Cyclosporin A Promotes Tumor Angiogenesis in a Calcineurin ...Region-1 (DSCR1), suppresses tumor growth and angiogenesis, in contrast to the effect observed after long-term cyclosporin

Signal Transduction

Cyclosporin A Promotes Tumor Angiogenesis in aCalcineurin-Independent Manner by IncreasingMitochondrial Reactive Oxygen Species

Alice Yao Zhou and Sandra Ryeom

AbstractThe widely used immunosuppressant cyclosporin A, a potent calcineurin inhibitor, significantly increases the

incidence of cancer in organ transplant patients. Calcineurin signaling is an important mediator of VEGF signalingin endothelial cells. Negative regulation of calcineurin by its endogenous inhibitor, Down Syndrome CandidateRegion-1 (DSCR1), suppresses tumor growth and angiogenesis, in contrast to the effect observed after long-termcyclosporin A treatment. Despite the significance of calcineurin signaling in endothelial cells, the consequences ofcyclosporin A on tumor angiogenesis have not been investigated. Using an in vivo model of skin carcinogenesis,prolonged treatment with cyclosporin A promoted tumor growth and angiogenesis. The addition of cyclosporin Ato endothelial cells in vitro increased proliferation and migration in a calcineurin-independent manner and isassociated with increased mitochondrial reactive oxygen species (ROS). Co-treatment with antioxidants signif-icantly abrogated cyclosporin A–induced endothelial cell activation. Furthermore, mice treated with antioxidantswere protected against cyclosporin A–mediated tumor progression. Taken together, these findings suggest thatcyclosporin A affects endothelial cells in a calcineurin-independent manner to potentiate tumor growth bypromoting tumor angiogenesis through increasing mitochondrial ROS production. This work identifies apreviously undescribed mechanism underlying a significantly adverse off-target effect of cyclosporin A and suggeststhat co-treatment with antioxidants would inhibit the tumor-promoting effects of cyclosporin A.

Implications:Targeting the proangiogenic effects of cyclosporin Amay be useful in themanagement of transplant-associated cancers. Mol Cancer Res; 12(11); 1663–76. �2014 AACR.

IntroductionOrgan transplantation has revolutionized the treatment

for end-stage organ failure. Cyclosporin A is a commonlyused immunosuppressant for organ transplant patients (1).Cyclosporin A binds to intracellular cyclophilins, complex-ing specifically with cyclophilin A to inhibit calcineurin, acalcium-responsive ser/thr phosphatase that is best knownfor its dephosphorylation and activation of the nuclear factorof activated T cells (NFAT) family of transcription factors.Activation of the T-cell receptor increases intracellular cal-cium and results in NFAT-induced transcription of keycytokines such as IL2, IFNg and IL4, critical for theexpansion and function of effector T cells. Inhibition of

calcineurin activity with cyclosporin A results in potentsuppression of the adaptive immune response (2). However,an adverse side effect of patients who remain on chroniccyclosporin A treatment is an elevated risk of malignancies.The incidence of skin cancers is significantly increased, witha �65-fold increase in risk when compared with the rest ofthe population (1, 3). The progression of skin cancers incyclosporin A–treated patients is significantly more aggres-sive, with an increased incidence of metastasis and a poorprognosis (3). With advances in post-transplant care,patients receiving allografts are living longer, and post-transplant malignancies are one of the major causes ofmorbidity and mortality in this population (4).The increased incidence in cancer observed in transplant

recipients has been attributed to the function of cyclosporinA as an immunosuppressant, leading to the loss of tumorimmunosurveillance. This is supported by an increasedincidence of human papillomavirus observed in transplantrecipients with skin cancers as compared with skin cancers inpatients not on immunosuppressive therapy (5). In contrast,patients on FK506 (tacrolimus) for immunosuppressionhave a significantly lower incidence of cancer post-transplantthan patients on cyclosporin A (1), suggesting that whileimportant, immunosuppression alone is not sufficient topromote tumorigenesis. Cyclosporin A treatment has also

Department of Cancer Biology, Abramson Family Cancer Research Insti-tute, University of Pennsylvania School of Medicine, Philadelphia,Pennsylvania.

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

Corresponding Author: Sandra W. Ryeom, Abramson Family CancerResearch Institute, University of Pennsylvania School of Medicine, 421Curie Blvd, 711 BRB II/III, Philadelphia, PA 19104. Phone: 215-573-5857;Fax: 215-573-2014; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0136

�2014 American Association for Cancer Research.

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been shown to potentiate tumor growth and metastasis ofxenograft tumors in SCID mice (6). These studies demon-strate that while cyclosporin A–mediated immunosuppres-sion contributes to tumorigenesis, cyclosporin A also hasprotumor effects independent of a functional adaptiveimmune system.A few studies have examined the immune-independent

mechanisms of cyclosporin A–induced tumorigenesis.Cyclosporin A has been shown to suppress both apoptosisand the expression of DNA repair proteins after UVexposure (7, 8), permitting the persistence of sporadicmutations while evading cell death. In another study,oncogenic Ras-induced senescence is circumvented bycyclosporin A inhibition of calcineurin–NFAT signalingin keratinocytes, leading to ATF3-dependent suppressionof p53 (9). Cyclosporin A has also been shown to increaseTGFb production by tumor cells, leading to enhancedtumor invasion and metastasis in SCID mice (6).The role of the calcineurin pathway in tumor progression

is complicated by the identification of the endogenouscalcineurin inhibitor DSCR1 (also known as RCAN1).Increased expression of DSCR1 has been shown by us andothers to inhibit tumor growth by blocking VEGF–calci-neurin–NFAT signaling in endothelial cells attenuatingtumor angiogenesis (10–15). The physiologic relevance ofthis pathway was illustrated in Down Syndrome mousemodels whereby chromosome 21–encoded inhibitors of thecalcineurin–NFAT pathway, including Dscr1, are overex-pressed blocking tumor angiogenesis and tumor progression(10). Despite the importance of calcineurin signaling inendothelial cells, the effect of cyclosporin A on endothelialcells in a tumorigenic context has not been extensivelyexamined.Cyclosporin A is an 11 amino acid fungal-derived peptide

and although it binds to cyclophilin A to inhibit calcineurin,it also binds to other cyclophilin family members. Cyclo-philins are a family of 16 conserved members with intracel-lular peptidyl-prolyl cis–trans isomerase activity functioningmostly as chaperone proteins (16). Cyclophilins have beenassociated with breast and lung cancers, but their role intumorigenesis is not understood (17). Cyclophilin D islocated in the mitochondrial inner membrane as a compo-nent of the mitochondrial permeability transition pore(MPTP). Binding of cyclosporin A to cyclophilin D hasbeen shown to be cytoprotective against several forms ofmitochondrial-mediated cell death (18, 19) and alters theflux of mitochondrial contents through theMPTP. Changesin mitochondrial physiology can have a range of effects onendothelial cells, as the endothelium is particularly sensitiveto changes in oxidative state, activating signaling pathwaysthat affect vascular tone, permeability, and angiogenesis(20, 21). An elevation in reactive oxygen species (ROS)levels due to altered mitochondrial metabolism has beenshown to promote tumorigenesis by increasing mitogen-activated protein kinase (MAPK) signaling to a proliferativelevel, directly promoting the cell division of cancer cells (22),or through stabilization of hypoxia-inducible factor andincreased tumor angiogenesis (23). The effect of cyclosporin

A binding to cyclophilin D on theMPTP in endothelial cellshas not yet been examined in a nonapoptotic context.Here we show that cyclosporin A promotes tumorigenesis

by increasing tumor angiogenesis in a calcineurin-indepen-dentmanner. Using an in vivo skin carcinogenesis model, wedemonstrate that cyclosporin A potentiates tumor growth byupregulating tumor angiogenesis as evidenced by increasedmicrovessel density. Our data demonstrate that cyclosporinA treatment stimulates a proliferative and migratory phe-notype in endothelial cells and is associated with elevatedmitochondrial ROS. Pharmacologic quenching of cyclo-sporin A–induced ROS with antioxidants is sufficient toabolish cyclosporin A–induced endothelial cell proliferationand migration in vitro and tumor growth in vivo. Ourfindings suggest that prophylaxis treatment with an antiox-idant to target ROS may decrease transplant-associatedmalignancies without affecting the immunosuppressivecapabilities of cyclosporin A.

Materials and MethodsReagentsNIM811was a gift fromNovartis. Cyclosporin A, FK506,

andN-acetyl cysteine (NAC), were from Sigma-Aldrich.Mn(III)tetrakis(4-benzoic acid)porphyrin Chloride was fromMillipore.

Animals and tumor studiesWT C57Bl/6 mice were from The Jackson Laboratory.

Dscr1-targeted transgenic mice (10) and Calcineurin Bf/f

mice were previously described (24). Mice were orallygavaged daily with 10 mg/kg of cyclosporin A oral solutionUSPmodified or 0.15mg/kg FK506 (Hospital of Universityof Pennsylvania Pharmacy, Philadelphia, PA) diluted inpeanut oil or simple syrup, respectively, starting 7 to 14days before tumor initiation, or supplemented with40 mmol/L NAC in the drinking water starting 14 daysbefore cyclosporin A treatment. Chemically induced papil-lomas (25) and B16-F10 melanoma xenograft tumors (10)were generated as previously described. B16-F10 melanomacells, originally from ATCC, were authenticated to be ofC57Bl/6 murine origin using microsatellite markers(RADIL) in 2011. Mice were euthanized if mice becamemoribund or tumors became ulcerated before experimentalendpoint. Mice were 8 to 12 weeks old. All animal experi-ments were performed according to the protocols approvedby the University of Pennsylvania IACUC.

Immunofluorescence and CD31 quantificationTumors were harvested from mice and frozen in OCT

freezing medium (Tissue-Tek) and then sectioned for stain-ing as previously described (26). Primary and secondaryantibodies were rat anti-mouse CD31 antibody (1:50, BDBiosciences) and Alexa 594 goat anti-rat (1:2,000, Invitro-gen), respectively. Five random 10�magnification pictureswere taken of each slide, and the area of CD31þ structures,visible lumens, total vessels, and vessels �100 mm wascounted. Images were taken with a 10� or 20� magnifi-cation objective lens and with a digital camera AxioCAM

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HRc (Zeiss) mounted on Zeiss Imager M1 Axio using ZeissAxioVision Acquisition software (version 4.5).

Primary endothelial cell isolationPrimary murine lung endothelial cells (LuEC) were iso-

lated from 3- to 4-week-old mice as previously described(27).

In vitro proliferation and TUNEL apoptosis assays.LuEC were plated in triplicate at 5� 103 cells per well in

0.1% gelatin-coated 24-well tissue culture plates. Cells werecounted by a coulter counter (Beckman). For bromodeox-yuridine (BrdUrd) labeling, 2.5 � 103 LuEC were platedonto 0.1% gelatin-coated glass coverslips serum starved for24 hours, incubated with drug treatment, and then pulsedwith 10 mmol/L BrdUrd (BD Biosciences) for 1.5 hours.Cells were then fixed with 4% paraformaldehyde, permea-bilized with 0.1% TBST, and denatured with 2N HCL.Endothelial cells were stained with anti-BrdUrd mouseantibody (1:50, Dako). Secondary antibody was Alexa594 goat anti-rat (1:2,000, Invitrogen), and nuclei wereidentified with Hoechst 33342 (1:1,000, Invitrogen). Sevenrandom 10�magnification pictures were taken of each slideusing�10 or�20magnification objective lens with a digitalcamera AxioCAM HRc (Zeiss), BrdUrd-positive cells andtotal cell number were counted. In situ terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling(TUNEL) was performed using the DeadEnd FluorometricTUNEL System (Promega) according to the manufacturer'sinstructions. Flow cytometry of TUNEL-stained cells wasperformed on a FACS Canto flow cytometer (BD Bio-sciences) and analyzed with FlowJo Software (TreeStar).

Migration assayBasal endothelial media (27) with 0.5% FBS and lacking

ECGswas placed in the lower chamber of amodifiedBoydenchamber (Corning) separated by an 8-mm pore filter. LuECwere serum starved overnight, treated with experimentalreagents for 2 hours, and 2 � 104 cells were plated in theupper chamber and allowed to migrate for 2 to 4 hours at37�C. Filters were stained with Diff-Quick solution (Bax-ter), and the cells that migrated across the filter were countedin 5 random images taken at �10 or �20 magnificationobjective lens and with a digital camera AxioCAM HRc(Zeiss).

AdCre infectionLuEC from CnBf/f mice were infected with Cre recom-

binase adenovirus (Ad5CMVCre; University of Iowa,Gene Transfer Vector Core) at 500 multiplicity of infec-tion (MOI) overnight, followed by PBS wash and mediachange.

Mitosox and TMRE stainingMitosox (Invitrogen) and tetramethylrhodamine ethyl

ester (TMRE; Life Technologies) staining on LuECwere performed following the manufacturer's instructions.Mitosox fluorescence was detected by flow cytometry using a

FACS Canto flow cytometry machine reading at 610 or613 nm, and TMRE fluorescence was detected reading at575 nm. Data were analyzed with FlowJo software.

Western blottingWhole LuEC lysates were separated by SDS-PAGE,

transferred to nitrocellulose membrane, and probed withthe following antibodies: p-ERK, total ERK, p-AKT, totalAKT (Cell Signaling Technology); calcineurin A (SantaCruz Biotechnology); b-actin (Sigma). Band densities ofphospho-proteins were quantified with densitometry anal-ysis using ImageJ software (NIH) and then normalized withthe total protein.

Statistical analysisData is represented as either the mean � SD or the

mean � SEM and indicated in figure legends. P valuesbetween 2 groups are calculated using the Student t test.Differences were considered significant when P < 0.05.

ResultsCyclosporin A treatment in vivo promotes skintumorigenesis and angiogenesisA well-characterized carcinogen-induced model of skin

carcinogenesis uses a single application on the skin of wild-type mice with 7,12-dimethylbenz[a]anthracene (DMBA)followed by twice weekly application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), leading to dermal papillomas andeventual carcinomas (25). Cyclosporin A treatment of miceduringDMBA andTPA application promotes tumor growth.Consistent with a previous study examining the applicativevalue of the DMBA-TPA carcinogenesis model in immuno-suppressive contexts (28), clinically relevant dosing of cyclo-sporin A significantly increases the number of dermal pap-illomas on wild-type mice (Fig. 1A). Similarly, cyclosporin Atreatment of wild-type mice after flank injection of syngeneicB16-F10 melanoma cells causes a significant increase intumor growth when compared withmice treated with vehiclealone (Fig. 1B). To examine the effect of cyclosporin A ontumor angiogenesis, we immunostained tumors with anti-CD31 identifying a pronounced increase in the microvesseldensity of cyclosporin A–treated tumors (Fig. 1C). The totalnumber of vessels was higher in cyclosporin A–treated tumorswith numerous short CD31þ vessels than in untreatedcontrols. However, the number of mature lumenized vesselstructures did not change and vessels larger than 100mmwereonly moderately increased in cyclosporin A–treated tumors(Fig. 1D). This characterization of tumor angiogenesis sug-gests that cyclosporin A treatment may promote the earlystages of vessel formation.

Cyclosporin A promotes endothelial cell proliferationand migration in vitroTo investigate the effect of cyclosporin A on endothelial

cell activation, primary microvascular LuEC were isolatedfrom wild-type mice and treated with cyclosporin A orvehicle alone. Cyclosporin A treatment increases LuECnumber andBrdUrd incorporation as comparedwith vehicle

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alone (Fig. 2A andB). Because cyclosporin A has been shownto decrease apoptosis under UV or calcium overload stress(8), we assayed cyclosporin A–treated LuEC for apoptosis.After treatment with either cyclosporin A or vehicle for72 hours in normal culture conditions and in the absence ofany apoptotic inducers, we found no differences in TUNELstaining (Fig. 2C). Pretreatment of LuECwith cyclosporin Aalso shows increased migration through a Transwell filtertoward cyclosporin A in comparison to vehicle-treated cellstoward basal media (Fig. 2D). Taken together, these dataindicate that cyclosporin A promotes endothelial cell pro-liferation and migration in vitro.

Calcineurin is not required for cyclosporin A–inducedproliferation and migrationBecause calcineurin is downstream of VEGF signaling

in endothelial cells, inhibition of the calcineurin pathwayis expected to decrease tumor angiogenesis, as demon-strated by overexpression of the endogenous calcineurininhibitor Dscr1 (10). Despite being a potent calcineurininhibitor, cyclosporin A treatment instead positively reg-ulates tumor angiogenesis and endothelial cell activation.Therefore, we set out to investigate the calcineurindependence of the endothelial phenotypes inducedby cyclosporin A. Cyclosporin A treatment of LuEC-

Figure 1. Cyclosporin A promotes tumor angiogenesis and tumor growth. A, quantification of chemically induced papillomas in mice treated with eithercyclosporin A (CsA) or vehicle. Representative images are shown on the right with papillomas highlighted in red. n ¼ 5 mice per group, results shownare representative of 2 independent experiments. B, tumor growth over time of subcutaneous B16-F10 melanoma tumors in mice treated with CsA orvehicle. Data are shown as the mean � SEM, with n ¼ 3–5 mice per group. Representative results of 2 independent experiments are shown.C, immunofluorescent images of B16-F10 tumor sections stained for the endothelial cell marker CD31 (red) and the DNA stain Hoechst (blue). Scale bar,100 mm. D, quantification of microvessel density, total vessels, lumenized vessels, and large (�100 mm) vessels in anti-CD31–stained tumor sections.Data are shown as mean � SD. Statistical analysis was performed by the Student t test. �, P < 0.05.

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overexpressing DSCR1 still increases endothelial cellgrowth, despite the fact that DSCR1 overexpression alonedecreases endothelial cell proliferation compared withwild-type (Fig. 3A). Furthermore, treatment of B16-F10 tumor–bearing mice with FK506, an independentcalcineurin inhibitor, showed no increase tumor growthcompared with mice treated with vehicle alone (Supple-

mentary Fig. S1A). FK506 treatment of endothelial cellsin vitro decreases endothelial cell growth (SupplementaryFig. S1B) and VEGF-induced migration (SupplementaryFig. S1C) as compared with cyclosporin A treatment orvehicle alone. These data suggest a differential and nonover-lapping mechanism by which these calcineurin inhibitorsregulate endothelial cell activity.

Figure 2. CyclosporinA (CsA)promotesendothelial cell proliferationandmigration in vitro. A, proliferationof primary LuECwithCsAor vehicle,n¼3per group.B,representative imagesofBrdUrduptakebyLuEC treatedwith 0.25mmol/LCsA, 5ng/mLEGF,or vehicle for 48hours.QuantificationofBrdUrdþ cells is shownonthe right. C, quantification of TUNELþ LuEC after treatment with vehicle or CsA for 72 hours or doxorubicin (Dox) for 24 hours, n¼ 5 per group. D, representativeimages of LuEC migration through a Transwell filter in response to vehicle or 0.25 mmol/L CsA treatment. 100 ng/mL FGF is used as a positive control. Scalebar, 100 mm. Quantification of cells per high-power field (hpf) from 8 to 10 randomly taken images is shown on the bottom. Representative results of3 independent experiments are shown. Data is presented as mean � SD. Statistical analysis was performed by the Student t test. �, P < 0.05.






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To dissect out the immunosuppressive effects of cyclo-sporin A from its regulation of the mitochondrial perme-ability transition pore, we used the nonimmunosuppressivecyclosporin analogue NIM811, which binds to cyclophilinsbut does not interact with calcineurin (29). We find thattreatment with either NIM811 or cyclosporin A results in asimilar dose-dependent response in endothelial cell numbers(Fig. 3B). Both compounds also induce comparable levels ofLuEC proliferation and migration (Fig. 3C and D), suggest-ing a calcineurin-independent mechanism by which cyclo-sporin A and NIM811 affect endothelial cell activation.To further examine the requirement for calcineurin in

cyclosporin A–mediated endothelial cell activation, we delet-ed the calcineurin B subunit in Calcineurin Bf/f LuECthrough adenovirus-Cre infection. Upon genetic deletionof Calcineurin B and the subsequent degradation of calci-neurin A (Supplementary Fig. S2) leading to loss of calci-neurin function (24), treatment with cyclosporin A stillincreases endothelial cell proliferation (Fig. 3E and F). Ourstudies show that deletion of Calcineurin B in LuEC resultsin a near-complete loss of cell migration; however, cyclo-sporin A treatment in combination with the growth factorFGF causes a slight but meaningful increase in migrationwhen compared with untreated cells (Fig. 3G andH). Takentogether, these data indicate that cyclosporin A promotesendothelial cell activation through a calcineurin-indepen-dent mechanism.

Cyclosporin A induces ROS release from themitochondriaIn addition to forming an intracellular complex with

cyclophilin A and inhibiting calcineurin function, cyclo-sporin A also binds to another cyclophilin family member,cyclophilin D (30), at a similar affinity as cyclophilin A (31).Because our data show comparable effects between cyclo-sporin A and NIM811, a nonimmunosuppressive cyclo-sporin analogue, we hypothesize that the mechanism bywhich cyclosporin A and NIM811 affect endothelial cellactivation is similar. NIM811 binds to other intracellularcyclophilins and in particular cyclophilin D, a regulatorysubunit of the MPTP (19). Thus, we examined whethercyclosporin A–mediated endothelial effects occur throughthe mitochondrial-localized cyclophilin D.Binding of cyclosporin A to cyclophilinD has been shown

to affect MPTP activity, including the release of ROS fromthe mitochondria in the cytosol, either directly through poreopening (31, 32) or indirectly through changes inmembranepotential (33). Release of mitochondrial ROS can stimulate

mitogenic pathways in tumor and stromal cells (22, 34).Staining of cyclosporin A–treated LuECwith the mitochon-drial superoxide marker Mitosox (8, 20) shows increasedfluorescence compared with vehicle-treated cells (Fig. 4A),indicating cyclosporin A treatment increases mitochondrialROS. Similarly, treatment withNIM811 results in increasedMitosox fluorescence (Fig. 4B), consistent with the notionthat cyclosporin A increase is acting on themitochondria in acalcineurin-independent manner to increase mitochondrialROS production in endothelial cells.Binding of cyclosporin A to cyclophilinD has been shown

to desensitize MPTP opening by increasing the stimulusthreshold required to open the pore, thus decreasingMPTP-mediated cell death (8, 30). In the absence of apoptoticstimuli, the MPTP has been described to transiently open,regulating mitochondrial membrane potential and affectingROS production, among other things (35). This transientMPTP opening is sensitive to cyclosporin A treatment (35).Thus, it is possible that cyclosporin A may increase mito-chondrial ROS production by altering MPTP-regulatedmitochondrial membrane potential. TMRE is a fluorescentlipophilic cation that accumulates in the mitochondria indirect proportion to the mitochondrial membrane potential.High levels of TMRE fluorescence would indicate a highmitochondrial membrane potential. We examined TMREstaining of LuEC after cyclosporin A treatment and found adramatic increase in TMRE fluorescence (Fig. 4C), indi-cating a change in the mitochondrial membrane potential.Treatment with LuEC with the nonimmunosuppressivecyclosporin analogue NIM811 also increases TMRE stain-ing similar to cyclosporin A treatment (Fig. 4D).Our studieswith Mitosox and TMRE lend support to our hypothesiswhereby cyclosporin A can activate endothelial cells throughits interaction with mitochondrial cyclophilin D and in acalcineurin-independent manner.

Antioxidant treatment abolishes cyclosporin A–inducedendothelial cell activationOne method to inhibit cellular or mitochondrial-derived

ROS is to treat cells with antioxidants, a class of drugs thatquench intracellular ROS by either boosting endogenousantioxidant systems or scavenging ROS directly. Co-treat-ment of cyclosporin A with the cellular antioxidantNAC decreases cyclosporin A–induced LuEC prolifera-tion in vitro (Fig. 5A). NAC treatment alone had no effecton either wild-type or Dscr1 transgenic LuEC. Similarly,NAC treatment also blocks cyclosporin A–inducedLuEC migration (Fig. 5B). Co-treatment with the

Figure 3. Calcineurin is not required for cyclosporin A (CsA)-inducedendothelial cell proliferation andmigration. A, proliferation ofwild-type (WT) alone orDscr1transgenic (Tg) LuEC after treatment with CsA or vehicle. B, proliferation of WT LuEC for 6 days in response to vehicle alone or increasing concentrations ofCsA or NIM811, n ¼ 4. C, proliferation of WT LuEC treated with CsA, NIM811, or vehicle. D, representative images of WT LuEC migration through aTranswell filter in response to 0.25 mmol/L CsA, 0.1 mmol/L NIM811, or vehicle alone. Scale bar, 100 mm. Quantification of cells per high-power field (hpf) isshown on the bottom right. Representative results are shown from 2 independent experiments. E and F, proliferation of uninfected (E) or adenovirus-Cre–infected (F)Calcineurin Bf/f (CnBf/f) LuEC in the presence of CsA or vehicle alone. G andH, representative images of uninfected (G) or adenovirus-Cre–infected(H) CnBf/f LuEC migration through a Transwell filter after treatment with 0.25 mmol/L CsA, 100 ng/mL FGF, CsA þ FGF, or vehicle alone. Scale bar,100 mm. Quantification of cells per hpf is shown on the bottom. A–G, n ¼ 3–4 per group, representative result of 2 independent experiments is shown.Data are shown as mean � SD. Statistical analysis was performed by the Student t test. �, P < 0.05.






mitochondrially permeant superoxide dismutase mimeticand peroxynitrate scavenger, manganese(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; 36) also

attenuates cyclosporin A–induced proliferation and migra-tion (Fig. 5C and D). These data support the notionof cyclosporin A treatment, triggering an increase in

Figure 4. Cyclosporin A (CsA)targets the mitochondria toincrease ROS in vitro. A and B,Mitosox fluorescence in LuECeither unstained or treated withvehicle, (A) 0.25 mmol/L CsA, or(B) 0.1mmol/LNIM811 for 24 hours.Mitosox fluorescence wasquantified by flow cytometry withmean fluorescence intensity (MFI)shown on the right (A). C and D,TMRE fluorescence in LuEC eitherunstained or treated with vehicle,(C) 0.25 mmol/L CsA or(D) 0.1 mmol/L NIM811 for24 hours. TMRE fluorescencewas quantified by flow cytometrywith MFI shown on the right(C). n ¼ 4–5 per group, data areshown as mean � SD. Statisticalanalysis was performed by theStudent t test.

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mitochondrial ROS. Mitosox staining confirms thatMnTBAP co-treatment with cyclosporin A reduces mito-chondrial-generated ROS (Fig. 5E). Both pretreatmentand simultaneous co-treatment of mice with the clinicallyused antioxidant NAC decreases B16-F10 tumor growthin animals on cyclosporin A therapy (Fig. 5F), demon-strating that quenching ROS levels protects against cyclo-sporin A–induced tumorigenesis in vivo.

Cyclosporin A–induced ROS activates ERK signalingModerate increases in cellular ROS levels have been

shown to activate mitogenic signals (22, 37, 38). Manytriggers of ROS have been identified, including oncogenicRas signaling, which has been shown to elevate mitochon-drial ROS, an important contributor for anchorage-inde-pendent growth (22). Conversely, various components ofthe Ras pathway have been demonstrated to be redox-sensitive. Phosphatases that regulate MAPK signaling, suchas the ERK1/2-directed phosphatases, can be inactivated byelevated intracellular ROS, thereby prolonging ERK1/2phosphorylation (38). Cyclosporin A treatment of LuECspecifically increases ERK1/2 phosphorylationwith no effecton AKT phosphorylation (Fig. 6A). Antioxidant pretreat-ment with NAC blocks cyclosporin A–induced ERK phos-phorylation (Fig. 6B). While ERK activation during normalgrowth is transient, cyclosporin A treatment shows sustainedERK1/2 phosphorylation as compared with vehicle alone(Fig. 6C). These data suggest that one mechanism by whichcyclosporin A–mediated increased ROS levels promoteendothelial activation is through increasedMAPK signaling.

DiscussionThe use of cyclosporin A in immunosuppressive therapy

for transplant recipients has long been associated with anincreased cancer incidence (1).Here, we identify a previouslyundescribed mechanism whereby cyclosporin A promotestumor growth through its activation of tumor angiogenesisin a calcineurin-independentmanner. Our data indicate thatcyclosporin A increases endothelial cell proliferation andmigration by increasing mitochondrial ROS levels. Antiox-idant treatment mitigated cyclosporin A–induced tumori-genesis in mouse models, identifying a potential therapeuticintervention for cyclosporin A–associated tumorigenesis.Previous studies investigating the mechanism underlying

the increased cancer incidence with cyclosporin A treatmentfocused on the effect of cyclosporin A on tumor cells. Inresponse to UV irradiation, cyclosporin A suppresses DNArepair genes and apoptosis in keratinocytes (7, 8), allowingUV-induced DNA damage to go unrepaired while escapingcell death. Another study examined oncogenic Ras-drivenskin lesions and found that cyclosporin A treatment inducedATF3 expression, blocking p53-dependent senescence thusevading a crucial tumor-suppressive mechanism in a cancercell autonomousmanner (9). These data offer an explanationfor the high incidence of skin cancers observed in patients oncyclosporin A regimens. Furthermore, in the presence ofcyclosporin A, renal carcinoma cells were found to secrete

TGFb, inducing an invasive cancer phenotype with pro-nounced metastasis (6). Cyclosporin A–associated cancers,in addition to their high metastatic potential, grow rapidlydespite the presumed tumor-initiating events, such as chron-ic sun exposure, occurring years or decades before treatmentonset (3). These data suggest that cyclosporin A may alsoplay a role in tumor promotion.Systemic cyclosporin A treatment has also been proposed

to affect tumor vasculature. Cyclosporin A treatment hasbeen shown to induce VEGF (39) and heme oxygenase-1(40, 41) production in tumor cells, which signals to thesurrounding tumor microenvironment to promote angio-genesis. Furthermore, treatment of endothelial cells in vitrowith low levels of cyclosporin A can be cytoprotective againstnutrient deprivation (42). The consequences of cyclosporinA treatment directly on endothelial cells in a tumorigeniccontext, however, have not been extensively investigated.In this work, we used a well-characterized and clinically

relevant skin tumor model with long-term cyclosporin Atreatment. Our data demonstrate a significant increase intumor angiogenesis with cyclosporin A treatment resultingin an abundance of small, short vessels in tumors, andpreservation of large lumenized vessels, suggesting thatcyclosporin A promotes the formation of immature vessels.The early stages of angiogenesis are characterized by degra-dation of the extracellular space, followed by migration andproliferation of endothelial cells to generate new vessels.Newangiogenic structures are pruned and stabilized throughpericyte coverage, resulting in mature vessels (43). Thisconcerted process is an infrequent event in adults and istightly regulated by angiogenic factors to ensure properneovascularization. Tumor vessels are often disorganized,abnormal, and inefficient due to imbalances in angiogenicregulators (44). An increase in tumor microvessel density,even when composed mostly of small or immature vessels,can aid in nutrient and oxygen delivery. In vitro studiesconfirmed a proproliferative and promigratory effect ofcyclosporin A treatment on primary endothelial cell cultures.Variations in batches of primary endothelial cell cultures aredue, in part, to isolation technique and passage numberamong other factors and can translate to differences in rawdata for endothelial cell activation assays. This was notable inour migration assays thus data were analyzed as fold changerelative to day 1 as well as by differences in raw numbers withconclusions drawn from consistent changes observed whendata were analyzed by multiple methods.Studies from our laboratory and others have shown that

inhibition of calcineurin by overexpression of its endogenousinhibitor DSCR1, or by deletion of calcineurin, negativelyregulates endothelial cell activation (10, 14, 45). The calci-neurin pathway in endothelial cells lies downstream ofVEGFR2 activation and transmits angiogenic signalsthrough one of its effectors, the NFAT family of transcrip-tion factors (10, 11, 27). We have previously shown thatcalcineurin inhibition in endothelial cells by DSCR1 blockstumor angiogenesis (10, 14). Given cyclosporin A is a potentcalcineurin inhibitor, we would predict that cyclosporin Atreatment would also block tumor angiogenesis. In contrast,






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our data show a significant increase in tumor angiogenesis incyclosporin A–treated tumor-bearing mice, implicatingthe possibility that the proangiogenic effect of cyclosporinA may be calcineurin independent. To prove this concept,we compared the effect of cyclosporin A on endothelialcells with a nonimmunosuppressive cyclosporin analogueNIM811 that does not bind calcineurin. Despite its inabilityto inhibit calcineurin, NIM811 acts similarly to cyclosporinA by increasing endothelial cell proliferation and migration.To further confirm the calcineurin-independent effects ofcyclosporinA on endothelial cell activation, we examined theproliferation and migration of calcineurin-null endothelialcells after cyclosporin A treatment. We found that endo-thelial activation after cyclosporin A treatment was similar inwild-type and calcineurin-null endothelial cells. Despitebeing a downstream mediator of VEGF signaling, prolifer-ation in vitro was preserved in calcineurin-null endothelialcells, possibly due to signaling through calcineurin-indepen-dent VEGF pathways as well as other proangiogenic path-ways. Calcineurin signaling has also been implicated intumor cell migration (46), and its deletion results in thenear-complete loss of migration in vitro, an outcome likelymade more pronounced by the serum and growth factordepletion conditions used in the migration assay.While cyclosporin A preferentially binds to cyclophilin A

forming a calcineurin inhibitory complex, it also binds toother intracellular cyclophilins, a family of peptidyl-prolylisomerases with diverse yet poorly understood functions(16, 17). Binding of cyclosporin A to other cyclophilinfamily members can result in additional cellular effects thatare calcineurin-independent. Cyclosporin A is known tobind to cyclophilin D, a mitochondrial-localized cyclophilinand part of the MPTP. During calcium and oxidative stress,cyclosporin A limits superoxide release by inhibiting high-conductive irreversible MPTP opening (30) but has alsobeen shown in other situations to increase mitochondrialROS levels (8, 33, 34). Under basal, or nonapoptoticconditions, the MPTP has also been found to open brieflyin transient "superoxide flashes" (35, 47), or "low-conduc-tive" openings, which do not result in mitochondrial swell-ing or massive release of mitochondrial contents. This "low-conductive" MPTP opening has possible roles in mitochon-drial-cytosol crosstalk or cellular metabolism, and its open-ing temporarily lowers themembrane potential, serving as anoverflow mechanism to maintain mitochondrial membranepotential (47, 48). Therefore, regulation of the MPTP bycyclosporin A can have very different outcomes dependingon pore state. Published studies show that cyclosporin Aincreases the apoptotic threshold in response to calcium or

oxidative stress by inhibiting irreversible MPTP opening (8)and prevents the release of damaging levels of mitochondrialcontent, including ROS, into the cytosol. In the absence ofapoptotic stimuli, cyclosporin A increases the mitochondrialmembrane potential (33), possibly through the inhibition of"low-conductance"MPTPopening, which is associatedwithincreased mitochondrial ROS generation (49). Under non-apoptotic conditions, our data shows that cyclosporin Atreatment of endothelial cells increases the mitochondrialmembrane potential and mitochondrial superoxide levels.At higher concentrations (>5.0 mmol/L), cyclosporin A hasalso been shown to inactivate a major mitochondrial super-oxide detoxifier, resulting in damaging levels of mitochon-drial ROS and consequent vascular damage (33). While thedose of cyclosporin A used in our study is within a clinicallyrelevant range and the effects are proangiogenic, it remains tobe determined whether the increased mitochondrial ROS isdue to increased membrane potential or inactivation ofan antioxidant enzyme. Future studies examining the cel-lular consequences of cyclosporin A binding to other intra-cellular cyclophilins will be important to determine the fullrange of cyclosporin A effects on endothelial cells.While high levels of ROS are associated with lipid perox-

idation,DNAdamage, and ultimately cell death, low levels ofROS can act as physiologic signaling molecules in a variety ofpathways (50). In particular, mitogenic pathways have beendirectly linked to ROS (37), a process that has been co-optedby cancer cells. In oncogenic Kras-driven tumors, mitochon-drial-derived ROS is necessary to maintain anchorage-inde-pendent growth and modulate ERK phosphorylation to aproliferative level (22). The vascular system, in particular, ishighly regulated by changes in oxidative and nitration states(20). A number of angiogenic pathways are redox-sensitive,including theMAPK pathway and endothelial cell migratorypathways. We show that cyclosporin A treatment in endo-thelial cells increases ERK1/2 phosphorylation in a ROS-dependent manner. Treatment with antioxidants abolishedcyclosporin A–induced endothelial ERK activation, prolifer-ation, and migration. Antioxidant treatment of tumor-bear-ing mice also decreased cyclosporin A–induced tumorgrowth. Collectively, our data suggest that neutralization ofROS with antioxidants during cyclosporin A therapy may bean effective prophylactic treatment to decrease cyclosporinA–associated malignancies.Our study highlights a previously undescribedmechanism

for increased tumorigenesis after long-term cyclosporin Atreatment. We show that cyclosporin A acts in a calcineurin-independent manner to induce endothelial cell activationand tumor angiogenesis by increasing mitochondrial ROS

Figure 5. Antioxidant treatment abolishes cyclosporin A (CsA)-induced endothelial cell activation and tumorigenesis. A and C, proliferation of LuEC aftertreatment with CsA plus (A) 5 mmol/L NAC, or (C) 10 mmol/L MnTBAP. n ¼ 3 per group, with representative results of 2 independent experiments shown.B and D, representative images of LuEC migration through a Transwell filter in response to 0.25 mmol/L CsA, (B) 5 mmol/L NAC, (D) 10 mmol/L MnTBAP,or vehicle. Scale bar: (B) 50 mm, (C) 100 mm. Quantification of cells per high-power field (hpf) is shown on the right. Representative results of 2 independentexperiments are shown. E, quantification of Mitosox fluorescence (mean fluorescence intensity; MFI) in LuEC treated with 0.25 mmol/L CsA or vehiclefor 24 hours, with or without 10 mmol/L MnTBAP co-treatment. Representative results from 2 independent experiments are shown, with n ¼ 4 per group.F, B16-F10 tumor growth in WT mice � CsA and �NAC. Representative results of 2 independent experiments are shown, with n ¼ 5–9 mice per group.A–E, data are shown as mean � SD. F, data are shown as mean � SEM. Statistical analysis was performed by the Student t test. �, P < 0.05.






Figure 6. Cyclosporin A (CsA)-induced ROS upregulatesMAPK signaling. A,Western blotting of phospho- and total ERK and AKT in LuEC after treatment withCsA or vehicle for 5 minute. Each lane is an individual sample. B and C, Western blotting of phospho- and total ERK in LuEC after treatment with CsA for5 minutes with pretreatment with NAC for 30 minutes followed by CsA or vehicle for 5 minutes (B) or with CsA or vehicle for the indicated time (C). A–C,phosphorylated proteins are normalized to their respective total protein levelswith relative expression levels quantified on the right. Results are representativeof 1 experiment (C, 2–24 hours) or pooled from 3 to 5 experiments (A–C, 5–45 minutes). Data are shown as mean � SD. Statistical analysis was performedby the Student t test. �, P < 0.05.


Zhou and Ryeom




release. Importantly, our work demonstrates that cyclospor-in A–induced tumor angiogenesis is calcineurin-indepen-dent, thus preserving calcineurin inhibition as a feasibletarget for antiangiogenic therapy. Furthermore, our datasuggest thatmanipulatingmitochondrial ROSmay be usefulin not only cyclosporin A–associated malignancies but alsofor the development of new antiangiogenic therapies for allsolid tumors.

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

Authors' ContributionsConception and design: A.Y. Zhou, S.W. RyeomDevelopment of methodology: A.Y. ZhouAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A.Y. ZhouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): A.Y. Zhou, S.W. Ryeom

Writing, review, and/or revision of the manuscript: A.Y. Zhou, S.W. RyeomStudy supervision: S.W. Ryeom

AcknowledgmentsThe authors thank all the members of the Ryeom laboratory for their input,

especially Keri Schadler, Dong Ha Bhang, David Poon, and Diana Blidarescu. Theyalso thank David Feldser, Alan Diehl, DougWallace, Todd Ridky,Meenhard Herlyn,and Mitchell Weiss for scientific discussion and input and Novartis Pharmaceuticalsfor providing NIM811.

Grant SupportThis work was funded by NIH grant R01 CA118374 (S.W. Ryeom), The

Garrett B. Smith Foundation (S.W. Ryeom), and the TedDriven Foundation(S.W. Ryeom).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received March 12, 2014; revised June 18, 2014; accepted June 22, 2014;published OnlineFirst July 9, 2014.

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2014;12:1663-1676. Published OnlineFirst July 9, 2014.Mol Cancer Res Alice Yao Zhou and Sandra Ryeom Reactive Oxygen SpeciesCalcineurin-Independent Manner by Increasing Mitochondrial Cyclosporin A Promotes Tumor Angiogenesis in a

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