Cancer Biology and Signal Transduction

FTY720 (Fingolimod) Inhibits HIF1 and HIF2Signaling, Promotes Vascular Remodeling, andChemosensitizes in Renal Cell CarcinomaAnimal ModelC�ecile Gstalder1,2,3, Isabelle Ader1,2,3, and Olivier Cuvillier1,2,3

Abstract

Clear cell renal cell carcinoma (ccRCC) is characterized byintratumoral hypoxia and chemoresistance. The hypoxia-induc-ible factors HIF1a and HIF2a play a crucial role in ccRCCinitiation and progression. We previously identified the sphin-gosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathwayas a newmodulator ofHIF1a andHIF2aunder hypoxia in variouscancer cell models. Here, we report that FTY720, an inhibitor ofthe S1P signaling pathway, inhibits both HIF1a and HIF2aaccumulation in several human cancer cell lines. In a ccRCCheterotopic xenograft model, we show that FTY720 transientlydecreases HIF1a and HIF2a intratumoral level and modifiestumor vessel architecture within 5 days of treatment, suggesting

a vascular normalization. In mice bearing subcutaneous ccRCCtumor, FTY720 and a gemcitabine-based chemotherapy alonedisplay a limited effect, whereas, in combination, there is asignificant effect on tumor size without toxicity. Noteworthy,administration of FTY720 for 5 days before chemotherapy isnot associated with a more effective tumor control, suggesting amode of action mainly independent of the vascular remodel-ing. In conclusion, these findings demonstrate that FTY720could successfully sensitize ccRCC to chemotherapy and estab-lish this molecule as a potent therapeutic agent for ccRCCtreatment, independently of drug scheduling. Mol Cancer Ther;15(10); 2465–74. �2016 AACR.

IntroductionClear cell renal cell carcinoma (ccRCC) accounts for around 3%

of adult cancers. Its prognosis is closely related with disease stage,with 5-year survival rates ranging from90% to 10% for stage I andstage IV disease, respectively. The last decade has witnessed majoradvances in the understanding of ccRCCbiology, and a number ofeffective treatments are now available, such as antiangiogenicagents andmTOR inhibitors (1, 2). However, the development ofacquired resistance to these targeted therapies has heightened theneed for continued investigation of novel approaches to ccRCCmanagement. Because numerous studies have exhibited themajorrole of hypoxia-inducible factors HIF1a and HIF2a, master reg-ulators of cell response to hypoxia, in ccRCC initiation andprogression (3–5), their inhibition may represent a potent ther-apeutic strategy. HIF transcription factors control the expressionof numerous target genes contributing to tumor angiogenesis,invasion, metastasis, and therapeutic failure (6). In tumors,HIF1a andHIF2a stimulate the production of angiogenic factors,such as VEGF, an independent prognostic marker in ccRCC (7),

leading to an unrestricted angiogenesis that generates a chaoticvascular network with compromised tumor blood perfusion,resulting in a lack of oxygen delivery, thereby exacerbating tumorhypoxia and fueling a self-reinforcing vicious circle (8). As thedirect inhibition of HIF transcription factors appears to be achallenging task (9), targeting upstream signaling could representa more practical strategy.

Sphingolipids constitute a class of bioactive lipids that regu-lates many fundamental biological processes (10). Among them,ceramide and sphingosine trigger antiproliferative and apoptoticresponses, whereas sphingosine 1-phosphate (S1P) stimulatescell proliferation, survival, migration, inflammation, and angio-genesis (10). The ceramide/S1P balance, which is crucial fordetermining whether a cell survives or dies (11), is mostly underthe control of sphingosine kinase 1 (SphK1) isoform that phos-phorylates sphingosine to form S1P. The S1P produced intracel-lularly is secreted to exert paracrine or autocrine effects as a ligandfor five specific high-affinity G protein–coupled receptors (GPCR)known as S1P1–5 (12), which differ in their tissue distribution,and the specific tumorigenic and angiogenic effects, depending onthe repertoire of S1P receptor subtypes expressed (10). AlternateGPCR-independent signaling of S1P also exists, with recent stud-ies establishing direct modulation of intracellular proteins (10).

In cancer, S1P metabolism is often dysregulated with over-expression of SphK1 that generally correlates with resistance totherapeutics and significant decrease in survival rate in patients(13). Recent studies have shown that patients with ccRCC showSphK1 overexpression in their tumors and elevated plasma S1Plevels (14, 15). These data support the hypothesis that targetingSphK1/S1P signaling pathway represents a relevant target forccRCC therapy (16). We previously identified the SphK1/S1Psignaling as a new modulator of HIF1a under hypoxia in vitro

1CNRS, Institut de Pharmacologie et deBiologie Structurale,Toulouse,France. 2Universit�e de Toulouse, UPS, Toulouse, France. 3EquipeLabellis�ee Ligue contre le Cancer, Paris, France.

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

Corresponding Authors: Olivier Cuvillier, CNRS, 205 Route de Narbonne, BP64182, Toulouse 31077, France. Phone: 335-6117-5513: Fax: 335-6117-5871; E-mail:olivier.cuvillier@ipbs.fr; and Isabelle Ader, STROMALab, EFS, INP-ENVT, InsermU1031, UPS, Toulouse 31432, France. E-mail: isabelle.ader-perarnau@inserm.fr

doi: 10.1158/1535-7163.MCT-16-0167

�2016 American Association for Cancer Research.

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and in vivo using several cancer cell models (17, 18). Lately, weestablished that the SphK1/S1P signaling regulates HIF2a underhypoxia in multiple cancer cell lines, including 786-O, CAKI-1,and A498 ccRCC, and demonstrated the specific involvement ofthe S1P1 receptor in the regulation of both HIF1a and HIF2aexpression and activity (19). Collectively, these data establish thatthe SphK1/S1P/S1P1 signaling is a key regulator of the adaptiveresponse to hypoxia in cancer, including ccRCC, and suggest thattargeting the SphK1/S1P/S1P1 pathway could represent a perti-nent strategy to control intratumoral hypoxia and tumor growthin cancer, especially in ccRCC.

FTY720 (fingolimod; ref. 20) is an analogue of sphingosinethat has been approved by the FDA in 2010 for the treatment ofrelapsing-remitting multiple sclerosis after two phase III clinicalstudies establishing its efficacy and global safety (21, 22). It canbe phosphorylated in vivo to form FTY720-phosphate (FTY720-P), a mimetic of S1P, interacting with several S1P receptorsbut preferentially inducing internalization and degradation ofS1P1 (23). Some of its actions are also attributed to its unpho-sphorylated form, notably inhibition of SphK1 activity assuggested by several independent studies (24–26). Antitumoralproperties of FTY720 have already been evidenced in severalcancer animal models (25, 27–30).

Here, we report that the inhibition of the SphK1/S1P/S1P1pathway using FTY720 decreases HIF1a and HIF2a intratumorallevel, which is associated with vascular remodeling and tumoroxygenation in a ccRCCmousemodel. We also show that FTY720sensitizes our ccRCC model to gemcitabine yet independently ofvascular remodeling. FTY720 is the only FDA-approved drugtargeting S1P metabolism; thus, we suggest that its use representsa promising therapeutic strategy to inhibit ccRCC tumor growthby sensitizing to chemotherapy.

Materials and MethodsChemicals and reagents

Culture medium was from Life Technologies. Serum was fromPerbio. FTY720 was from Enzo Life Science. Gemcitabine wassupplied by Hopital Rangueil. hVEGF plasma levels were quan-tified using a human VEGF ELISA Kit (Life Technologies) accord-ing to the manufacturer's instructions. All other reagents werefrom Sigma.

Cell linesHumanPC-3 prostate cancer cell linewas obtained fromDSMZ

in 2002. Human CAKI-1 ccRCC cell line was purchased fromATCC in 2009. Human A498 ccRCC cell line was kindly suppliedbyDrG.Melillo (NCI, Frederick,MD) in2008.Cellswere culturedin RPMI containing 10% FBS at 37�C in 5% CO2-humidifiedincubators. Cell lines were routinely verified by the followingtests: morphology examination, growth analysis, and mycoplas-ma detection (MycoAlert, Lonza). All experiments were startedwith low-passaged cells (<15 times). Hypoxia (0.1%O2, 5%CO2,94.5% N2) was achieved using an Invivo2 Hypoxic Workstation.

Western blot analysis and antibodiesMouse anti-HIF1a (BDBiosciences), rabbit anti-HIF2a (Novus),

and rabbit anti-S1P1 (Novus) were used as primary antibodies.Proteinswere visualizedbyan enhanced chemiluminescence detec-tion system (GEHealthcare) using anti-mouse or anti-rabbit horse-radish peroxidase–conjugated IgG (Bio-Rad). Equal loading ofprotein was confirmed by probing the blots with anti-a-tubulin

antibody (Santa Cruz Biotechnology). Densitometry quantitationwas determined using ImageJ software (NIH, Bethesda, MD).

SphK1 enzymatic assayThe protocol for the determination of SphK1 enzymatic activity

has been described in details previously (31).

AnimalsFemale NMRI/Nu (nu/nu) 6-week-old mice were obtained

from Elevage Janvier. Mice were housed in a barrier facility ofhigh-efficiency particulate air–filtered racks. At 7 weeks of age, theanimals were used in accordance with the principles and proce-dures outlined in Council Directive 86/809/EEC. The InstitutF�ed�eratif de Recherche Bio-m�edicale de Toulouse (Toulouse,France) and the Animal Care and Use Committee approved allanimal studies.

Heterotopic implantation of CAKI-1 ccRCC cells and miceexperimentation

Subcutaneous ccRCC xenografts were established in nudemiceby heterotopic implantation. Donor mice were anesthetized byisoflurane inhalation, and 106 CAKI-1 cells were injected intotheir right flank. After 6 weeks of tumor growth, donor mice weresacrificed, tumors harvested and divided in 2 mm3 CAKI-1 tumorfragments, and then implanted into the right flank of recipientmice. Tumor volume was measured weekly using a caliper. Twoweeks after tumor fragment implantation, recipient mice wererandomized into different groups and were treated with FTY720(or DMSO as a control) and/or gemcitabine (or PBS as a control).

Blood and plasma samplesBlood samples were analyzed using the ABX Micros 60 Hema-

tology Analyzer, and analytic parameters were determined inplasma by routine laboratory methods using an autoanalyzer(Cobas Mira) at the Plateforme Ph�enotypage ANEXPLO.

Immunostaining, IHC, and immunofluorescenceStaining was conducted on paraformaldehyde (PFA)-fixed and

paraffin-embedded tissue using 5-mm sections. Intratumoral hyp-oxia was assessed using a commercially available Hypoxyprobe-1Kit for the detection of tissue hypoxia (Hypoxyprobe Inc.). Pimo-nidazole hydrochloride was given at a dose of 60 mg/kg in PBS viaintraperitoneal injection 45 minutes before euthanasia, and het-erotopic tumors were harvested and fixed in 4% PFA. Tumornecrosis was assessed after hematoxylin–eosin staining accordingto the necrotic anatomopathologic criteria that include an eosin-ophilic cytoplasm and a fragmented, compacted, or dissolvednucleus. Details regarding antibodies, dilutions, and antigen-retrieval methods used are provided in Supplementary Table S1.

Image acquisition, processing, and quantificationFor bright-field and fluorescence, slides were scanned with

Nanozoomer 2.0 RS Hamamatsu by the Service Anatomie-Patho-logique et Histologie-Cytologie (Hopital Rangueil, Toulouse,France). A fluorescence imaging module was used for tumor slidesstained with anti-CD34, anti-aSMA, anti-desmin, and anti-Ki67. Absolute numbers of CD34þ vessels present within0.75 mm2 of the tumor area, absolute numbers of cleavedcaspase-3þ cells per 0.75 mm2 area, percentage of aSMA/CD34þ staining per 0.75 mm2 area, percentage of desmin/CD34þ staining per 0.75 mm2 area, and percentage of necroticareas were quantified by optical counting. Automatic cell

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counts of Ki67þ and pimonidazole intensity were determinedper 0.75 mm2 of the tumor area with ImageJ software.

Statistical analysisThe statistical significance of differences between the means

of two groups was evaluated by unpaired Student t test. Allstatistical tests were two-sided, and the level of significance wasset at P < 0.05. Calculations were done using Prism 7 (Graph-Pad Software).

ResultsFTY720 decreases S1P1 expression, SphK1 activity, and inhibitsHIF1a and HIF2a expression under hypoxia in renal andprostate cancer cell lines

We previously reported that the SphK1/S1P/S1P1 pathway is aregulator of both HIF1a and HIF2a during hypoxia in multiple

cancer cell lineages, including prostate cancer and ccRCC(17–19).To evaluate the relevance of inhibiting this pathwaywith regard toHIF1a andHIF2a expression in cancer cells, we relied on FTY720,a drug that antagonizes the S1P1 receptor (23) and inhibitsSphK1 (24–26, 32). In all cell lines tested, FTY720 significantlyreduced both S1P1 protein expression (range, 30%–90%, Fig. 1A)and SphK1 activity (range, 30%–70%, Fig. 1B) within 24 hoursof treatment. In the pseudohypoxic A498 cell line that lacks afunctional VHL gene and expresses only HIF2a (33), FTY720strongly decreased HIF2a expression in normoxic conditions(Fig. 1C). In VHL wild-type CAKI-1 and PC-3 cell lines thatproduce both HIF1a and HIF2a, FTY720 treatment markedlyreduced the expression of these transcription factors underhypoxic conditions (Fig. 1C). These data indicate that inhibi-tion of the SphK1/S1P/S1P1 signaling pathway by FTY720 isassociated with the downregulation of HIF1a and HIF2a con-tent in cancer cells.

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Figure 1.

FTY720 decreases S1P1 expression, SphK1 activity, and HIF1a and HIF2a intracellular level in vitro. A, human A498, CAKI-1, and PC-3 cells were incubated undernormoxia (21% O2) and treated with FTY720 (10 mmol/L) or DMSO (control) for 6 and 24 hours. S1P1 expression was analyzed by immunoblotting using ananti-S1P1 antibody. Similar resultswere obtained in at least three independent experiments, and equal loadingwasmonitoredusing antibody to tubulin. Densitometryquantification is indicated. B, SphK1 enzymatic activity in A498, CAKI-1, and PC-3 cells was measured after 24 hours of treatment with FTY720 (10 mmol/L)or DMSO (control) under normoxia (21%O2). Columns, mean of at least four independent experiments; bars, SEM. �� , P <0.01. C,A498, CAKI-1, and PC-3were treatedwith FTY720 (10 mmol/L) or DMSO (control) for 18 hours, then incubated under normoxia (21% O2) or hypoxia (0.1% O2) for an additional 6 hours. HIF1aand/or HIF2a expression was analyzed by immunoblotting using an anti-HIF1a or anti-HIF2a antibody. Similar results were obtained in at least three independentexperiments, and equal loading was monitored using antibody to tubulin. Densitometry quantification is indicated.

Antagonism of S1P Signaling in ccRCC

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FTY720 transiently decreases HIF1a and HIF2a intratumoralexpression and tumor-dependent VEGF production in vivo

Because FTY720 is already used in human clinics, we nextevaluated its ability to inhibit HIF1a and HIF2a expression invivo. ccRCC initiation and progression are particularly dependenton these transcription factors (5); we thus established a hetero-topic model of ccRCC in nude mice, using CAKI-1 tumor frag-ments. Two weeks after implantation, when tumor volumereached 100 mm3, mice were treated with 2.5 mg/kg per dayFTY720 or DMSO (control) by intraperitoneal injection. Bloodand primary tumors were collected from animals sacrificed at day0 or after 3, 5, 7, or 9 days of treatmentwith FTY720orDMSO. Theexcised tumors were first evaluated by Western blot analysis forHIF1a (Fig. 2A) and HIF2a (Fig. 2B) expression. A remarkabledecrease of both HIF1a and HIF2a protein expression wasobserved when animals are treated with FTY720 for 5 to 7 days(range, 70%–95%) but returned to the control value at day 9,suggesting a transitory effect of FTY720 (Fig. 2A and B).

In addition, animals treated with FTY720 had significantlyreduced levels of circulating proangiogenic human VEGF(hVEGF) produced by the CAKI-1 xenografts (Fig. 2C). Thesein vivo findings demonstrate that FTY720 not only downregulatesHIF1a and HIF2a content but also decreases the secretionof VEGF by the tumor, the expression of which depends on bothHIF-1 and HIF-2 transcriptional activity in ccRCC (34).

FTY720 transiently remodels tumor vasculature and increasestumor oxygenation

As HIF1 and HIF2 control the expression of numerous proan-giogenic factors, such as VEGF, and because ccRCC are known to

be highly vascularized, we next analyzed the effect of FTY720 ontumor vasculature by characterizing the expression of CD34 as abiological marker of blood vessels. Immunohistochemical anal-yses indicated a significant decrease in microvessel density intumors treatedwith FTY720 fromday 5 today 9 (Fig. 3A andB). Inline with a previous study using the mouse corneal pocket assay(28), our data establish, herein, the antiangiogenic activity ofFTY720 in a ccRCCmouse model. We next evaluated the effect ofFTY720 treatment on intratumoral hypoxia. The quantification ofpimonidazole staining revealed that FTY720 triggers a markeddecrease of intratumoral hypoxia from day 5 to day 7 (Fig. 3Cand D). We next characterized the morphology and architectureof tumor blood vessels. Our data show that after 5 days oftreatment with FTY720, the morphology of remaining bloodvessels was clearly different from their untreated tumor counter-parts (Fig. 3A). Hallmarks of abnormal vessels are disruption ofpericyte and endothelial cell contacts, and hypoxic vessels areparticularly characterized by absent or detached pericytes (35).Perivascular pericytes play a critical role in vessel maturation andstabilization (8), and antiangiogenic therapies (e.g., anti-VEGF)have been shown to induce characteristic features of vessel nor-malization, including reduced numbers and size of immaturetumor vessels and increased pericyte coverage (35). A doublestaining for pericytes (aSMA or desmin) and endothelial cells(CD34)was performed toquantify the extent of pericyte coverage.A significant increase of the percentage ofaSMA (Fig. 4A and B) ordesmin (Fig. 4C and D)-positive intratumoral blood vessels wasfound inmice treated with FTY720 for 5 to 7 days, demonstratingthat FTY720 transiently improves the maturation of tumor bloodvessels and potentially their functionality. Collectively, these data

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Figure 2.

Effect of FTY720 treatment on HIF1a and HIF2a intratumoral expression and tumor-dependent VEGF production in vivo. CAKI-1 tumor fragments weresubcutaneously implanted in the right flank of nude mice to form heterotopic xenografts. Fourteen days after injection, mice were treated daily with FTY720(2.5 mg/kg) or DMSO (control) during 10 days. After 0, 3, 5, 7, or 9 days of treatment, mice were sacrificed, and primary tumors and blood were collected. A and B,analysis of HIF1a (A) and HIF2a (B) intratumoral expression by Western blot analysis on fresh tumors harvested after 0, 3, 5, 7, or 9 days of treatment with2.5 mg/kg/day FTY720 (mice # 5–7, 10–12, 15–17, 20–22) or DMSO (mice # 1–4, 8, 9, 13, 14, 18, 19). a-Tubulin was used as a loading control. C, determination ofplasmatic human VEGF concentration (pg/mL) by ELISA assay after 0, 3, 5, 7, or 9 days of treatment with 2.5 mg/kg/day FTY720 (black) or DMSO (white).Columns, mean of 2 (day 0) or 4 (day 3, 5, 7, 9) mice per group; bars, SEM. � , P < 0.05.

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establish for the first time that FTY720 can induce a transientvascular remodeling or vessel normalization, which might beassociated with enhanced efficacy of chemotherapy (8).

FTY720 decreases tumor proliferation and increases cell deathin vivo

As the antiproliferative and proapoptotic properties of FTY720have beenwell established in vivo (25, 27–30, 36), we assessed theability of FTY720 to impact tumor cell proliferation and deathin our ccRCC mouse model. The Ki67 proliferation marker wasused to estimate the fraction of viable cells undergoing activeproliferation within the tumor. FTY720-treated tumors exhibiteda marked decrease in cell proliferation rate throughout the exper-iment time course (Fig. 5A and B). Tumor cell death was firstassessed after hematoxylin–eosin staining by the quantification ofnecrotic areas within the tumor. In agreement with previous invitro studies (37), FTY720 induces a pronounced increase innecrotic cell death at day 5 (Fig. 5C and D). Apoptosis withinthe tumor was determined by quantifying cleaved caspase-3. Asshown in Fig. 5E and F, FTY720 induced apoptosis as reflected bythe increased level of cleaved caspase-3 at day 5 in accordancewith

the reported in vitro studies (25). Collectively, these data dem-onstrate that FTY720 displays antiproliferative properties andtransiently stimulates necrotic and apoptotic cell death in ccRCCxenografts.

FTY720 sensitizes a ccRCC mouse model to gemcitabineindependently of drug scheduling

Abnormal vasculature and intratumoral hypoxia play a criticalrole in therapeutic failure and, notably, chemoresistance, a hall-mark of ccRCC. Preclinical and clinical data support the conceptthat a sound use of antiangiogenic compounds in terms of doseand timing may normalize tumor vascular network, henceimproving tumor oxygenation and drug delivery (8). We hypoth-esized that the vascular normalization induced by FTY720 after 5to 7 days of treatment could sensitize a ccRCC mouse model tochemotherapy. Using a heterotopic model of CAKI-1 tumorfragments in nude mice, we combined FTY720 to gemcitabine,one of the few cytotoxic agents that have shown amodest activityfor RCC in human clinic (38, 39). We studied the therapeuticrelevance of FTY720-induced transient vascular normalization togemcitabine efficacy by varying the sequence of FTY720 and

Figure 3.

Effect of FTY720 treatment onmicrovascular density andintratumoral hypoxia. A,characterization of blood vesselquantity and morphology byimmunofluorescence staining of anendothelial cell marker (CD34, red)and nuclear counterstain (DAPI, blue).Images are representative of tumorsections from 8 animals treated 5 dayswith 2.5 mg/kg/day FTY720 or DMSO.Scale bar, 200 mm. B, number of bloodvessels (CD34þ structures) per 0.75mm2 area after 0, 3, 5, 7, or 9 daysof treatment with 2.5 mg/kg/dayFTY720 (black) or DMSO (white).Columns, mean of 4 (day 0) or 8 (day3, 5, 7, 9) mice per group; bars, SEM.� , P < 0.05; �� , P < 0.01. C, detection ofintratumoral hypoxia areas byimmunohistochemical staining ofpimonidazole adducts (brown) andhematoxylin nuclear counterstain(blue). Images are representative oftumor sections from 8 animals treated5 days with 2.5 mg/kg/day FTY720 orDMSO. Scale bar, 250 mm. D, relativepimonidazole intensity per 0.75 mm2

area after 0, 3, 5, 7, or 9 days oftreatment with 2.5 mg/kg/dayFTY720 (black) or DMSO (white).Columns, mean of 4 (day 0) or 8 (day3, 5, 7, 9) mice per group; bars, SEM.� , P < 0.05.

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gemcitabine administration (Fig. 6A). Our results show thatheterotopic CAKI-1 tumors are not sensitive to gemcitabine(mean size at 28 days ¼ 420 mm3; Fig. 6B and C). Similarly,administration of FTY720 alone was not accompanied with astatistical reduction of tumor volume (mean size at 28 days ¼370 mm3). Of note, significantly smaller tumors were observedin animals treated with a combination of FTY720 and gemci-tabine (arm 4, mean size at 28 days ¼ 237 mm3; arm 5, meansize at 28 days ¼ 252 mm3) as compared with control animals(mean size at 28 days ¼ 532 mm3; Fig. 6B and C). However, nodifferences were noted between animals treated with FTY720prior to gemcitabine (arm 5) and animals treated simulta-neously with FTY720 and gemcitabine (arm 4). These datasuggest that increased tumor perfusion is not a prerequisitefor chemosensitization by FTY720 and do not support thenotion that chemotherapy efficacy could be optimum whengiven during a vascular normalization period in our model. Inaddition to efficacy studies described above, we conducted atoxicology analysis at the end of the experiment to evaluatehow well FTY720 was tolerated in mice. Complete blood countrevealed less toxicity when FTY720 was given prior to gemci-tabine compared with the classical combination. Notably, astatistically significant lymphopenia was only observed in micetreated with the classical combination of FTY720 and gemci-tabine (Supplementary Fig. S1). We also monitored animal

weight throughout the experiment time course and concludedthat treatments were well tolerated as animal weights weresimilar before and after treatments (Fig. 6D).

DiscussionIntratumoral hypoxia is a hallmark of solid tumors and a poor

prognosis marker that promotes tumor aggressiveness by activat-ing the HIF transcription factors (40). In contrast to healthytissues, hypoxia-triggered overproduction of VEGF and otherproangiogenic factors in tumors lead to a structurally and func-tionally abnormal vascular network that enhances intratumoralhypoxia, fueling a self-reinforcing vicious circle and promotingchemoresistance (8). Thus, targeting the HIF-induced abnormalvasculature represents a potent therapeutic strategy. As the direct-ed inhibition of HIF transcription factors is a challenging task (9),targeting upstream signaling, such as the SphK1/S1P pathway,represents a more practical strategy (41). Indeed, we previouslyidentified the SphK1/S1P/S1P1 signaling as a new modulator ofboth HIF1a and HIF2a accumulation and activity under hypoxiain several human tumor cell lines, including ccRCC (17–19).

The therapeutic drug FTY720 (also known as fingolimod andmarketed by Novartis as Gilenya) is a structural analogue ofsphingosine, displaying two major activities depending on itsphosphorylation status (42). When phosphorylated by SphK2

Figure 4.

Effect of FTY720 treatment onpericyte coverage. A and C,characterization of pericyterecruitment on blood vessels byimmunofluorescence staining of twopericyte markers, aSMA (A, green)and desmin (C, green) and anendothelial cell marker (CD34, red).Nuclear counterstain (DAPI, blue).Images are representative of tumorsections from 8 animals treated 5 days(A) or 7 days (C) with 2.5 mg/kg perday FTY720 or DMSO. Scale bar,200 mm. B and D, quantification ofpericyte coverage per 0.75 mm2 areaafter 0, 3, 5, 7, or 9 days of treatmentwith 2.5 mg/kg/day FTY720 (black) orDMSO (white). Columns, mean of4 (day 0) or 8 (day 3, 5, 7, 9) mice pergroup; bars, SEM. � , P < 0.05.

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isoform, FTY720-P binds to S1P receptors (with the exception ofS1P2) yet preferentially to S1P1 (23). Although FTY720-P has aninitial agonist activity, it subsequently causes internalization,thereby reducing S1P1 levels on the cell surface (43). In partbecause of its structural similarity to sphingosine, FTY720 alsoimpacts other component of the sphingolipid pathway, notablySphK1 isoform as being a competitive inhibitor with sphingosineand a noncompetitive inhibitor with ATP (44). In this work,FTY720 treatment in vitro clearly led to downregulation of S1P1expression and inhibition of SphK1 enzymatic activity, althoughthe ratio of FTY720 versus FTY720-P in our cancer cell models isunknown. Previous studies have reported major differences inthis ratio depending on the cell type utilized, likely reflecting thedifferences in levels of SphK2, FTY720-P phosphatases, andFTY720-P transporters (45, 46). As both SphK1 and S1P1 havebeen previously reported to be involved in regulation of HIF1aand HIF2a expression in various cancer lines (17, 19), we sug-gest that the dual effect on SphK1 and S1P1 is responsible forthe strong inhibitory effect of FTY720 on both HIF1a and

HIF2a expression in prostate cancer and ccRCC cells we observ-ed. The mechanistic link between S1P1 signaling and HIF1 andHIF2 transcription factors likely involves Akt signaling as previ-ously established in multiple cancer cell lines (17, 19, 47). Itmight also implicate STAT3 signaling, which has been shownto be a crucial regulator of HIF1-mediated VEGF production,notably, in ccRCC (48–50) and a recently established target ofSphK1/S1P/S1P1 signaling (51).

Importantly, in a heterotopic ccRCC mouse model, treatmentwith FTY720 led to a less hypoxic environment, as shown by thedecrease in HIF1a and HIF2a intratumoral level and tumor-dependent VEGF secretion, an HIF1 and HIF2 target gene, whosebaseline level has been identified as an independent prognosticmarker in two ccRCC-randomized phase III trials (7). As a result,vascular remodeling occurred. As reported for anti-VEGF strate-gies and more recently with anti-S1P strategy (18), FTY720 wasable to normalize intratumoral vasculature yet for a short periodof time. At the concentration of 2.5 mg/kg used in our in vivostudies, FTY720 and FTY720-P are in equal levels in plasma

Figure 5.

Effect of FTY720 treatment on tumor proliferation and cell death in vivo.A, detection of proliferative tumor cells by immunofluorescence staining of Ki67þ cells (red)and nuclear counterstain (DAPI, blue). Images are representative of tumor sections from 8 animals treated 5 days with 2.5 mg/kg/day FTY720 or DMSO.Scale bar, 200 mm. B, percentage of Ki67þ cells per 0.75 mm2 area after 0, 3, 5, 7, or 9 days of treatment with 2.5 mg/kg/day FTY720 (black) or DMSO (white).Columns, mean of 4 (day 0) or 8 (day 3, 5, 7, 9) mice per group; bars, SEM. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. C, detection of tumor necrotic areas byhematoxylin and eosin staining. Images are representative of tumor sections from 8 animals treated 5 days with 2.5 mg/kg/day FTY720 or DMSO. Scale bar,250 mm. D, percentage of necrotic areas, calculated as the necrotic areas compared with the tumor section areas after 0, 3, 5, 7, or 9 days of treatment with2.5mg/kg/dayFTY720 (black) orDMSO(white). Columns,mean of 4 (day0) or 8 (day3, 5, 7, 9)mice per group; bars, SEM. � ,P<0.05.E, characterization of tumor cellapoptosis by immunohistochemical staining of cleaved caspase-3 (brown) and hematoxylin nuclear counterstain (blue). Images are representative of tumorsections from4 animals treated 5 dayswith 2.5mg/kg/day FTY720 or DMSO. Scale bar, 200mm. F, quantification of cleaved caspase-3þ cells per 0.75mm2 area after0, 3, 5, 7, or 9 days of treatment with 2.5 mg/kg/day FTY720 (black) or DMSO (white). Columns, mean of 2 (day 0) or 4 (day 3, 5, 7, 9) mice per group; bars,SEM. �� , P < 0.01.

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(52, 53), suggesting a dual effect on SphK1 and S1P1 in the tumorcompartment, limiting hypoxia and its effects on the vascularcompartment through diminution of VEGF levels. However, S1Phas been shown to stabilize blood vessels in development andhomeostasis through S1P1 (43, 54–56), and FTY720 in its phos-phorylated form might therefore directly limit vascular stabiliza-tion triggered by the antihypoxic effect of FTY720. Anothermechanism, which could explain in our model why vascularnormalization is not as strong and prolonged as with anti-S1Pstrategy we recently reported (18), is the fact that the S1P2 subtypeS1P receptor is upregulated under hypoxic stress (57) and coun-terbalances the effect of S1P1 by increasing vascular permeability

(58). Although neutralization of S1P produced by hypoxic tumorcells by anti-S1P antibody could have a direct impact on vessels byswitching off S1P2-mediated vascular leakage, we could not counton this effect because FTY720-P does not target S1P2.

ccRCCs are notoriously known to be chemoresistant and gen-erally develop an acquired resistance to targeted therapies (59).Because vascular normalization and tumor oxygenation canimprove tumor response to chemotherapy (8), we evaluatedwhether the FTY720-induced vascular normalization could sen-sitize a ccRCC mouse model to gemcitabine. Our data show thatFTY720 displays a chemosensitizing effect yet probably indepen-dent of vascular normalization as FTY720 pretreatment is as

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Figure 6.

FTY720 sensitizes a ccRCC mousemodel to gemcitabine. A, two weeksafter heterotopic implantation ofCAKI-1 tumor fragments, mice wererandomized into five groups of 5 to 7animals each. These animalswere thentreated with daily DMSO (group 1, n ¼6), FTY720 2.5 mg/kg/day starting atday 14 (group 2, n ¼ 6), gemcitabine2 mg/kg twice a week startingday 14 (group 3, n ¼ 5), FTY7202.5 mg/kg/day and gemcitabine 2mg/kg twice a week starting day 14(group 4, n ¼ 7), or FTY7202.5 mg/kg/day prior to gemcitabine2 mg/kg twice a week starting day 19(group 5, n¼ 6). At day 28, mice wereanesthetized and weighed, tumorvolume was measured using a caliper,and blood was taken for completeblood count. B and C, evolution oftumor growth from day 0 to day 28(B), and quantification of final tumorvolume at day 28 (C). ns, notsignificant. Columns, mean of 5 to 7mice per group; bars, SEM. � , P < 0.05.D, evolution of tumor weight from day0 to day 28. Symbols, mean of 5 to 7mice per group; bars, SEM.

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effective as the classical combination. As discussed above, theFTY720-mediated vascular normalization is likely either too shortor tooweak to allow anoptimal delivery of gemcitabine, althoughpretreatment with FTY720 allows to treat with gemcitabine for ashorter time to achieve a similar effect. The in vivo chemosensitiz-ing effect of FTY720 might thus rely on the inhibition of theSphK1/S1P signaling that can impact HIF1a andHIF2a signalingas well as other signaling pathways (not related to HIF1a andHIF2a) known to promote chemoresistance (60, 61) in a tumormodel (ccRCC), where the SphK1/S1P signaling is known to beupregulating in patients (14, 15). Furthermore, FTY720-mediatedeffects in blocking prosurvival signaling by 14-3-3, PKC or PP2Aare likely to substantially augment these effects (41, 62).

Overall, our findings provide a rationale to target the SphK1/S1P/S1P1 signaling pathway with FTY720 by increasing theefficacy of gemcitabine in ccRCC. Indeed, SphK1/S1P inhibitionmight represent an alternative therapeutic strategy for ccRCC thatis resistant to antiangiogenic agents or mTOR inhibitors (14), asS1P is considered as a growth-like factor and a potent protectoragainst apoptosis in addition to its specific regulatory role onHIF1a and HIF2a. FTY720 (fingolimod) is routinely used inhuman clinics for multiple sclerosis, and its toxicity profile iswell described. Because it can target a broad range of processesinvolved in tumorigenesis, including hypoxia as described in thiswork, FTY720 remains a promising anticancer agent that meets anumber of criteria accepted for drug repurposing.

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

Authors' ContributionsConception and design: C. Gstalder, I. Ader, O. CuvillierDevelopment of methodology: C. Gstalder, I. AderAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Gstalder, I. AderAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Gstalder, I. Ader, O. CuvillierWriting, review, and/or revision of the manuscript: C. Gstalder, I. Ader,O. CuvillierAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. Ader, O. CuvillierStudy supervision: I. Ader, O. Cuvillier

Grant SupportThis work was supported by funding from the Ligue Nationale Contre le

Cancer (Equipe Labellis�ee LIGUE 2011; to O. Cuvillier), the Fondation deFrance (to O. Cuvillier), the Bourse Angiogen�ese from Roche (to O. Cuvillier),the Universit�e de Toulouse (to I. Ader), the Fondation pour la RechercheM�edicale (PhD fellowship to C. Gstalder), and the Fondation ARC (PhDfellowship to C. Gstalder).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 23, 2016; revised June 23, 2016; accepted July 7, 2016;published OnlineFirst August 9, 2016.

References1. Albiges L, Salem M, Rini B, Escudier B. Vascular endothelial growth factor-

targeted therapies in advanced renal cell carcinoma. Hematol Oncol ClinNorth Am 2011;25:813–33.

2. Voss MH, Molina AM, Motzer RJ. mTOR inhibitors in advanced renal cellcarcinoma. Hematol Oncol Clin North Am 2011;25:835–52.

3. Kondo K, KimWY, Lechpammer M, KaelinWG Jr. Inhibition of HIF2alphais sufficient to suppress pVHL-defective tumor growth. PLoS Biol 2003;1:E83.

4. Fu L,WangG, ShevchukMM,NanusDM,Gudas LJ. Generation of amousemodel of Von Hippel-Lindau kidney disease leading to renal cancers byexpression of a constitutively active mutant of HIF1alpha. Cancer Res2011;71:6848–56.

5. Keith B, Johnson RS, SimonMC. HIF1alpha and HIF2alpha: sibling rivalryin hypoxic tumour growth and progression. Nat Rev Cancer 2012;12:9–22.

6. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell2012;148:399–408.

7. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Staehler M, et al.Sorafenib for treatment of renal cell carcinoma: final efficacy and safetyresults of the phase III treatment approaches in renal cancer globalevaluation trial. J Clin Oncol 2009;27:3312–8.

8. Carmeliet P, Jain RK. Principles and mechanisms of vessel normalizationfor cancer and other angiogenic diseases. Nat Rev Drug Discov 2011;10:417–27.

9. Onnis B, Rapisarda A, Melillo G. Development of HIF-1 inhibitors forcancer therapy. J Cell Mol Med 2009;13:2780–6.

10. MaceykaM,HarikumarKB,Milstien S, Spiegel S. Sphingosine-1-phosphatesignaling and its role in disease. Trends Cell Biol 2012;22:50–60.

11. Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, et al.Suppression of ceramide-mediated programmed cell death by sphingo-sine-1-phosphate. Nature 1996;381:800–3.

12. Takabe K, Paugh SW, Milstien S, Spiegel S. "Inside-out" signaling ofsphingosine-1-phosphate: therapeutic targets. Pharmacol Rev 2008;60:181–95.

13. Zhang Y, Wang Y, Wan Z, Liu S, Cao Y, Zeng Z, et al. Sphingosine kinase 1and cancer: a systematic review and meta-analysis. PLoS One 2014;9:e90362.

14. Zhang L, Wang X, Bullock AJ, Callea M, Shah H, Song J, et al. Anti-S1Pantibody as a novel therapeutic strategy for VEGFR TKI-resistant renalcancer. Clin Cancer Res 2015;21:1925–34.

15. Salama MF, Carroll B, Adada M, Pulkoski-Gross M, Hannun YA, ObeidLM, et al. A novel role of sphingosine kinase-1 in the invasion andangiogenesis of VHL mutant clear cell renal cell carcinoma. FASEB J2015;29:2803–13.

16. Cuvillier O. Downregulating sphingosine kinase-1 for cancer therapy.Expert Opin Ther Targets 2008;12:1009–20.

17. Ader I, Brizuela L, Bouquerel P, Malavaud B, Cuvillier O. Sphingosinekinase 1: a new modulator of hypoxia inducible factor 1alpha duringhypoxia in human cancer cells. Cancer Res 2008;68:8635–42.

18. Ader I, Gstalder C, Bouquerel P, Golzio M, Andrieu G, Zalvidea S, et al.Neutralizing S1P inhibits intratumoral hypoxia, induces vascular remo-delling and sensitizes to chemotherapy in prostate cancer. Oncotarget2015;6:13803–21.

19. Bouquerel P, Gstalder C, Muller D, Laurent J, Brizuela L, Sabbadini R, et al.Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor2alpha expression and activity in cancer. Oncogenesis 2016;5:e209.

20. Kahan BD. FTY720: a new immunosuppressive agent with novel mecha-nism(s) of action. Transplant Proc 1998;30:2210–3.

21. Cohen JA, Barkhof F, Comi G, Hartung HP, Khatri BO, Montalban X, et al.Oral fingolimod or intramuscular interferon for relapsing multiple scle-rosis. N Engl J Med 2010;362:402–15.

22. Kappos L, Radue EW,O'Connor P, PolmanC,Hohlfeld R, Calabresi P, et al.A placebo-controlled trial of oral fingolimod in relapsing multiple scle-rosis. N Engl J Med 2010;362:387–401.

23. Graler MH, Goetzl EJ. The immunosuppressant FTY720 down-regulatessphingosine 1-phosphate G-protein-coupled receptors. FASEB J 2004;18:551–3.

24. Vessey DA, Kelley M, Zhang J, Li L, Tao R, Karliner JS, et al. Dimethyl-sphingosine and FTY720 inhibit the SK1 form but activate the SK2 form ofsphingosine kinase from rat heart. J BiochemMol Toxicol 2007;21:273–9.

25. Pchejetski D, Boehler T, Brizuela L, Sauer L, Doumerc N, Golzio M, et al.FTY720 (fingolimod) sensitizes prostate cancer cells to radiotherapy byinhibition of sphingosine kinase-1. Cancer Res 2010;70:8651–61.

Antagonism of S1P Signaling in ccRCC

www.aacrjournals.org Mol Cancer Ther; 15(10) October 2016 2473

on January 30, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 9, 2016; DOI: 10.1158/1535-7163.MCT-16-0167

26. Lim KG, Tonelli F, Li Z, Lu X, Bittman R, Pyne S, et al. FTY720 analogues assphingosine kinase 1 inhibitors: enzyme inhibition kinetics, allosterism,proteasomal degradation, and actin rearrangement inMCF-7 breast cancercells. J Biol Chem 2011;286:18633–40.

27. Azuma H, Takahara S, Ichimaru N, Wang JD, Itoh Y, Otsuki Y, et al.Marked prevention of tumor growth and metastasis by a novel immu-nosuppressive agent, FTY720, in mouse breast cancer models. CancerRes 2002;62:1410–9.

28. LaMontagne K, Littlewood-Evans A, Schnell C, O'Reilly T, Wyder L,Sanchez T, et al. Antagonism of sphingosine-1-phosphate receptors byFTY720 inhibits angiogenesis and tumor vascularization. Cancer Res2006;66:221–31.

29. Li MH, Hla T, Ferrer F. FTY720 inhibits tumor growth and enhances thetumor-suppressive effect of topotecan in neuroblastoma by interferingwith the sphingolipid signaling pathway. Pediatr Blood Cancer 2013;60:1418–23.

30. Rosa R, Marciano R, Malapelle U, Formisano L, Nappi L, D'Amato C, et al.Sphingosine kinase 1overexpression contributes to cetuximab resistance inhuman colorectal cancer models. Clin Cancer Res 2013;19:138–47.

31. Brizuela L, Cuvillier O. Biochemical methods for quantifying sphingoli-pids: ceramide, sphingosine, sphingosine kinase-1 activity, and sphingo-sine-1-phosphate. Methods Mol Biol 2012;874:1–20.

32. Lim KG, Tonelli F, Berdyshev E, Gorshkova I, Leclercq T, Pitson SM, et al.Inhibition kinetics and regulation of sphingosine kinase 1 expression inprostate cancer cells: functional differences between sphingosine kinase 1aand 1b. Int J Biochem Cell Biol 2012;44:1457–64.

33. Bertout JA, Majmundar AJ, Gordan JD, Lam JC, Ditsworth D, Keith B, et al.HIF2alpha inhibition promotes p53 pathway activity, tumor cell death,and radiation responses. Proc Natl Acad Sci U S A 2009;106:14391–6.

34. Raval RR, Lau KW, Tran MG, Sowter HM, Mandriota SJ, Li JL, et al.Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol2005;25:5675–86.

35. Goel S, Duda DG, Xu L, Munn LL, Boucher Y, Fukumura D, et al. Nor-malization of the vasculature for treatment of cancer and other diseases.Physiol Rev 2011;91:1071–121.

36. Ubai T, Azuma H, Kotake Y, Inamoto T, Takahara K, Ito Y, et al. FTY720induced Bcl-associated and Fas-independent apoptosis in human renalcancer cells in vitro and significantly reduced in vivo tumor growth inmousexenograft. Anticancer Res 2007;27:75–88.

37. Zhang N, Qi Y, WadhamC,Wang L, Warren A, Di W, et al. FTY720 inducesnecrotic cell death and autophagy in ovarian cancer cells: a protective roleof autophagy. Autophagy 2010;6:1157–67.

38. MertensWC, Eisenhauer EA,MooreM, Venner P, StewartD,Muldal A, et al.Gemcitabine in advanced renal cell carcinoma. A phase II study of theNational Cancer Institute of Canada Clinical Trials Group. Ann Oncol1993;4:331–2.

39. HaasNB, Lin X,Manola J, PinsM, LiuG,McDermott D, et al. A phase II trialof doxorubicin and gemcitabine in renal cell carcinoma with sarcomatoidfeatures: ECOG 8802. Med Oncol 2012;29:761–7.

40. Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinicaloutcome. Cancer Metastasis Rev 2007;26:225–39.

41. Cuvillier O, Ader I, Bouquerel P, Brizuela L, Gstalder C, Malavaud B, et al.Hypoxia, therapeutic resistance, and sphingosine 1-phosphate. AdvCancerRes 2013;117:117–41.

42. Pitman MR, Woodcock JM, Lopez AF, Pitson SM. Molecular targets ofFTY720 (fingolimod). Curr Mol Med 2012;12:1207–19.

43. Oo ML, Chang SH, Thangada S, Wu MT, Rezaul K, Blaho V, et al. Engage-ment of S1P(1)-degradative mechanisms leads to vascular leak in mice. JClin Invest 2011;121:2290–300.

44. Lim KG, Sun C, Bittman R, Pyne NJ, Pyne S. (R)-FTY720 methyl ether isa specific sphingosine kinase 2 inhibitor: Effect on sphingosine kinase 2

expression in HEK 293 cells and actin rearrangement and survival ofMCF-7 breast cancer cells. Cell Signal 2011;23:1590–5.

45. Anada Y, Igarashi Y, Kihara A. The immunomodulator FTY720 is phos-phorylated and released fromplatelets. Eur J Pharmacol 2007;568:106–11.

46. Hisano Y, KobayashiN, Yamaguchi A, Nishi T.Mouse SPNS2 functions as asphingosine-1-phosphate transporter in vascular endothelial cells. PLoSOne 2012;7:e38941.

47. Kalhori V, Kemppainen K, Asghar MY, Bergelin N, Jaakkola P, TornquistK, et al. Sphingosine-1-phosphate as a regulator of hypoxia-inducedfactor-1alpha in thyroid follicular carcinoma cells. PLoS One 2013;8:e66189.

48. Xu Q, Briggs J, Park S, Niu G, Kortylewski M, Zhang S, et al. Targeting Stat3blocks both HIF-1 and VEGF expression induced by multiple oncogenicgrowth signaling pathways. Oncogene 2005;24:5552–60.

49. Jung JE, Lee HG, Cho IH, Chung DH, Yoon SH, Yang YM, et al. STAT3 is apotential modulator of HIF-1-mediated VEGF expression in human renalcarcinoma cells. FASEB J 2005;19:1296–8.

50. Dodd KM, Yang J, Shen MH, Sampson JR, Tee AR. mTORC1 drives HIF-1alpha and VEGF-A signalling viamultiple mechanisms involving 4E-BP1,S6K1 and STAT3. Oncogene 2015;34:2239–50.

51. Liang J, Nagahashi M, Kim EY, Harikumar KB, Yamada A, HuangWC, et al.Sphingosine-1-phosphate links persistent STAT3 activation, chronic intes-tinal inflammation, and development of colitis-associated cancer. CancerCell 2013;23:107–20.

52. Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, et al.Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptoragonists. Science 2002;296:346–9.

53. Sensken SC, Bode C, Graler MH. Accumulation of fingolimod (FTY720) inlymphoid tissues contributes to prolonged efficacy. J Pharmacol Exp Ther2009;328:963–9.

54. Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, Hobson JP, et al. Edg-1, the Gprotein-coupled receptor for sphingosine-1-phosphate, is essential forvascular maturation. J Clin Invest 2000;106:951–61.

55. Chae SS, Paik JH, Furneaux H, Hla T. Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNAinterference. J Clin Invest 2004;114:1082–9.

56. Gaengel K, Niaudet C, Hagikura K, Lavina B,Muhl L, Hofmann JJ, et al. Thesphingosine-1-phosphate receptor S1PR1 restricts sprouting angiogenesisby regulating the interplay between VE-cadherin and VEGFR2. Dev Cell2012;23:587–99.

57. Skoura A, Sanchez T, Claffey K,Mandala SM, Proia RL,Hla T, et al. Essentialrole of sphingosine 1-phosphate receptor 2 in pathological angiogenesis ofthe mouse retina. J Clin Invest 2007;117:2506–16.

58. Du W, Takuwa N, Yoshioka K, Okamoto Y, Gonda K, Sugihara K, et al.S1P(2), the G protein-coupled receptor for sphingosine-1-phosphate,negatively regulates tumor angiogenesis and tumor growth in vivo inmice. Cancer Res 2010;70:772–81.

59. Diamond E, Molina AM, Carbonaro M, Akhtar NH, Giannakakou P,Tagawa ST, et al. Cytotoxic chemotherapy in the treatment of advancedrenal cell carcinoma in the era of targeted therapy. Crit Rev Oncol Hematol2015;96:518–26.

60. Bonhoure E, Pchejetski D, Aouali N, Morjani H, Levade T, Kohama T,et al. Overcoming MDR-associated chemoresistance in HL-60 acutemyeloid leukemia cells by targeting sphingosine kinase-1. Leukemia2006;20:95–102.

61. Pchejetski D, Doumerc N, Golzio M, Naymark M, Teissie J, Kohama T,et al. Chemosensitizing effects of sphingosine kinase-1 inhibition inprostate cancer cell and animal models. Mol Cancer Ther 2008;7:1836–45.

62. White C, Alshaker H, Cooper C, Winkler M, Pchejetski D. The emergingrole of FTY720 (Fingolimod) in cancer treatment. Oncotarget 2016;7:23106–27.

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2016;15:2465-2474. Published OnlineFirst August 9, 2016.Mol Cancer Ther   Cécile Gstalder, Isabelle Ader and Olivier Cuvillier  Carcinoma Animal ModelVascular Remodeling, and Chemosensitizes in Renal Cell FTY720 (Fingolimod) Inhibits HIF1 and HIF2 Signaling, Promotes

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