inhibition of pai-1 limits tumor angiogenesis regardless of … · pai-1 might regulate the...

Therapeutics, Targets, and Chemical Biology

Inhibition of PAI-1 Limits Tumor AngiogenesisRegardless of Angiogenic Stimuli in MalignantPleural MesotheliomaYusuke Takayama1, Noboru Hattori1, Hironobu Hamada2, Takeshi Masuda1,Keitaro Omori1, Shin Akita3, Hiroshi Iwamoto1, Kazunori Fujitaka1, andNobuoki Kohno1

Abstract

Malignant pleural mesothelioma (MPM) is an aggressivemalignant tumor that secretes various angiogenic factors. Themain inhibitor of plasminogen activators, PAI-1 (SERPINE1),has been implicated in tumor progression and angiogenesis,and high PAI-1 expression has been associated with poorprognosis in MPM patients. In this study, we examined theantiangiogenic effects of PAI-1 inhibition in MPM. We admin-istered the PAI-1 inhibitor, SK-216, to orthotopic mouse mod-els in which MPM cells expressing high levels of VEGF (VEGFA)or bFGF (FGF2) were intrapleurally transplanted. SK-216administration reduced tumor weights and the degree of angio-

genesis in intrapleural tumors, irrespective of their angiogenicexpression profiles. In addition, a combination of SK-216 andthe chemotherapeutic agent cisplatin significantly reducedtumor weights compared with monotherapy, prolonging thesurvival of animals compared with cisplatin treatment alone.Furthermore, SK-216 inhibited migration and tube formationof cultured human umbilical vein endothelial cells induced byvarious angiogenic factors known to be secreted by MPM. Thesefindings suggest that PAI-1 inactivation by SK-216 may repre-sent a general strategy for inhibiting angiogenesis, including forthe treatment of MPM. Cancer Res; 76(11); 3285–94. �2016 AACR.

IntroductionMalignant pleural mesothelioma (MPM) is an aggressive malig-

nant tumor, and its incidence is increasing worldwide (1–3).Although a combination of therapies, including surgery, radio-therapy, and chemotherapy, is performed, the prognosis of MPMpatients is very poor. Therefore, novel therapeutic strategies arerequired to improve the prognosis of this disease.

Because angiogenesis is a critical factor for the progression ofsolid tumors (4–6), inhibition of tumor angiogenesis representsan attractive therapeutic approach for the treatment of malig-nancies. Antiangiogenic therapy with bevacizumab, which tar-gets VEGF, has been shown to improve survival in patients withlung cancer (7) and colorectal cancer (8). Although a recentFrench study reported that addition of bevacizumab to peme-trexed plus cisplatin improved survival in patients with MPM

(9), several clinical trials (10, 11) have demonstrated thatbevacizumab fails to provide clinical benefits in patients withMPM. This inconsistent response to anti-VEGF therapy might bedue to the property of MPM cells to express various angiogenicfactors other than VEGF, such as basic FGF (bFGF; ref. 12),platelet-derived growth factor (PDGF; ref. 13), and hepatocytegrowth factor (HGF; ref. 14). Given the clinical application ofantiangiogenic therapy for MPM, development of a strategy toinhibit tumor angiogenesis irrespective of the primary angio-genic stimulus is necessary.

The plasminogen activation system, represented by uroki-nase-type plasminogen activator (uPA), the cellular receptor foruPA (uPAR), and its specific inhibitor, plasminogen activatorinhibitor-1, PAI-1 (SERPINE1), plays a crucial role in tumorprogression and angiogenesis (15, 16). In particular, a largenumber of animal and/or in vitro studies have revealed thatPAI-1 promotes tumor progression (17–19) and, in fact, asignificant association between high expression of PAI-1 andpoor prognosis in various types of tumors, including MPM, hasbeen confirmed (20–23). Several possible mechanisms havebeen postulated to explain the linkage between PAI-1 andtumor progression; among them, the involvement of PAI-1 intumor angiogenesis receives the most attention. Experimentsusing PAI-1–deficient (PAI-1�/�) mice have demonstrated thesignificance of host PAI-1 for tumor angiogenesis (24–26),most likely through modulation of plasmin-mediated proteol-ysis (27), migration (28, 29), and apoptosis (30) of endothelialcells. Recently, we demonstrated that inhibition of PAI-1 by aspecific inhibitor of PAI-1, SK-216, limited tumor angiogenesisin vivo and migration and tube formation of human umbilicalvein endothelial cells (HUVEC) induced by VEGF in vitro (31).

1Department of Molecular and Internal Medicine, Graduate School ofBiomedical &Health Sciences, HiroshimaUniversity, Hiroshima, Japan.2Department of Physical Analysis andTherapeutic Sciences,GraduateSchool of Biomedical & Health Sciences, Hiroshima University, Hir-oshima, Japan. 3Department of Hematology and Respiratory Medi-cine, Kochi University, Nankoku, Kochi, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Noboru Hattori, Department of Molecular and InternalMedicine, Graduate School of Biomedical & Health Sciences, Hiroshima Univer-sity, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: 818-2257-5196; Fax: 818-2255-7360; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1796

�2016 American Association for Cancer Research.

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Published OnlineFirst April 13, 2016; DOI: 10.1158/0008-5472.CAN-15-1796

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These results prompted us to hypothesize that inhibition ofPAI-1 might regulate the occurrence of tumor angiogenesisregardless of angiogenic stimuli.

Taking all of this into consideration, we decided to deter-mine whether inhibition of PAI-1 by SK-216 could exert anantitumor effect on MPM through antiangiogenic activityregardless of the type of primary angiogenic factor. To thisend, we first established orthotopic implantation mouse mod-els of MPM using VEGF-high–expressing or bFGF-high–expres-sing MPM cells and examined the effect of systemic adminis-tration of SK-216 on tumor progression and angiogenesis. Inthese models, we also assessed whether SK-216 affected thetherapeutic effect of cisplatin. Then, in vitro, we examined theeffects of SK-216 on proliferation, migration, and tube forma-tion of HUVECs induced by angiogenic factors other than VEGFthat are known to be secreted by MPM.

Materials and MethodsCells and cell culture

The humanMPM cell line EHMES-10 was established from thepleural effusion of a patient with MPM at Ehime University(Matsuyama, Japan; refs. 32, 33), andMSTO-211Hwaspurchasedfrom the ATCC. These cell lines were cultured in RPMI1640medium (Gibco) supplemented with 10% FBS, penicillin (100U/mL), and streptomycin (100 mg/mL). HUVECs authenticatedby Lifeline Cell Technology were purchased from Kurabo andcultured following the manufacturer's instructions. All cells werecultured at 37�C in a 5% CO2 humidified atmosphere. MSTO-211H cells and HUVEC were used within 6 months after resus-citation. EHMES-10 cells were authenticated by short tandemrepeat analyses (Promega).

Reagents and animalsMatrigel was purchased from BD Biosciences. Recombinant

human bFGF (FGF2) was obtained from Wako Pure Chemical,and recombinant VEGF (VEGFA), PDGF-BB (PDGFB), and HGF(HGF) were purchased from PeproTech. SK-216 (SupplementaryFig. S1) was chemically synthesized and supplied by ShizuokaCoffein Co., Ltd. Inhibitory activity of SK-216 on PAI-1 wasinvestigated using previously published methods (34), and theIC50 was determined to be 44 mmol/L, as reported in internationalpatentWO04/010996. Bevacizumab(Avastin)waspurchased fromChugai Pharmaceutical Co., Ltd. AZD4547 was purchased fromSelleck Chemicals. Cisplatin was purchased from Nippon Kayaku.Male SCIDmice (6weeks old)were obtained fromCLEA Japan andwere maintained according to guidelines for the ethical use ofanimals in research at Hiroshima University (Hiroshima, Japan).

Quantification of VEGF, bFGF, PDGF-BB, and HGF proteinConcentrations of VEGF, bFGF, PDGF-BB, and HGF in cul-

ture supernatants and concentrations of VEGF and bFGF in thelysates of intrapleural tumors were measured using an ELISAKit according to the manufacturer's instructions (R&D Sys-tems). Culture supernatants were prepared from EHMES-10or MSTO-211H cells (2 � 105) seeded in 6-well plates andcultured in 2 mL RPMI1640 with 10% FBS for 48 hours. Lysatesof intrapleural tumors were prepared as follows: intrapleuraltumors (approximately 50 mg each) consisting of EHMES-10or MSTO-211H cells were homogenized in 1 mL lysis buffer(Thermo Scientific Pierce) at 4�C for 2 minutes, centrifuged at

10,000 � g in 4�C for 5 minutes, and the supernatants werecollected.

Orthotopic implantation modelThe cultured EHMES-10 cells or MSTO-211H cells were har-

vested, washed twice, and resuspended in PBS. Thereafter, 3 � 106

cells (EHMES-10) or 1 � 106 cells (MSTO-211H) in 100 mL PBSwere injected into the pleural cavity of SCID mice as describedpreviously (33). Mice were randomly assigned to a control groupand drug treatment group, each consisting of 6 or 7 animals.SK-216 (100 or 500 ppm) was orally administered through drink-ing water at the same time as tumor cell inoculation. Bevacizumab(3 or 10 mg/mouse) was given by intraperitoneal injection on days7, 10, 14, and 17. AZD4547 (3 mg/kg) was given twice daily fromday 7 to day 19 by oral gavage. Cisplatin (1.5 mg/kg) was given byintraperitoneal injection on days 7, 14, and 21 in the mice bearingEHMES-10 cells, and cisplatin (1.0 mg/kg) was given on days 7and 14 in the mice bearing MSTO-211H cells. The mice weresacrificed 28 days (EHMES-10) or 21 days (MSTO-211H) aftertumor cell inoculation. The thoracic tumorswere carefully removedand weighed, pleural effusions were harvested using a 1-mLsyringe, and their volumes were measured.

Evaluation of microvessel density in intrapleural tumorsFrozen tissue sections were incubated with a rat polyclonal

antibody against mouse CD31 (BD Pharmingen) at 4�C over-night. After washing with PBS, the slides were stained with abiotinylated rabbit anti-rat IgG antibody (Vector Laboratories).The immunoreaction was amplified with a VECTASTAIN ABC Kit(Vector Laboratories) and visualized by incubation with a 3,3-diaminobenzidine solution acting as a chromogen. The sectionswere then counterstained with hematoxylin and dehydrated.Images were captured using a microscope at a magnification of�200 (model BZ-9000; Keyence), and the area of CD31þ vessel-like structures was measured in five random microscopic fieldsper section using Dynamic cell count software BZ-HIC (Keyence).

Proliferation assayEHMES-10 or MSTO-211H cells (1.5 � 104/100 mL) were

seeded in 96-well plates and incubated with various concentra-tions of SK-216 and cisplatin for 48 hours. HUVECs (1.5 � 104/100 mL) were incubated with angiogenic factors (20 ng/mLbFGF, 100 ng/mL PDGF-BB, or 30 ng/mL HGF) in the presenceof SK-216 at various concentrations for 16 and 36 hours. Cellproliferation was assessed using the Cell Counting Kit-8(Dojindo) by measuring the absorbance of the medium.

Cell migration assayHUVEC migration was assessed using an Oris Universal Cell

Migration Assembly Kit (Platypus Technologies). HUVECs (2 �104) suspended in 100 mLmediumwere seeded into each test wellof the Oris plate with the well inserts (stoppers) and thenincubated to allow cell attachment. After 4 hours, the stoppersin each well were removed. HUVECs were incubated with angio-genic factors (20 ng/mL bFGF, 100 ng/mL PDGF-BB, or 30 ng/mLHGF) in the presence of SK-216 at various concentrations for36 hours and were then stained with calcein acetoxymethyl-ester (Calcein AM) stock solution (2 mmol/L; Dojindo). Imageswere captured at a magnification of �40 using a fluorescencemicroscope (model BZ-9000; Keyence). The areas occupied by

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HUVECs (migration area) were measured and quantified usingDynamic cell count software BZ-HIC (Keyence).

Capillary-like tube formation assaySeventy microliters of Matrigel was applied to each well of a

96-well plate and incubated for 30minutes. HUVECs (7.5� 103)suspended in 100 mL medium were plated onto the Matrigel andincubated with angiogenic factors (20 ng/mL bFGF, 100 ng/mLPDGF-BB, or 30 ng/mLHGF) in the presence of SK-216 at variousconcentrations for 16 hours and then were stained with CalceinAM stock solution (2 mmol/L). Images were captured at a mag-nification of �20 using a fluorescence microscope, and the totalarea of the tube-like space was quantified using Dynamic cellcount software BZ-HIC.

Statistical analysisStatistical analyses were performed using SPSS 20.0 for

Windows (SPSS Japan). The results are expressed as mean� SEMor median with ranges. The statistical significance of differencebetween two datasets obtained from the in vitro and in vivoexperiments was analyzed by one-way ANOVA with Dunnettpost hoc test or Student t test, where applicable. The Kaplan–Meiermethod was used to evaluate the survival analysis, and compar-isons were made using a log-rank test. P > 0.05 was consideredstatistically significant.

ResultsProfiles of angiogenic factors differed between two MPM celllines, EHMES-10 and MSTO-211H, in vitro and in vivo

To determine the profiles of angiogenic factors in twoMPM celllines, EHMES-10 and MSTO-211H, we evaluated the expressionlevels of VEGF, bFGF, PDGF-BB, and HGF in these cells. Neithercell line was observed to secrete PDGF-BB or HGF in the culturesupernatants (data not shown). Regarding VEGF and bFGF,EHMES-10 cells secreted much higher amounts of VEGF andmuch lower amounts of bFGF compared with MSTO-211H cells(Fig. 1A and B). To confirm whether this expression pattern ofangiogenic factorswas retained in vivo, the concentrations of VEGFand bFGF were measured in the lysates of intrapleural tumorsconsisting of EHMES-10 or MSTO-211H cells generated in SCIDmice. Consistent with the in vitro secretion profiles of angiogenicfactors, EHMES-10 cells were shown to produce much higheramounts of VEGF andmuch lower amounts of bFGF thanMSTO-211H cells even in vivo (Fig. 1C and D).

SK-216 reduced tumor weights in both orthotopicimplantation mouse models using VEGF-high–expressingEHMES-10 cells and bFGF-high–expressing MSTO-211H cells

To determine whether inhibition of PAI-1 by a PAI-1–specificinhibitor, SK-216, affects intrapleural tumor progression, SK-216

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Figure 1.Differences in profiles of angiogenicfactors between EHMES-10 andMSTO-211H cells. A andB, EHMES-10 orMSTO-211H cells (2� 105)were seededin 6-well plates and cultured for 48hours. Concentrations of VEGF andbFGF in culture media were measuredby ELISA. C and D, intrapleural tumorswere excised 28 days for EHMES-10cells or 21 days for MSTO-211H cellsafter the intrapleural inoculation ofMPM cells into SCID mice.Concentrations of VEGF and bFGF intumor lysates were measured byELISA. Data, mean values (�SEM) oftriplicate samples.

Inhibition of PAI-1 Suppresses Tumor Angiogenesis

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was administered through drinking water at 100 or 500 ppmto the SCID mice intrapleurally inoculated with the two MPMcell lines. The weights of intrapleural tumors and the volume ofpleural effusions were evaluated 28 days (EHMES-10) or 21 days(MSTO-211H) after the inoculation. Intrapleural tumors con-sisting of VEGF-high–expressing EHMES-10 cells were accom-panied by much larger amounts of pleural effusions comparedwith those of bFGF-high–expressing MSTO-211H cells (Sup-plementary Fig. S2B and S2D). Administration of SK-216 at100 and 500 ppm significantly reduced the weights of tu-mors consisting of EHMES-10 cells, whereas administration ofSK-216 at 500 ppm, but not at 100 ppm, demonstrated a signi-ficant reduction in weights of tumors consisting of MSTO-211H cells (Supplementary Fig. S2A and S2C). Of note, SK-216 did not affect the amount of pleural effusions associatedwith the tumors of EHMES-10 or MSTO-211H cells (Supple-mentary Fig. S2B and S2D). On the basis of these results, wedecided to use 500 ppm as the administration dosage of SK-216in drinking water for further experiments. To further confirm

the antitumor effects of SK-216, SK-216 was then administeredthrough drinking water at 500 ppm to SCID mice subcutane-ously inoculated with EHMES-10 and MSTO-211H cells. Inthis experiment, the administration of SK-216 was startedon day 0 at the same time as cell inoculation or day 7 whenmeasurable (100–200 mm3) subcutaneous tumors were observ-ed. As shown in the Supplementary Fig. S3, SK-216 reducedthe sizes of subcutaneous tumors consisting of EHMES-10and MSTO-211H cells, regardless of whether the administra-tion of SK-216 was started on day 0 or day 7. In addition, wealso found that administration of SK-216 did not affect thesizes of subcutaneous tumors 7 days after cell inoculation. Theseresults suggested that the antitumor activity of SK-216 wasmore effective against tumor progression following the establish-ment of tumor growth rather than during the initial phase oftumor growth.

To confirm whether the profile of antitumor activity ofSK-216 differed from that of VEGF-targeted therapy, the ther-apeutic effects of SK-216 and bevacizumab on intrapleural

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Figure 2.Effects of SK-216 and bevacizumab onintrapleural tumors (A and C) andpleural effusions (B and D) of SCIDmice inoculated with MPM cells. SCIDmice were intrapleurally inoculatedwith EHMES-10 cells (3� 106) orMSTO-211H cells (1 � 106). The mice weretreated with SK-216 (500 ppm indrinking water) or bevacizumab(intraperitoneal injection at 3 or10 mg/mouse on days 7, 10, 14, and 17)andwere sacrificed 28days for EHMES-10 cells (A and B) or 21 days for MSTO-211H cells (C and D) after theinoculation. The weight of intrapleuraltumors and the amount of pleuraleffusions were evaluated as describedin Materials and Methods. Twoexperiments were performed for eachcell line, with identical results. Each barrepresents the mean value of 6 miceper group. The data were analyzed byone-way ANOVA with Dunnett posthoc test. � , P < 0.05 versus controlgroup; �� ,P<0.01 versus control group.NS, not significant.

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tumor progression were compared. Intraperitoneal injection ofbevacizumab at doses of 3 or 10 mg/mouse on days 7, 10, 14,and 17 significantly reduced tumor weights and the amountof pleural effusions from the mice inoculated with VEGF-high–expressing EHMES-10 cells (Fig. 2A and B), whereas neitherdosage of bevacizumab showed inhibitory effects on the tumorweights and the amount of pleural effusions from the miceinoculated with bFGF-high–expressing MSTO-211H cells(Fig. 2C and D). In contrast, administration of SK-216 at 500ppm through drinking water significantly reduced the weights ofintrapleural tumors consisting of EHMES-10 or MSTO-211Hcells (Fig. 2A and C); however, it was again confirmed that SK-216 did not exert an inhibitory effect on the amount of pleuraleffusions (Fig. 2B and D).

Finally, to assess whether SK-216 suppressed tumor progres-sion beyond its effects on inhibition of the tumor-promotingaction of VEGF and bFGF, we evaluated the antitumor effectsof SK-216 under conditions in which VEGF or bFGF wasblocked by anti-VEGF antibodies (bevacizumab) in mice bear-ing EHMES-10 cells or by an FGFR inhibitor (AZD4547) inmice bearing MSTO-211H cells. As shown in SupplementaryFig. S4, the antitumor effects of bevacizumab alone and SK-216alone were similar in mice bearing EHMES-10 cells. In addi-tion, the antitumor effects of AZD4547 alone and SK-216 alonewere similar in mice bearing MSTO-211H cells. Furthermore,SK-216 in combination with bevacizumab or AZD4547 wasfound not to enhance the antitumor effects of bevacizumab inmice bearing EHMES-10 cells or AZD4547 in mice bearing

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Figure 3.Evaluation of angiogenesis inintrapleural tumors consisting ofEHMES-10 (A and B) or MSTO-211H(C and D) cells. Mice were treated withSK-216 (500 ppm) or bevacizumab(3 mg/mouse). A and C, the area ofCD31þ vessels was calculated asdescribed in Materials and Methods.Two experiments were performed foreach cell line, with identical results.Data represent themean values (SEM)of 6 mice in each group and wereanalyzed by one-way ANOVA withDunnett post hoc test. � , P < 0.001versus control group. NS, notsignificant. B and D, representativeimmunohistochemical staining ofCD31 in thoracic tumors. Scale bar,100 mm.






MSTO-211H cells. These results suggested that SK-216 sup-pressed tumor progression through its effects on inhibition ofthe tumor-promoting action of VEGF in mice bearing VEGF-overexpressing cells or bFGF in mice bearing bFGF-overexpres-sing cells.

SK-216 reduced the degree of angiogenesis in intrapleuraltumors consisting of VEGF-high–expressing EHMES-10 cellsand bFGF-high–expressing MSTO-211H cells

To determine whether SK-216 could affect the degree ofangiogenesis in intrapleural tumors, microvessel density was

evaluated by immunohistochemical staining of the intrapleuraltumors consisting of EHMES-10 or MSTO-211H cells using ananti-CD31 mAb. As shown in Fig. 3, SK-216 reduced themicrovessel density in both intrapleural tumors consisting ofEHMES-10 and MSTO-211H cells; however, as expected, bev-acizumab successfully decreased the microvessel density in thetumor of EHMES-10 cells but failed to in that of MSTO-211Hcells (Fig. 3).

Then, we examined whether SK-216 altered the expressionlevel of PAI-1 in intrapleural tumors by IHC. As shown inSupplementary Fig. S5, SK-216 was found not to affect the

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Figure 4.Effects of cisplatin and/or SK-216 on invitroproliferation ofMPM cell lines andthe additive therapeutic effect of SK-216 combined with cisplatin onintrapleural tumors and pleuraleffusions of SCIDmice inoculated withMPM cells. A and B, proliferation ofEHMES-10 (A) andMSTO-211H (B) cellswas quantified after 48 hours in thepresence of SK-216 alone, cisplatinalone at various concentrations (0.1–50 mmol/L), or SK-216 (30 mmol/L) incombination with cisplatin (0.1–50mmol/L). Data, mean values (SEM) oftriplicate samples. C–F, SCID micewere intrapleurally inoculated withEHMES-10 (3 � 106) or MSTO-211Hcells (1 � 106). The mice were treatedwith SK-216 (500 ppm in drinkingwater) alone, cisplatin alone(intraperitoneal injection at 1.5 mg/kgfor EHMES-10–bearing mice or at 1.0mg/kg for MSTO-211H–bearing miceper week), or SK-216 in combinationwith cisplatin and were sacrificed 28days for EHMES-10 cells (C andD) or 21days for MSTO-211H cells (E and F)after the inoculation. The weight ofintrapleural tumors and amount ofpleural effusions were evaluated asdescribed in Materials and Methods.Two experiments were performed foreach cell line, with identical results.Each bar, mean value of 6 mice pergroup. The data were analyzed byone-way ANOVA with Dunnett posthoc test. � , P < 0.05 versus controlgroup; �� , P < 0.01 versus controlgroup; †, P < 0.05 versus the grouptreated with SK-216 in combinationwith cisplatin; z, P < 0.01 versus thegroup treated with SK-216 incombination with cisplatin. NS, notsignificant.

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expression level of PAI-1 in intrapleural tumors consisting ofEHMES-10 and MSTO-211H cells.

Combined SK-216 and cisplatin enhanced the antitumor effectof each agent alone in MPM mouse models

Because antitumor activity of SK-216 as a single agent wasdemonstrated, we next questioned whether SK-216 affected theantitumor activity of cisplatin. First, we assessed the effect ofSK-216 and/or cisplatin on the proliferation of twoMPMcell linesin vitro. The proliferation assay showed that cisplatin inhibited thegrowth of both EHMES-10 and MSTO-211H cells in a dose-dependent manner; however, SK-216 neither showed an inhib-itory effect on the proliferation of these cells nor enhanced theantiproliferative activity of cisplatin in vitro (Fig. 4A and B).

Next, themice inoculatedwith EHMES-10 orMSTO-211H cellswere treated with SK-216 alone, cisplatin alone, or a combinationof SK-216 and cisplatin. In this experiment, a lower dose ofcisplatin was used for MSTO-211H cell–bearing mice (1.0 mg/kg)than for EHMES-10 cell–bearing mice (1.5 mg/kg) becausecisplatin showed a much stronger inhibitory effect on the proli-feration of MSTO-211H cells than that of EHMES-10 cells in vitro.As shown in Fig. 4C and E, the cisplatin and SK-216 combinationtreatment significantly reduced the tumor weights compared withtreatmentwith each agent alone in bothmouseMPMmodelswithEHMES-10 and MSTO-211H cells. Although treatment with SK-216 or cisplatin alone failed to inhibit the formation of pleuraleffusions in EHMES-10 cell–bearingmice, the combination treat-ment significantly reduced the amount of pleural effusions(Fig. 4D). In addition, the cisplatin and SK-216 combinationtreatment tended to prolong the survival time of MPM modelmice treated with each agent alone (Fig. 5).

SK-216 inhibited migration and tube formation of HUVECsinduced by bFGF, PDGF-BB, and HGF in vitro

Because the antiangiogenic activity of SK-216 was demonstrat-ed in vivo irrespective of the primary angiogenic factor producedby the tumor, we assessed whether SK-216 affected proliferation,migration, and tube formation of endothelial cells induced bybFGF, PDGF-BB, and HGF, as in our previous study that usedinduction by VEGF (31). The proliferation assay showed that SK-216 did not affect the proliferation rate of HUVECs in thecopresence of each angiogenic factor in culture (Fig. 6A and B).

As shown in Fig. 6C and E, however, SK-216 was found to inhibitbothmigration and tube formation ofHUVECs induced by bFGF,PDGF-BB, or HGF in a dose-dependent manner. A similar inhib-itory effect of SK-216 on migration and tube formation ofHUVECs was demonstrated in our previous study using VEGFas the inducer (31).

To further confirm the involvement of PAI-1 in migration andtube formation of HUVECs, we prepared HUVECs in which PAI-1expression was knocked down by RNA interference (Supplemen-tary Fig. S6). As shown in Supplementary Fig. S7, silencing of PAI-1 in HUVECs also inhibited migration and tube formationinduced by VEGF, bFGF, PDGF-BB, and HGF.

Finally, to assess whether SK-216 affected downstream signal-ing targets of angiogenic factors in HUVECs, we examined theeffects of SK-216 onphosphorylation of ERK1/2 andAKT inducedby VEGF, bFGF, PDGF-BB, and HGF. As shown in SupplementaryFig. S8, SK-216 did not inhibit the phosphorylation of ERK1/2and AKT induced by these angiogenic factors, except for HGF-induced phosphorylation of ERK1/2.

DiscussionIn this study, we found that systemic administration of a

specific PAI-1 inhibitor, SK-216, could suppress tumor progres-sion through limiting angiogenesis in orthotopic implantationmouse models of MPM using two MPM cell lines in which theprimary angiogenic factors differ, namely, VEGF-high–expressingEHMES-10 cells and bFGF-high–expressing MSTO-211H cells. Ineachmousemodel ofMPMusing EHMES-10 cells orMSTO-211Hcells, the antitumor effect of cisplatin was augmented by com-bining it with SK-216. In vitro, inhibition of PAI-1 by SK-216suppressed migration and tube formation of HUVECs inducedby bFGF, PDGF-BB, and HGF, as well as by VEGF, as reportedpreviously (31).

The most striking finding of this study is that inhibition ofPAI-1 by a PAI-1 inhibitor, SK-216, suppressed migration andtube formation of HUVECs induced by bFGF, PDGF-BB, andHGF, as well as by VEGF, as reported previously (31). Theseresults imply that inactivation of PAI-1 in the tumor environ-ment has the potential to limit the progression of tumorangiogenesis regardless of angiogenic factors produced by thetumor and, therefore, can become an antiangiogenic strategy

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Figure 5.Effects of cisplatin and/or SK-216 onsurvival of MPM cell–bearing mice.SCID mice (6 mice/group) inoculatedwith EHMES-10 (A) or MSTO-211H (B)cells were treated with SK-216 (500ppm), cisplatin (1.5 mg/kg; EHMES-10or 1.0 mg/kg; MSTO-211H per week), orSK-216 (500 ppm) in combination withcisplatin (1.5 mg/kg; EHMES-10 or 1.0mg/kg; MSTO-211H). Two experimentswere performed for each cell line, withidentical results. � , P < 0.05 versuscontrol group; †, P < 0.01 versus controlgroup; z, P < 0.05 versus the grouptreated with SK-216.






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Figure 6.Effects of SK-216 on the degrees of proliferation, migration, and tube formation of HUVECs. A and B, proliferation of HUVECs induced by bFGF, PDGF-BB,and HGF was quantified after 16 hours (A) and 36 hours (B) in the presence of SK-216 at various concentrations. C and E, migration (C) and tubeformation (E) of HUVECs induced by bFGF, PDGF-BB, and HGF in the presence of SK-216 at various concentrations were evaluated as described in Materialsand Methods. D and F, representative images of HUVECs in a cell migration assay (D) and capillary-like tube formation assay (F). HUVECs were stained withCalcein AM stock solution. Data represent the mean values (SEM) of triplicate samples and were analyzed by one-way ANOVA with Dunnett post hoc test. � , P< 0.01 versus control; †, P < 0.05 versus control; scale bar, 250 mm.

Takayama et al.





against any type of angiogenic factor. In addition, these resultsindicate that PAI-1 plays a crucial role in migration and tubeformation of endothelial cells stimulated by any angiogenicfactors. Regarding a role of PAI-1 in the migration of endothe-lial cells induced by several different angiogenic factors, Pragerand colleagues demonstrated that VEGF, bFGF, and HGF stim-ulated the migration of endothelial cells by activating pro-uPAand by forming uPAR–uPA–PAI-1 complexes followed byinternalization of uPAR (35, 36). Of note, the formation ofuPAR complexes and internalization of uPAR are PAI-1 depen-dent (35). These observations suggest a key role of PAI-1 in themigration of endothelial cells induced by various angiogenicfactors. In addition, we assessed whether PAI-1 inhibition bySK-216 affected the phosphorylation of ERK1/2 and AKT, twomajor downstream signaling targets of VEGF (37), FGF (38),PDGF (39), and HGF (40). Our results showed that the phos-phorylation of ERK1/2 and AKT in HUVECs induced by theseangiogenic factors, except for HGF-induced phosphorylation ofERK1/2, was not inhibited by SK-216. Although we cannotexplain why SK-216 inhibited HGF-induced phosphorylationof ERK1/2, our results suggested that inhibition of PAI-1 wasnot largely associated with these signaling pathways. We spec-ulate that inhibition of PAI-1 may act at a distal point wherethese signaling pathways converge.

MPM is known to secrete various angiogenic factors, such asVEGF, bFGF, PDGF, andHGF. TwoMPM cell lines available to us,EHMES-10 and MSTO-211H, were found to dominantly expressVEGF and bFGF, respectively, but secrete neither PDGF-BB norHGF into the culture supernatants (data not shown). Using thesetwo cell lines, we could establish orthotopic implantation mousemodels of MPM. As previously reported (41), we confirmed thatbevacizumab could limit tumor progression through inhibitionof angiogenesis in a mouse MPMmodel with VEGF-high–expres-sing EHMES-10 cells, but not with bFGF-high–expressing MSTO-211H cells. In contrast to bevacizumab, we found that SK-216could limit tumor progression through inhibition of angiogenesisin both MPM models using EHMES-10 and MSTO-211H cells.These results suggest that inhibition of PAI-1 can exert antitumorand antiangiogenic effects even in vivo irrespective of the primaryangiogenic factor produced by the tumor and are also supportedand explained by its ability to suppress migration and tubeformation of HUVECs induced by various types of angiogenicstimuli. Regarding the formation of pleural effusion, we foundthat VEGF-high–expressing EHMES-10 cells produced a largeamount of pleural effusions, but bFGF-high–expressing MSTO-211H did not. In addition, bevacizumab was very effective ininhibiting the formation of pleural effusion, whereas SK-216 wasnot. These results suggest that VEGF was responsible for thedevelopment of pleural effusions by inducing vascular perme-ability as described in a previous report (42), and SK-216 mighthave little effect on VEGF-induced vascular permeability.

In this study, the antitumor effect of SK-216 in orthotopicimplantation mouse models of MPM was clearly demonstrated.Because this effect is likely mediated by its antiangiogenic prop-

erties and SK-216 did not show any cytotoxic activity on MPMcells in vitro, we then tried to determinewhether inhibition of PAI-1 by SK-216 affected the antitumor activity of cytotoxic agents,such as cisplatin, in vitro and in vivo. In vitro, SK-216 did notaugment the cytotoxic activity of cisplatin. However, in theorthotopic implantation mouse models, the cisplatin and SK-216 combination treatment significantly enhanced the antitumoractivity of each agent alone and tended to prolong the survivaltime of MPM model mice treated with cisplatin alone. Theseresults suggest that SK-216 can enhance the antitumor effects ofcytotoxic agents, and we propose that SK-216 should be used incombination with cytotoxic agents rather than as a single agentwhen used as an antitumor agent.

In conclusion, systemic administration of SK-216, a specificinhibitor of PAI-1, could suppress tumor progression of MPMcells in vivo through inhibition of angiogenesis regardless ofwhichangiogenic factors were produced by MPM tumors. In addition,SK-216 was shown to have an inhibitory effect on migration andtube formation of HUVECs induced by various angiogenic factorsthat are known to be secreted by MPM, such as VEGF, bFGF,PDGF-BB, and HGF in vitro. Furthermore, the combination ofSK-216 and cisplatin was shown to enhance the antitumor effectof each agent alone and tended to prolong the survival time ofMPM model mice treated with each agent alone. Taken together,these results suggest that inactivation of PAI-1 by SK-216 maybecome a novel strategy for inhibiting angiogenesis regardlessof the type of angiogenic stimulus and that the combinationof SK-216 and cisplatin may become a promising therapy forMPM, which is known to secrete various angiogenic factors.

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

Authors' ContributionsConception and design: N. Hattori, T. MasudaDevelopment of methodology: N. Hattori, H. Hamada, K. Omori, S. AkitaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Takayama, T. Masuda, S. AkitaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Takayama, K. OmoriWriting, review, and/or revision of the manuscript: Y. Takayama, N. Hattori,H. Hamada, H. Iwamoto, N. KohnoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Takayama, S. Akita, H. IwamotoStudy supervision: N. Hattori, H. Hamada, K. Fujitaka, N. Kohno

Grant SupportThis study was supported by grants-in-aid for Scientific Research from the

Ministry of Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 3, 2015; revised March 4, 2016; accepted March 8, 2016;published OnlineFirst April 13, 2016.

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2016;76:3285-3294. Published OnlineFirst April 13, 2016.Cancer Res Yusuke Takayama, Noboru Hattori, Hironobu Hamada, et al. Angiogenic Stimuli in Malignant Pleural MesotheliomaInhibition of PAI-1 Limits Tumor Angiogenesis Regardless of

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