc.. Growth factor–reduced basement membrane proteins(Matrigel), human fibronectin, collagen type 1, and IntegrinsSampler Kits [#611435] were purchased from BD Biosciences.Antibodies against VEGF (A-20), integrin a1 (R-164), matrixmetalloproteinase-2 (MMP-2; H-76), and CD31 (M-20) werefrom Santa Cruz Biotechnology. Antibodies against p-IKKa/b(Ser176/180), IkB-a, b-actin, and cleaved caspase-3 (Asp175)were obtained from Cell Signaling Technology. Antibody againstKi-67 (RM-9106) was from Thermo Scientific.

Cell cultureSKOV3ip1 cells (serous adenocarcinoma, mutation; PIK3CA,

ARID1A, amplification; ERBB2; ref. 21) were kindly provided byDr. Ernst Lengyel (University of Chicago, Chicago, IL) in 2007.RMUG-S cells (mucinous adenocarcinoma, mutation; TP53;ref. 21)wereobtained from theHealth ScienceResearchResourcesBank (Osaka, Japan) in 2008. CaOV3 (serous adenocarcinoma,mutation; TP53) and ES-2 cell lines (clear-cell carcinoma, muta-tion; TP53, BRAF; ref. 21) were purchased from ATCC (CaOV3; in2007, ES-2; in 2014). Cellswere cultured inDMEMsupplementedwith 10% FBS and 1,000 U/mL penicillin/streptomycin andincubated in 95% air/5% CO2 at 37�C. Cells were authenticatedby short tandem repeat DNA profiling at Takara-Bio Inc. and wereused for this study within 6 months of resuscitation. Humanumbilical vein endothelial cells (HUVEC) were collected as pre-viously reported (22).

TMA preparation and immunohistochemistryOvarian carcinoma samples were collected from patients trea-

ted at Gifu University Hospital (Gifu, Japan) between 2006 and2011 and used to construct the tissue microarray slides. From thecorresponding regions in paraffin blocks, tissue cores (diameter,3 mm) were removed using a hollow needle, arrayed in paraffinblocks and sliced (4 mm) onto slides. Institutional Review Boardapproval was obtained. Satisfactory tissue cores were finallyobtained from 94 patients, and the corresponding clinical datawere collected. The slides were deparaffinized in xylene andrehydrated with 100% ethanol before antigen unmasking wasperformed by boiling in Target Retrieval Solution (pH 9.0;Nichirei Biosciences). After being placed in 3% H2O2 and beingblocked with blocking solution (Dako), they were incubated withthe primary p-IKKa/b antibody at 1:150 and VEGF-A antibody at1:40 for 1 hour at room temperature. After washing with TBScontaining 0.1% Tween-20 (TBST), they were stained using theEnvision system (Dako) and then counterstained with Carrazzi

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hematoxylin. Slides were intensively examined by two indepen-dent qualified pathologists (E. Morii and S. Mabuchi) withoutknowledge of the clinical outcomes. The phospho-IKK stainingsample was scored on the basis of the intensity of the staining(0, none; 1, weak; 2, strong). "Positive" expression of p-IKK wasdefined if the score of intensity was 1 or 2. "Negative" expressionwas defined if the score of intensity was 0. Human VEGF-Astaining was scored on the basis of the intensity of the staining(1, weak; 2, medium; 3, strong) and the percentage of positivecells (1, <10%; 2, 10%–40%; 3, >40%). "High" VEGF expressionwas defined if the total score of intensity and density was �5."Low" VEGF expression was defined if the total score was �4.

Cell viability assessmentOvarian cancer cells (3 � 103 cells) were seeded in 96-well

plates and cultured in DMEM supplemented with 10% FBSwith increasing concentrations of IMD-0354 ranging from 0.1 to1.0 mmol/L for 48 hours. Cell viability was assessed using theCyQUANT cell proliferation assay kit (Molecular Probes). Cellviability was expressed as the ratio of the number of viable cellswith IMD-0354 treatment to the number without treatment.

Western blot analysisA total of 5� 105 cells were plated onto 6-well plates and lysed

with 1 � Cell Lysis Buffer (Cell Signaling Technology). Lysates(15 mg) were separated by 5%–20% SDS-PAGE and transferredto polyvinylidene difluoride membranes, followed by incuba-tion with the primary antibodies (p-IKKa/b, 1:1,000 in 5% BSA;IkB-a, 1:1,000 in5%BSA; integrina1, 1:1,000 in5%BSA; integrina2, 1:1,000 in 5% BSA; integrin a5, 1:2,500 in 5% BSA; integrinb1, 1:2,500 in 5% BSA; integrin b3, 1:2,500 in 5% BSA; MMP-2,1:1,000 in 5% BSA; b-actin, 1:2,000 in 5% BSA, and then with acorresponding secondary horseradish peroxidase–conjugatedIgG). The proteins were visualized with an electrochemilumines-cent system (PerkinElmer Life Science).

Immunofluorescent analysisA total of 5 � 103 cells were plated on 8-well chamber slides

and allowed to attach overnight. After pretreatment with0.1 mmol/L of IMD-0354 for 24 hours, the cells were stimulatedby TNFa (10 ng/mL, 30 minutes), fixed with 4% paraformalde-hyde and stained with 1:200 rabbit anti-human NF-kB p65 (CellSignaling Technology) at room temperature for 1 hour. Afterwashing, samples were incubated with 1:50 Alexa Fluor 555-labeled goat anti-rabbit IgG (A-21429; Life Technologies) andfinally stained with DAPI. The samples were observed using anFV1000-D Laser Scanning Confocal Microscope (Olympus).

In vitro adhesion assayOvarian cancer cells (SKOV3ip1 or RMUG-S, 5 � 104) were

plated in a 96-well plate precoated with 50 mg/mL fibronectin or50mg/mL type I collagen. After incubation for 45minutes at 37�C,cells were washed three times with PBS, fixed with methanol, andstained with Giemsa solution. The number of adhesive cells wascounted under a light microscope.

Matrigel invasion assayIn vitro cellular invasion assay was performed as described

previously (23).

Luciferase activity assayThe pGL4-phVEGFA plasmid bearing human VEGF-A promot-

er sequence [position; 3853–5157 of human VEGF sequence(NG_008732)] into a firefly luciferase vector was obtained fromRIKEN BioResource Center. 50-deletion mutant were made bydeleting fragments using internal restriction sites for NheI (mutpGL4-phVEGFA, position; 5043–5157). A total of 1 � 105 SKO-V3ip1 cells were seeded in 12-well plates. After the replacement ofculture media by Opti-MEM I Reduced Serum Media (Life Tech-nologies), 1.0 mg of pGL4-phVEGFA, and 0.1 mg of pRL-TK Renillaluciferase vector were cotransfected with Lipofectamine 2000(Life Technologies). On the next day, various concentrations ofIMD-0354 were treated and, 24 hours later, luciferase activity wasdetermined using the Dual Luciferase Reporter Assay System(Promega). Firefly luciferase values were normalized to Renillaluciferase values.

ELISA of human VEGF-AA total of 1� 105 SKOV3ip1 cells were plated on 6-well plates.

The cells were then starved in serum-free medium with variousconcentrations of IMD-0354 or the equivalent volume of DMSOfor 24hours. Thereafter, conditioned culturemediawere collectedand stored at �80�C until analysis. Human VEGF-A PlatinumELISA Kit (eBiosecience) was used to determine the concentrationof VEGF-A.

Wound healing assayHUVECs were cultured to subconfluence in 24-well culture

plates and one wound of approximately 0.4 mm per well wasmade with a P-200 plastic tip. The cells were further cultured inserum-free conditions in the absence or presence of 5ng/mLVEGFor concentrated SKOV3ip1 cells culture medium. ConcentratedSKOV3ip1 cells culturemediumwas collected by culturing cells inserum-free DMEM with or without IMD-0354 in a subconfluentcondition for 24 hours and concentrated using Amicon Centrif-ugal Filter Units (Merck Millipore). Forty-eight hours after theincubation, the cells were fixed in 3.7% paraformaldehyde andstained with Giemsa solution. Migration was assessed by exam-ining photographs of the cells that hadmigrated inside thewoundarea.

Animal experimentsFemale athymic BALB/c nude mice (aged 4–5 weeks) were

purchased from CLEA Japan Inc. and were bred in aseptic con-ditions and kept at constant humidity and temperature (25–28�C). All animal experiments were approved by the InstitutionalAnimal Care and Use Committee of Osaka University (Osaka,Japan), in accordance with institutional and NIH guidelines.Ovarian cancer cells were suspended as single cells in a volumeof 0.5mLof PBS (SKOV3ip1; 1�106 cells, ES-2; 2�106 cells) andinjected intraperitonally into the mice. Mice were assessed dailyfor general health and development of ascites. After the inocula-tion (SKOV3ip1; 7 days, ES-2; 3 days), the mice were intraper-itonally administered either 0.5 mL of 0.5% carboxymethylcel-lulose sodium salt (CMC-Na; Wako, Osaka, Japan) or IMD-0354(30 mg/kg) every day for a total of 6 weeks to the SKOV3ip1inoculatedmice or 11 days to the ES-2 inoculatedmice and finallysacrificed. The number of metastases and the volume of ascitesin each mouse were counted, carefully dissected, and the remov-ed tumors weighed. Tumor tissues were immediately fixed and

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embedded in paraffin. The slides were prepared and incubatedwith p-IKKa/b antibody at 1:150, human VEGF antibody at 1:40,mouse CD-31 antibody, Ki-67 antibody, and cleaved caspase-3antibody at 1:300 for 1 hour at room temperature. After washingwith TBST, they were stained usingN-Histofine Simple StainMAXPO(R) (Nichirei Biosciences) and then counterstained withCarrazzi hematoxylin.

Statistical analysisStatcel version 3 (OMS-Publishing Inc., Saitama, Japan) and

JMP version 10.0.2 (SAS Institute Japan Ltd.) were used forstatistical analyses. Data were expressed as means � SEM. Differ-ences were analyzed using the Mann–Whitney U test. Survivalestimates were computed using the Kaplan–Meier method, andcomparisons between groups were analyzed using the log-ranktest. Multivariable analysis was performed using a Cox propor-tional-hazards regression model. Differences were consideredstatistically significant at P < 0.05.

ResultsPhosphorylation of IKK is an independent prognostic markerof ovarian cancer

First, TMA slides from patients with ovarian cancer wereestablished. The characteristics of 94 patients are summarizedin Supplementary Table S1. Phospho-IKK expression was eval-uated by immunohistochemistry and each sample was scored

on the basis of the intensity of staining. Typically, clear cyto-plasmic staining was seen in the cases of positive p-IKK expres-sion (Fig. 1A). Of 94 patients, 58 (61.7%) showed strong p-IKKexpression, 23 (24.4%) weak, and 13 (13.8%) cases werenegatively expressed. In total, 81 (86.2%) cases showed pos-itive p-IKK expression. Among patients with ovarian cancer,those who had positive p-IKK staining showed significantlyworse PFS than those who had negative expression (PFS, 26.1vs. 49.8 months, P ¼ 0.011; Fig. 1B). Positive p-IKK stainingalso showed a trend toward worse OS compared with thosewith negative expression (OS, 34.0 vs. 50.9 months, P ¼0.073; Fig. 1C). For a multivariate analysis, a backward elim-ination approach was used to select a model for survival withmultiple predictors. Age, FIGO stage, histologic type, residualtumor at the time of surgery, and positive p-IKK expressionwere entered in this model. The final model included positivep-IKK expression as a significant independent predictor forreduced PFS in patients with ovarian cancer [P ¼ 0.019; HR,5.85 (1.26–104.26); Supplementary Table S2].

High VEGF expression is an independent prognostic marker inovarian cancer patients and positively correlates with IKKphosphorylation

Second, VEGF expression was evaluated by immunohis-tochemistry using anti-VEGF-A antibody. VEGF staining wasscored on the basis of the intensity of the staining and thepercentage of positive cells. Representative pictures from four

Negative controlClear-cell carcinoma

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Figure 1.Phospho-IKK expression correlateswith poor prognosis in patients withovarian cancer. Immunohistochemicalstaining of tissue microarrays withmalignant ovarian tissue sections (A).Representative areas of four differentovarian cancers stained using an anti-human phospho-IKK antibody andscored as 0, 1, and 2. No positive signalwas observed by nonimmune area.Original magnification, �200. Bar,100 mm. Kaplan–Meier curves ofprogression-free survival (B) andoverall survival (C) of patients withovarian cancer treated at GifuUniversity Hospital (n ¼ 94).

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different histologies are shown in Fig. 2A. Forty-seven (50.0%)cases were defined as "high" VEGF expression. Patients with highVEGF expression had a significantly worse PFS than those withlow VEGF expression (26.4 vs. 32.4 months, P ¼ 0.014; Fig. 2B).High VEGF expression also showed a trend toward worse OScompared with those with low VEGF expression (35.6 vs. 37.0months, P ¼ 0.086; Fig. 2C). Forty-four of 81 cases (54.3%) inwhich IKKa/b was phosphorylated showed high VEGF expres-sion, whereas only 3 (23.1%) of 13 cases in which p-IKKa/bexpression was negative showed high VEGF expression. Thecorrelation of IKK phosphorylation and VEGF expression wasexamined with the c2 test, revealing that these expressions weresignificantly positively correlated (Fig. 2D; P ¼ 0.037).

Constitutive activation of NF-kB in ovarian cancer cells wasinhibited by IMD-0354

As IKK phosphorylation was correlated with VEGF expres-sion in clinical samples and its expression was associated withpoor prognosis, we were encouraged to analyze the effect of anIKKb inhibitor on ovarian cancer cells. IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide], with

MW 383.7 (Fig. 3A), a specific IKK-b inhibitor selectivelyblocking IkBa phosphorylation (IC50; � 250 nmol/L; ref. 24)was used for the experiments. Although IMD-0354 at up to1 mmol/L did not affect the viabilities of SKOV3ip1 or RMUG-Sovarian cancer cells (Fig. 3B), it inhibited the constitutivephosphorylation of IKK (Fig. 3C). To determine the effect ofIMD-0354 on NF-kB activation, the localization of NF-kB p65was examined by immunofluorescence analyses. SKOV3ip1cells were immunostained with an anti-p65 antibody and cellnuclei were identified by costaining with DAPI. NF-kB p65 wasdominantly located in the cytoplasm in the control and uponTNFa stimulation, p65 drastically translocated into nuclei. Thepretreatment of 0.1 mmol/L IMD-0354 almost inhibited itsnuclear translocation (Fig. 3D).

IMD-0354 inhibits adhesion and invasion of ovarian cancercells

Next, we evaluated the effects of IMD-0354 on cell adhesionand invasion. IMD-0354 significantly inhibited the adhesiononto fibronectin (0.3 mmol/L: SKOV3ip1, 51%; RMUG-S, 55%,respectively) as well as onto collagen type I (0.3 mmol/L:

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Figure 2.VEGF expression correlates with poorprognosis in patients with ovarian cancer.Immunohistochemical staining of TMAs withmalignant ovarian tissue sections (A).Representative areas of four differentovarian cancers stained using an anti-humanVEGF-A antibody and scored as 1, 2, and 3.No positive signal was observed bynonimmune area. Original magnification,�200. Bar, 100 mm. Kaplan–Meier curves ofPFS (B) and OS (C) of patients with ovariancancer (n ¼ 94). High VEGF expression wassignificantly more frequent in tumors thatshowedphosphorylated IKK expression thanin those that had negative phosphorylatedIKK staining (D). Statistical differences wereexamined by c2 test(P ¼ 0.037).






SKOV3ip1, 62%; RMUG-S, 58%, respectively) in a dose-depen-dent manner (Fig. 4A). To analyze how IMD-0354 inhibited celladhesion, the expressions of various integrins were examined byWestern blot analysis (Fig. 4C). b1 Integrin expression was inhib-ited by IMD-0354 treatment in each ovarian cancer cell line tested(SKOV3ip1, RMUG-S, and CaOV3), while the expressions of the

other integrins tested (a1, a2, a5, and b3-integrin) were notaltered. Cell invasion follows adhesion. In vitro invasion assayrevealed that IMD-0354 significantly impaired the invasion ofcancer cells (0.3 mmol/L: SKOV3ipi, 27%; RMUG-S, 33%,respectively; Fig. 4D). The expression of MMP-2, known as animportant molecule in ovarian cancer invasion, was inhibited bythe treatment with IMD-0354 (Fig. 4E).

IMD-0354 inhibits VEGF production from ovarian cancer cells,leading to the inhibition of angiogenesis

Next, we examined the effect of IMD-0354 on VEGF produc-tion from cancer cells. Luciferase assay revealed that VEGF-Atranscriptional activity was significantly inhibited by IMD-0354in a dose-dependent manner (Fig. 5A). Several possible NF-kB–like binding sites within the VEGF 50-promoter region werepreviously reported (12) and pGL4-phVEGFA plasmid has twopossible binding sites (4332–4341, 4992–4931; Fig. 5B). Thus,50-deletion mutant (mut pGL4-phVEGFA) was made by deletingfragments containing these sites and a luciferase assay wasperformed (Fig. 5C). Luciferase activity was drastically abol-ished and IMD-0354 did not show any inhibitory effects,indicating that NF-kB directly regulates VEGF promoter activity.Accordingly, IMD-0354 treatment of SKOV3ip1 cells resultedin a decrease in the levels of VEGF-A protein from 1.61 ng/mLto 0.97 ng/mL as evidenced by quantitative ELISA (Fig. 5D). Toclarify the antiangiogenic effect of IMD-0354 in vitro, the migra-tory activity of HUVECs was examined by a wound healingassay. Representative images are shown in Fig. 5E. VEGF-A5 ng/mL was used as a positive control. The direct treatmentof IMD-0354 to HUVECs did not affect the migration. WhileSKOV3ip1 culture media induced HUVECs migration andalmost restored the scratched wound after 48 hours of incuba-tion, the pretreatment of IMD-0354 to SKOV3ip1 cells inhibitedHUVECs migration (Fig. 5F). The cotreatment of VEGF-A almostabolished the inhibitory effect of IMD-0354, indicating thatIMD-0354 inhibited the migratory activity of HUVECs by inhi-biting VEGF production from SKOV3ip1 cells.

IMD-0354 inhibits peritoneal metastasis in an ovarian cancerxenograft model by inhibiting VEGF production from cancercells

Finally, we examined the therapeutic potential of IMD-0354 inan ovarian cancer xenograft model. SKOV-3ip1 (1 � 106 cells)were injected intraperitoneally into female BALB/c nu/nu mice.One week after the inoculation, mice showed multiple tumordissemination on the peritoneal surface, the omentum, the sur-face of the liver and the small bowel mesentery. As preliminaryexperiments, dose-finding study (7.5mg, 15mg or 30mg/kg)wasperformed and 30 mg/kg showed the maximum effect withoutapparent adverse events on mice (Supplementary Fig. S1). Sixweeks after treatment, both the tumor weight and the number ofperitoneal implants were significantly inhibited in mice treatedwith IMD-0354 compared with controls (tumor weight: IMD-0354; 176.2 � 136.9 vs. control; 518.6 � 338.4 mg, numberof peritoneal implants: 1.9 � 2.7 vs. 26.1 � 17.2, respectively,P < 0.001; Fig. 6A and B). Similarly, in another ovarian cancerxenograft model with ES-2, IMD-0354 significantly exerted anti-tumor effects on tumor weight, number of peritoneal metastases,and ascites formation compared with the control (tumor weight:IMD-0354; 227.6 � 109.8 vs. control; 576.5 � 180.6 mg,

AMolecular formula : C15H8ClF6NO2Molecular weight : 383.67

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Figure 3.IMD-0354 inhibits NF-kB activation of ovarian cancer cells. Molecular formulaof IMD-0354 (A). Culturing of ovarian cancer cells (left, SKOV3ip1; right,RMUG-S) with up to 1 mmol/L IMD-0354 had no effect on their viability (B).Data represent mean � SEM, n ¼ 5 from triplicate independent experiments.Western blot analysis (C). SKOV3ip1 and RMUG-S cells were incubated withIMD-0354 for 24 hours. Cell lysates were immunoblotted with anti-phosphorylated IKKa/b antibody (top). The membranes were stripped andrehybridized with antibodies detecting IkBa (middle) or b-actin (lower).IMD-0354 inhibits NF-kB nuclear translocation induced by TNFa (D).Representative confocal images of SKOV3ip1 cells stimulated by TNFa (þ)(10 ng/mL, 30 minutes) with or without pretreatment of 0.1 mmol/L IMD-0354are shown.Cellswere immunostainedwithAlexa Fluor 555–labeledNF-kBp65(red). In controls, NF-kBwas dominantly located in the cytoplasm (top). TNFastimulation (middle) induced the nuclear translocation of NF-kB p65.IMD-0354 inhibited its nuclear translocation (lower). Bar, 50 mm.

Kinose et al.





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Figure 4.IMD-0354 causes significant inhibition of adhesion and invasion of ovarian cancer cell lines. In vitro adhesion assay (A). A total of 5� 104 ovarian cancer cells (left,SKOV3ip1; right, RMUG-S) were plated onto 50 mg/mL fibronectin- or collagen type 1–coated 96-well plates. After incubation for 45 minutes at 37�C, plateswerewashed to discard nonadherent cells and the number of adherent cellswas counted under a lightmicroscope. Data representmean� SEM, n¼ 5 from triplicateindependent experiments. Representative image of in vitro adhesion assay of SKOV3ip1 cells is shown (B). Bar, 200 mm. Western blot analysis (C). SKOV3ip1,RMUG-S, andCaOV3 cellswere incubatedwith 0.3mmol/L of IMD-0354 or control for 24 hours. Cell lysateswere immunoblottedwith an antibody against integrina1,a2, a5, b1, and b3. b-Actin was used as a loading control. Each sample was run in triplicate. Numbers show the ratio between integrins indicated and b-actinexpression. In vitro invasion assay (D). A total 5� 104 SKOV3ip1 (left) or RMUS-S (right) cells were placed on the top chamber in serum-freemediumwith IMD-0354,and allowed to invade for 24 hours. Noninvading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated.Representative images are shown. Bar, 100 mm. Data represent mean � SEM, n ¼ 5 from triplicate independent experiments. Western blot analysis (E). Celllysates were immunoblotted with an antibody against MMP-2. b-Actin was used as a loading control. Each sample was run in triplicate. Numbers show the ratiobetween MMP2 and b-Actin expression. ��, P < 0.01.






P < 0.001, number of peritoneal implants: 15.4 � 6.7 vs. 31.8 �10.1, P < 0.001, ascites: 1.06 � 0.72 vs. 2.84 � 1.28 g, P < 0.01,respectively; Fig. 6C–E). To analyze how IMD-0354 worked invivo, immunohistochemical analyses from inoculated tumorswere performed (Fig. 6F). Not only the phosphorylation of IKKbut the expression of human VEGF was evidently inhibited intumors treated with IMD-0354 compared with controls. Accord-ingly, the number of intratumoral vessels as presented by anti-mouse CD31 staining was significantly inhibited by the treatmentwith IMD-0354 (27.2 � 5.7 vs. 96.0 � 36.1/� 200 field, respec-tively, P < 0.01; Fig. 6G). In contrast, the staining of peritoneal

tumors with the proliferation marker Ki-67 and the apoptoticmarker cleaved caspase-3 did not show significant differencebetween IMD-0354–treated mice and controls (Fig. 6H and I),suggesting that IMD-0354 inhibited ovarian cancer progressionby inhibiting angiogenesis through the suppression of VEGFproduction from cancer cells.

DiscussionIn this study, we revealed that, in more than 80% of ovarian

cancer cases, IKK is phosphorylated and its phosphorylation is an

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Figure 5.IMD-0354 inhibits VEGF productionfrom ovarian cancer cells, which leadsto the inhibition of HUVECs migrationinduced by cancer cells. Effect of IMD-0354 on VEGF-A transcriptionactivation (A). pGL4-hVEGFA fireflyluciferase vector and Renillaluciferase reporter, pRL-TK, werecotransfected into SKOV3ip1 cells.VEGF-A transcriptional activity wasreduced in a dose-dependent mannerby the treatment with IMD-0354.Data represent mean � SEM, n ¼ 5from triplicate independentexperiments. The schema of originaland mutant pGL4-phVEGFA vector(mut pGL4-phVEGFA; B). PossibleNF-kB binding sites of VEGFApromoter are shown. Luciferaseactivity was drastically abolishedwithmut pGL4-phVEGFA and IMD-0354did not show any inhibitory effects(C). Data represent mean� SEM, n ¼4 from triplicate independentexperiments. ELISA assay of VEGF-A(D). A total of 1 � 105 SKOV3ip1 cellswere cultured with 2 mL of 0.1%bovine serum albumin (BSA)/DMEMwith or without IMD-0354 for 24hours. Conditioned media werecollected and the concentration ofhuman VEGF-A was measured. Datarepresent mean � SEM, n ¼ 4 fromtriplicate independent experiments.Effect of IMD-0354 on HUVECsmigration examined by a woundhealing assay (E). Endothelial cellmonolayers were wounded at time 0,and cultures were incubated withserum-free media (top) orconditioned media from SKOV3ip1cells (lower). Forty-eight hours later,cells were fixed and representativepictures were taken. A total of 5 ng/mL of human VEGF-A was used as apositive control. Bar, 200 mm.Percentage wound recovery wasmeasured and compared with that attime 0 (F). Experiments wererepeated seven times and values aremeans � SEM; n.s., not significant;� , P < 0.05; ��, P < 0.01; ��, P < 0.001;n.s., not significant.

Kinose et al.





independent prognosis factor for patients and positively corre-lates with high VEGF expression. Furthermore, using a new IKKbinhibitor, IMD-0354, we showed that this drug not only sup-pressed the adhesion and invasion of ovarian cancer cells butalso transcriptionally inhibited VEGF production from cancercells, which led to attenuation of the migration of endothelialcells induced by cancer cells. In a xenograft model, the treatmentwith IMD-0354 significantly inhibited peritoneal disseminationwith a marked reduction of blood vessel formation. Thus, wesuggest that an IKKb inhibitor such as IMD-0354 can be consid-ered to be an antiangiogenic agent and a potential treatment for

ovarian cancer, given that VEGF is one of the most potent andspecific angiogenic factors of tumor-induced angiogenesis.

As it is clear that angiogenesis is an indispensable contributorto ovarian carcinogenesis and progression, we believe thatresearch and development into new antiangiogenic compoundsshould continue, to maximize the benefits andminimize the sideeffects as well as the costs. Following the success of bevacizumab,new molecules active against angiogenesis have been developedand several studies have been conducted or are underway. Somesmall-molecule inhibitors of multitargeted tyrosine kinase (TKI),including VEGFR, have been shown to be active in ovarian cancer

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Figure 6.Treatment of IMD-0354 inhibitsperitoneal dissemination of ovariancancer cells through the inhibition ofVEGF production from cancer cells.Ovarian cancer cells (SKOV3ip1;1 � 106 cells, ES-2; 2 � 106 cells) wereinjected intraperitoneally into femaleBALB/c nu/nu mice. After theinjection (SKOV3ip1, 7 days; ES-2, 3days), IMD-0354 (30 mg/kg bodyweight) or an equal amount of 0.5%CMC-Na (control) was injectedintraperitoneally daily for a total of 6weeks to the SKOV3ip1 inoculatedmice or for 11 days to the ES-2inoculated mice. Effect of IMD-0354on intraperitoneal tumor weight (A),number ofmetastases (B) in SKOV3ip1inoculated mice. Effect of IMD-0354on intraperitoneal tumor weight (C),number ofmetastases (D), and ascitesformation (E) in ES-2–inoculatedmice. Results are expressed as mean� SEM, each n ¼ 18, respectively.Representative SKOV3ip1 tumor areaswere stained with H&E, p-IKKa/b,human VEGF, the angiogenesismarker anti-mouse CD31, theproliferation marker Ki-67, and theapoptotic marker cleaved caspase-3(F). Bar, 50 mm. Number ofmicrovessels per field by CD31staining (G), the percentage of Ki-67–positive nuclei (H), and thepercentage of cleaved caspase-3–positive cells (I). Results areexpressed asmean�SEM,n¼ 5, each.n.s., not significant; �� , P < 0.01;�� , P < 0.01; n.s., not significant.






(9). For example, pazopanib is an oral angiogenesis inhibitorthat inhibits VEGF receptor, platelet-derived growth factor recep-tor and c-Kit. In a phase II study evaluating pazopanib in patientswith recurrent ovarian cancer, the overall response rate was 18%and themedian response durationwas 113 days (25, 26). A recentphase III clinical trial (AGO-OVAR16) showed that pazopanibextended PFS by an average of 5.6 months (17.9 months vs. 12.3months), compared with a placebo, in women with advancedovarian cancer who underwent initial successful treatment, whilethe interim analysis showed no difference in OS between thegroups (27).Other TKIs such as sunitinib or sorafenib have shownlimited results (28, 29). Angiopoietins are circulating proteingrowth factors that promote angiogenesis by interacting withTie2 receptors. The antiangiopoietin 1/2 peptibody, AMG386,given in combination with weekly paclitaxel demonstrated pro-longation of PFS in a randomized phase II trial with recurrentovarian cancer patients (30). Although several candidates areunder evaluation, none of the molecules inhibiting NF-kB path-ways have been used as an angiogenesis inhibitor in the clinicalsetting so far. Herein, we suggested the potential of an IKKinhibitor, IMD-0354, for ovarian cancer treatment.

Angiogenesis in cancer has been shown to be associated withthe chemokines (like monocyte chemoattractant protein, IL8)or the growth factors (like TNFa). NF-kB activation is wellknown to play a role in the regulation of these angiogenesis-inducing products (31). Although VEGF is a potent angiogenicfactor, the mere inhibition of VEGF with bevacizumab hasfailed to demonstrate substantial prolongation of OS inpatients with ovarian cancer, indicating that there are severalalternative pathways of angiogenesis other than the VEGFpathway. Thus, targeting NF-kB has the potential to inhibitvarious angiogenesis pathways and is likely to be promising asan antiangiogenic therapy. While small-molecule compoundinhibitors of NF-kB have been proposed as a promising therapyfor cancers with aberrant NF-kB activity, most classical NF-kBinhibitors are poorly selective and are known to have off-targeteffects and so few of them have progressed to phase III clinicaltrials (32). Because proteasome-mediated degradation of IkB isa required step in NF-kB signaling, the proteasome inhibitor,bortezomib, has been proposed as a general inhibitor of NF-kB;it is the only one clinically available and approved as the first-line treatment for advanced multiple myeloma (33). However,there remains a concern that proteasome inhibition may affectother signaling pathways. IMD-0354 is a synthesized lowmolecular weight compound that specifically inhibits IKKb,inducing the inhibition of NF-kB activation only in inflamma-tory conditions, and it was proven that this drug does notinhibit other kinases, proteases, or proteasome-related immuneresponses (34). No IKKb inhibitor is known to be in the processof clinical application at present, except for IMD-1041, aprodrug of IMD-0354; a phase I study with the IMD-0354capsule confirmed sufficient safety (20). Since it appears obvi-ous that the mere inhibition of NF-kB would be insufficient fora pronounced response unless combined with apoptosis-induc-ing drugs for ovarian cancer treatment, NF-kB inhibitorsshould be used as adjuvants along with cytotoxic chemothera-pies in the clinical setting. For these reasons, we assume thatIMD-0354 would be an ideal candidate because it is feasible forcombination with current chemotherapies. Several previousstudies have reported the effect of IMD-0354 on adult T-cellleukemia cells (20), breast cancer cells (35) or chronic lym-

phocytic leukemia cells (36). In these studies, at higher con-centrations (1–50 mmol/L), IMD-0354 induced apoptosis ofcancer cells. Indeed, in our experience, 10 mmol/L of IMD-0354comprehensively induced apoptosis of ovarian cancer cellstested by G0–G1 phase cell-cycle arrest (data not shown).However, given that the IC50 value for IKKb of IMD-0354 is�250 nmol/L, these apoptotic effects appear to be caused by anonspecific toxicity of the drug. Further elucidation would berequired to identify how IMD-0354 works on cancer cells.

In immunohistochemical analyses, 81 of 94 (86%) ovariancancer samples showed positive phosphorylated IKK expression.This is similar to previous findings (15–17). Although some invitro studies using ovarian cancer cell lines suggested that NF-kBregulates VEGF expression (37), to our knowledge, none hasreported the direct correlation of NF-kB activation and VEGFexpression in clinical samples. In the current study, we reportedthat ovarian cancer tissues with positive p-IKK staining signifi-cantly expressed high levels of VEGF, proposing a clear rationalethat targeting IKK phosphorylation can be considered an anti-angiogenic therapy for ovarian cancer treatment.

In conclusion, IKK phosphorylation is an independent prog-nostic factor and is associated with VEGF expression in cancertissues. IMD-0354 not only inhibited ovarian cancer invasionbut suppressed VEGF production from cancer cells in vitro andin vivo. In light of multiple publications highlighting the impor-tance of angiogenesis in ovarian tumor biology and the resultsdescribed here, we are supportive of clinical trials to studywhether antagonizing IKKb phosphorylation is a viable treat-ment strategy for ovarian cancer.

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

Authors' ContributionsConception and design: K. Sawada, H. Makino, T. Fujikawa, A. ItaiDevelopment of methodology: H. Makino, T. Fujikawa, K. Hashimoto,A. Isobe, A. ItaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Kinose, H. Makino, T. Ogura, E. Morii,K. Nakamura, I. Sawada, A. Toda, K.-I. MorishigeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Kinose, K. Sawada, H. Makino, E. Morii,K.-I. MorishigeWriting, review, and/or revision of the manuscript: Y. Kinose, K. Sawada,H. Makino, H. Kurachi, T. KimuraAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Makino, T. Mizuno, N. Suzuki, T. Fujikawa,S. Mabuchi, T. Ohta, H. KurachiStudy supervision: H. Makino, T. Fujikawa, A. Itai, T. Kimura

AcknowledgmentsThe authors thank Yuko Nishimura for her secretarial assistance and Ayako

Okamura for her technical assistance.

Grant SupportThis work was supported by a Grant-in-Aid for Scientific Research from the

Ministry of Education, Science, Sports and Culture of Japan (24592515,26670725, and 26293360 to K. Sawada; 24659723, to H. Kurachi).

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

Received August 22, 2014; revised December 10, 2014; accepted January 14,2015; published OnlineFirst January 30, 2015.

Kinose et al.





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Published OnlineFirst January 30, 2015.Mol Cancer Ther Yasuto Kinose, Kenjiro Sawada, Hiroshi Makino, et al. TreatmentTherapeutic Target for Ovarian Cancer as an Antiangiogenic

Regulates VEGF Expression and Is a PotentialβIKK

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