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Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-1388

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apeutics, Targets, and Chemical Biology

720 (Fingolimod) Sensitizes Prostate Cancer Cells to

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iotherapy by Inhibition of Sphingosine Kinase-1

i Pchejetski1,4,5, Torsten Bohler2, Leyre Brizuela4, Lysann Sauer1, Nicolas Doumerc3, Muriel Golzio4,5,
l Salunkhe1, Justin Teissié4,5, Bernard Malavaud3,4,5, Jonathan Waxman1, and Olivier Cuvillier3,4,5
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iotherapy is widely used as a radical treatment for prostate cancer, but curative treatments are elusiveorly differentiated tumors where survival is just 15% at 15 years. Dose escalation improves local responseut is limited by tolerance in normal tissues. A sphingosine analogue, FTY720 (fingolimod), a drug cur-in phase III studies for treatment of multiple sclerosis, has been found to be a potent apoptosis inducerstate cancer cells. Using in vitro and in vivo approaches, we analyzed the impact of FTY720 on sphingo-etabolism in hormone-refractory metastatic prostate cancer cells and evaluated its potential as a radio-izer on cell lines and prostate tumor xenografts. In prostate cancer cell lines, FTY720 acted as aosine kinase 1 (SphK1) inhibitor that induced prostate cancer cell apoptosis in a manner independentingosine-1-phosphate receptors. In contrast, γ irradiation did not affect SphK1 activity in prostatecells yet synergized with FTY720 to inhibit SphK1. In mice bearing orthotopic or s.c. prostate cancers, we show that FTY720 dramatically increased radiotherapeutic sensitivity, reducing tumor growth and
tumor
metastasis without toxic side effects. Our findings suggest that low, well-tolerated doses of FTY720 could offersignificant improvement to the clinical treatment of prostate cancer. Cancer Res; 70(21); 8651–61. ©2010 AACR.

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he western world prostate cancer is now the mostonly diagnosed noncutaneous cancer in men and iscond leading cause of cancer-related death (1). In theStates the lifetime probability of developing prostateis 1 in 6, and it is estimated that 192,280 new cases ofte cancer were diagnosed during 2009, and there weredeaths.management of prostate cancer is complex, but forajority of patients radiotherapy remains a definitiveent for early-stage disease. However, up to 85% of pa-with poor histologic subtypes of localized prostater relapse (2). In radiotherapy research the current

computerized planning to deliver increaseds. There is an opportunity to approach the

have bthermsociat(11), aThe

a thersine, wcancefirmeditor, Sproapour grSphK1apy ancer thFTY

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ns: 1Department of Surgery and Cancer, Imperialondon, United Kingdom; 2INSERM U858/I2MR EQil, 3Hôpital Rangueil, Service d'Urologie et deénale, 4CNRS, Institut de Pharmacologie et de

rale; and 5Université de Toulouse, UPS, IPBS,

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

thors: Dmitry Pshezhetskiy, 807 Cyclotron Building,ital, DucaneRoadW120NN, London,UnitedKingdom.929; Fax: 02083835830; E-mail: d.pshezhetskiy@ivier Cuvillier, 205Route deNarbonne, 31077 Toulouse,[email protected].

5472.CAN-10-1388

ssociation for Cancer Research.

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Research. on February 28, 20cancerres.aacrjournals.org ed from

t of dose intensification from a different angle, and thisugh the use of radiosensitizer treatment.state cancer cell radioresistance has been linked withned activation of sphingosine kinase-1 (SphK1; ref. 3).is a lipid converting enzyme responsible for the con-

n of sphingosine into sphingosine-1-phosphate (S1P).nd sphingosine and its precursor ceramide are lipidd messengers. In response to various stimuli ceramidehingosine mediate cell death, whereas S1P abrogatesosis and mediates cell proliferation and migration (4).SphK1/S1P pathway contributes to cancer progressionads to increased cell proliferation (5), impairment ofosis (6), and oncogenic transformation (7). SphK1 is a-associated enzyme: high levels of SphK1 expressioneen shown in various human tumor tissues (8, 9). Fur-ore high levels of SphK1 expression and activity are as-ed with a poor prognosis in breast cancer (10), gliomand gastric cancer (12).se findings have highlighted the potential of SphK1 asapy target. A sphingosine analogue, dimethylsphingo-as the first reported SphK inhibitor to induce prostater cell apoptosis (3). These findings were further con-by ourselves (13), showing a new selective SphK inhib-KI-II (first reported by French et al.; ref. 9), to haveoptotic properties in prostate cancer cells. Work fromoup in prostate cancer indicates that inhibition of the/S1P pathway has a synergistic effect with chemother-d has the potential to act as a molecular target for can-erapy (14–16).
720 is a sphingosine analogue and a potent immuno-essive drug that induces lymphopenia via an inhibition
8651

21. © 2010 American Association for Cancer

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phocytes' egress from lymphoid organs through its an-st function on the lymphocytes' S1P receptors (17, 18).tly the results of phase III trials have become available,ng significant potential of oral FTY720 (fingolimod) inle sclerosis patients (19, 20).risingly, at higher doses FTY720 has been shown to bent apoptosis inducer in prostate, liver ,and bladderr cell lines (21–23). In a mouse model of melanoma0 inhibited tumor growth and metastasis (24) withoutg detectable toxicity in vital organs. Direct mitochon-amage (25), activation of caspases (21), and dephos-ation of Akt (26) have all been proposed as potentialnisms for FTY720-induced apoptosis. The effects of0 were reported be both S1P receptor dependent (24)dependent (27). In prostate cancer cells FTY720 hashown to be a potent apoptosis inducer (21), whereasal prostate cells exhibited resistance to the drug.0 has been shown to inhibit prostate cancer cell inva-ia downregulation of GTP-bound active form of RhoAnd administration of FTY720 at 10 mg/kg/day reducedowth of prostate CWR22R xenografts in castrated nude29).his current study we identified a novel mechanism of0-induced prostate cancer cell apoptosis. Here wethat FTY720 can act as a SphK1 inhibitor in vitro

n vivo and that SphK1 inhibition is critical for0-induced apoptosis. Sublethal concentrations of0 act as a radiosensitizer in cell lines and in implanteds, reducing tumor metastasis.preliminary data point to the need for clinical testingablish whether FTY720 therapy might provide antage in terms of increased local tumor control tots with prostate cancer treated with radiotherapy.

rials and Methods

nes3; 22Rv1, and DU145 cells were obtained fromche Sammlung von Mikroorganismen und Zellkulturen, and LNCaP-C4-2B cells were from Viromed; PC-3Mhighly metastatic derivative of PC-3 cells as previouslybed (30). Cells were cultured between passages 4 andPMI 1640 containing 10% fetal bovine serum. Cell linesroutinely verified by morphology and growth curveis and were routinely screened for mycoplasma infec-MP0035 Lookout, Sigma). SphK1-overexpressing cells have been described previously (13). All experimentsonducted in the absence of serum at 50% confluenceen treated as indicated in the figure legends.

ntsture medium, serum, antibiotics and Syto 13 were ob-from Invitrogen. Eschecheria coli diacylglycerol kinase,-glucopyranoside, and SKI-II compound were from. [γ-32P]-ATP was purchased from Perkin-Elmer, andgel 60 high-performance TLC plates were from VWR.
0 was obtained from Novartis. Annexin V–FITC wasD. All other chemicals were from Sigma Aldrich.
Graphsented

r Res; 70(21) November 1, 2010

Research. on February 28, 20cancerres.aacrjournals.org ownloaded from

radiation was performed using the IBL 637 irradiatorio International) using a Cesium 137 γ-ray sourceactivity ∼222 TBq) at the dose rate of 1 Gy/minute.

transfectionK1 and S1P1-5 were targeted using siRNA as previ-described (13, 31), along with relevant control siRNA.down was assessed by real-time quantitative reverseription-PCR (qRT-PCR) as previously described (16).

iability, flow cytometry, and staining ofotic nucleil viability, flow cytometry, and staining of apoptotici were measured as previously described using theassay (13), Annexin V–FITC/propidium iodide (PI)g, and Syto13/PI staining (14).

gosine kinase-1 assay and mass measurements ofide, sphingosine 1-phosphate, and sphingosineingosine kinase-1 assay and mass measurements ofide, sphingosine 1-phosphate, and sphingosine weremed as described previously (14, 32).

ime qRT-PCRl-timeqRT-PCRwasperformedas describedpreviously (16).

se-3/7 activitypase-3/7 activitywasmeasured as previously described (14).

rn blot analysisstern blot analysis was performed as described pre-y (16).

al studymal study was performed as previously described (13)., intraprostatic and s.c. human prostate cancer xeno-were established in NMRI/Nu (nu/nu) 7-week-old maley surgical orthotopic implantation or s.c. injection of 1 ×-3/GFP cells. Three weeks after implantation, mice weremized into different groups and treated for two weeks.p. injections of PBS (control), 2.5 mg/kg/day FTY720,ons of γ-irradiation (4 Gy) every 3rd day, or γ-irradiationned with FTY720. Two days after the last treatment, allere euthanized with carbon dioxide asphyxiation forinternal imaging. Tumor area (a) and the small diameterre used to assess tumor volume (v) using the formula× d × 2/3. Primary tumors were then removed, saved,utinely processed for H&E histology to confirm the na-f the disease or were processed for sphingolipid analyses.counts were determined as previously described (33).

representation and statistical analysisstatistical significance of differences between theof two groups was evaluated by unpaired two-sidedt's t test, and the overall significance of multiple groupsaluated by ANOVA. Calculations were performed using
pad Prism Software. Representative images are pre-for the experiments carried out with fluorescence.
Cancer Research



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gosine analogue FTY720 induces apoptosis inate cancer cells by inhibition of SphK1720 induces dose-dependent loss of cell viability in me-c prostate cancer PC-3 cells (Fig. 1A), achieving an IC50

hours of exposure at 5 μmol/L dose. This loss of cellty was preceded by an activation of caspase 3 and/or.5 fold at 6 hours; Fig. 1B) and was followed by anse in apoptotic cells (51 ± 5% at 48 hours; Fig. 1C). Ar decrease in cell viability (Fig. 1D) and an increase inosis and caspase-3 activation (not shown) were ob-in DU145 metastatic prostate cancer cells, in agree-

with previous reports (21, 34).720 is a sphingosine analogue and was previouslyto inhibit enzymatic activity of recombinant SphK1ere we report that FTY720 induced a rapid inhibitionK1 enzymatic activity in PC-3 and DU145 cells (-28 ±d -40 ± 5% in PC-3 and DU145 cells, respectively, atrs; Fig. 2). This inhibition was followed by a decreaseracellular S1P (-18 ± 6% and -31 ± 5% in PC-3 andcells, respectively, at 6 hours) and a more delayed

se in intracellular ceramide (51 ± 10% and 58 ± 5% inand DU145 cells, respectively, at 24 hours). Of note,CR analysis showed that treatment with FTY720 didduce significant changes in the expression of the SphK1uring first 24 hours (Supplementary Fig. S1).
rexpression of SphK1 in PC-3 cells rendered them lessve to FTY720 (65 ± 8% versus 35 ± 6% of l
to a sthe co

ed in triplicate; bars, SE.

acrjournals.org


ty in PC-3/NEO versus PC-3/SphK1 cells, respectively;C). Conversely, the silencing S1P1 to S1P5 receptorsindividually or altogether did not alter FTY720-d PC-3 cell death (Supplementary Fig. S2), suggestingptor-independent mode of action. Treatment with0 also decreased the levels of intracellular phospho-hich, however, was downstream of FTY720-induced1 downregulation because SphK1 overexpressioned the levels of phospho-Akt in FTY720-treated cellse SphK inhibitor SKI-II could per se reduce the levelsspho-Akt (Supplementary Fig. S3). As shown in Fig. 2,0 treatment induced only a late modest increase inellular ceramide. Pretreatment of PC-3 cells withisin B1 (inhibitor of de novo ceramide synthesis),69 (neutral sphingomyelinase inhibitor), or imipra-(acid sphingomyelinase inhibitor) could not abrogate0-induced apoptosis, suggesting that ceramide accu-ion was a consequence of SphK1 inhibition (Supple-ry Fig. S4).

diation induces prostate cancer cell deathendently of SphK1en treated with a single dose of γ-irradiation both PC-3U145 cells were sensitive to 10 Gy treatment, achieving ass of cell viability at ∼72 hours and 48 hours for PC-3U145 cells, respectively (Fig. 3A). In both cell lines ar increase in γ-irradiation dose to 20 Gy did not lead
ignificant increase in cell death (data not shown). Onntrary, both cell lines exhibited a partial resistance when oss of cell
1. FTY720 induces prostate cancerptosis. PC-3 (A–C) and DU145 (D)ere treated with FTY720 at indicatedtrations for the indicated times.lity of PC-3 cells was quantified by MTTn assay. B, caspase-3/7 activity wased using luminescent caspase-GLOte. C, number of apoptotic cells wased by flow cytometry after staining withV/PI. D, cell viability of DU145 cells.mean of four independent experiments

Cancer Res; 70(21) November 1, 2010 8653



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d with lower doses of γ-irradiation (only∼20% loss afterur treatment with 5 Gy in both cell lines; Fig. 3A).ontrast to FTY720, treatment of prostate cancer cells-irradiation did not result in SphK1 inhibition. Con-in DU145 cells, γ-irradiation induced rather an upre-n of SphK1 activity (43 ± 7% and 45 ± 6% by 5 Gy and
respectively, P < 0.05; Fig. 3B), which, however, was notted by SphK1 transcription (Fig. 3C).
succes(Fig. 4



1 inhibition by FTY720 sensitizes prostate cancero γ-irradiationne with previous reports showing that SphK1 inhibitionethylsphingosine or SKI-II could respectively sensitizete cancer cell models to γ-irradiation (3) and chemo-y (17, 18), here we report that 1 to 5 μmol/L FTY720

Figindin irevapotreatimS1PtomandstaempreFTYfoudupexp*, P(P >

sfully sensitized PC-3A and B). Fluoresce

21. © 2010 Americ

2. Sphingosine analogue FTY720in vitro inhibition of SphK1, a decrease

cellular S1P, and SphK1 overexpressionFTY720-induced prostate cancer cellsis. PC-3 (A) and DU145 (B) cells werewith 5 μmol/L FTY720 for the indicatednd SphK1 activity (top), intracellulariddle), and intracellular ceramide (bot-ere measured as described in Materialsthods. C, cell viability of PC-3 cellsverexpressing SphK1 (PC-3/SphK1) orvector (PC-3/NEO) as describedsly (13), and treated with 5 μmol/Lfor indicated time. Columns, mean ofependent experiments performed inte; points, mean of three independentents performed in triplicate; bars, SE..05; **, P < 0.01; ns, not significant

and DU145 cells to γ-irradiationnt activated cell sorter analysis

Cancer Research

an Association for Cancer


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C) and fluorescent microscopy (Supplementary Fig. S5)ed that this radiosensitization was mediated by anion of apoptosis. Isobologram analysis revealed that inell lines FTY720 and γ-irradiation act in synergy (Sup-ntary Fig. S6). Similarly to FTY720, the SphK inhibitor(Fig. 4D) and SphK1 siRNA, but not SphK2 siRNA (Sup-ntary Fig. S7), sensitized PC-3 and DU145 (not shown)te cancer cells to γ-irradiation.re 5 shows that the addition of FTY720 to PC-3 cellsted to γ-irradiation significantly exacerbated the inhi-of SphK1 activity (-46.0 ± 5.7% versus 0.2 ± 6.5%, P =), comparable with the levels in cells treated with0 alone (-27.5 ± 3.1%; Fig. 5A). Similarly, the addition720 decreased the levels of intracellular S1P in irradi-C-3 cells (Fig. 5A). There was no significant differenceen the levels of intracellular ceramide in cells subjectedtments alone or in combination (Fig. 5A).U145, γ-irradiation induced an increase in both SphK1y and intracellular S1P (43.0 ± 6.1% and 36.3 ± 3.6%,tively), which was completely reversed by the addition720 (-16.6 ± 4.8% and -19.7 ± 8.2%; Fig. 5B). In contrast
-3 cells, combination of FTY720 and γ-irradiationy increased intracellular ceramide (Fig. 5B).
with γ

.05; ns, not significant (P > 0.05).

acrjournals.org


data show that in contrast to a modest increase inide, 6 hours of treatment with FTY720 induced a sig-t, transient increase in intracellular sphingosine (Sup-ntary Fig. S8A). Furthermore, although γ-irradiationt influence sphingosine production, it acted in synergyTY720 (Supplementary Fig. S8A). We then verified thetial of proapoptotic sphingolipids as radiosensitizers.ementary Figure S8B shows that both sphingosinea smaller extent ceramide could sensitize PC-3 cells

rradiation. Although extracellular ceramide acted as aensitizer (Supplementary Fig. S8B), blocking the gener-of intracellular ceramide had no effect on PC-3 cellal (Supplementary Fig. S9).ilarly to blocking PC-3 cell apoptosis induced by FTY720(Fig. 2), SphK1 overexpression abrogated FTY720-ed radiosensitization (Supplementary Fig. S10A). Thisated with a significant decrease in SphK1 inhibitiond by FTY720 in PC-3 cells overexpressing SPhK1 (Sup-ntary Fig. S10B). Similarly to SphK1 overexpression,dition of extracellular S1P partially blocked PC-3 cellafter treatment with either FTY720 or FTY720 together
-irradiation (Supplementary Fig. S11). Although FTY720
alone induced a rapid increase in caspase-3/7 activity (Fig. 1B),

3. γ-Irradiation induces PC-3 and DU145th independently of SphK1. PC-3 andcells were irradiated once with 5 ornd were left to grow for the indicated, cell viability of PC-3 (left) and DU145ells was assessed by MTT assay.K1 activity of PC-3 (left) and DU145ells treated with 5 and 10 Gy. C, relativeion of the SphK1 gene in PC-3 andcells treated with 5 and 10 Gy forrs was analyzed using qRT-PCR.columns, mean of three independentents performed in triplicate; bars, SE;




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ned treatment with FTY720 and γ-irradiation did notin a further activation of executioner caspases (Supple-ry Fig. S12A). Moreover, addition of a pan-caspase in-r, Z-vad.fmk, did not result in a significant protectionTY720- or combination treatment-induced cell deathlementary Fig. S12B). Finally, to verify the universality720-induced radiosensitization, we treated androgen-ve metastatic LNCaP-C4-2B, PC-3M, and nonmetastaticprostate cancer cells with FTY720 with or withoutdiation (Supplementary Fig. S13). Our data show that0 acted as a radiosensitizer in all three cell lines.

0 radiosensitizes human fluorescent PC-3 tumorsished in nude micecutaneous and orthotopic PC-3 tumors were inocu-in nude mice and grown for three weeks. These ani-were then treated for two weeks with i.p. injectionsS (control), 2.5 mg/kg/day FTY720, 5 sessions ofdiation (4 Gy) every 3rd day, or γ-irradiation com-with FTY720.r 5 weeks s.c. tumors in nontreated animals reached± 388 mm3, whereas in animals treated with FTY720their volume was 1,355 ± 149 mm3 (P = 0.0043).diation alone diminished the tumor volume to 870 ±

3 (P < 0.0001 versus control; P = 0.0007 versus0), whereas combined treatment led to the most signif-

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eduction in tumor volume at 390 ± 70 mm3 (P < 0.0001γ-irradiation; ANOVA; P < 0.0001; Fig. 6A).

hotopic PC-3 tumors showed a similar response toeatments: 469 ± 41 mg, 281 ± 26 mg, 185 ± 13 mg,4 ± 12 mg in mice treated with control, FTY720,diation, or combined treatment respectively (P <, ANOVA; P < 0.0001, t-test γ-irradiation versus com-treatment; Fig. 6B).re 6C shows that although γ-irradiation alone did notcantly affect the levels of tumor SphK1 activity (100 ±sus 89.5 ± 7.9, not significant; in animals receiving con-γ-irradiation, respectively), treatment with FTY720 orbination of FTY720 with γ-irradiation significantly re-SphK1 activity (76.3 ± 6.6 versus 56.2 ± 9.4 in animalsd with FTY720 or a combination of FTY720 withdiation, respectively). Levels of tumor S1P were lowerals treated with combination therapy than in animals

ing γ-irradiation alone. The difference between intra-r ceramide levels in all four groups did not reachcance (Fig. 6C).effect of combination therapy on primary tumor

h was paralleled by a significant reduction of distanttasis (adrenal, liver, and lungs, excluding aortic lymphas being a primary site for prostate cancer metastasis;

FigandCelwithindexpCelindFTYPCwasC,afteassAnnandindperSE.***,(P >aloP vcom

e in all animals) with 63% (5 ot metastases (compared with 1

21. © 2010 American Associ

4. FTY720 sensitizes PC-3145 cells to γ-irradiation.ere pretreated for 1 hourwithout FTY720 or SKI-II atd concentrations andd to 5 Gy γ-irradiation.ere then cultured for thed time in presence ofor SKI-II. Cell viability of, D) and DU145 (B) cellsasured by MTT assay.ber of apoptotic PC-3 cellshours of incubation was

ed by flow cytometry aftern V/PI staining. Columnsints, mean of threedent experimentsed in triplicate; bars,< 0.05; **, P < 0.01;0.001; ns, not significant5). Top P values, FTY720ersus irradiation; lowers, FTY720 alone versus

f 8) of animals free of00% in both untreated

Cancer Research

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Y-treated groups, and 25% in irradiated animals, withmetastases-free; Fig. 6D).rescent imaging revealed that FTY720 alone did not af-e number of secondary tumors (3.33 ± 0.44 versus 3.14 ±etastases/mouse in control and FTY720 groups, respec-P not significant), whereas γ-irradiation significantlyd the number of distant metastases (1.43 ± 0.29 metas-mouse). Combination treatment further reduced theer of distant tumors (0.55 ± 0.23 metastases/mouse,366 in comparison with γ-irradiation alone; Fig. 6D).

0 and γ-irradiation reduce WBC counts inmiceude mice a single 2.5 mg/kg dose of FTY720 induced aeduction of leukocyte blood counts which within 3 dayslized to control levels (Supplementary Fig. S14A). Toy the impact of prolonged FTY720 treatment on bloodunts, blood was analyzed at the time of sacrifice (twofter the last treatment with FTY720). Mice treated with0 alone had a moderate ∼30% reduction in total circu-leukocytes and lymphocytes, and no changes were
ved in peripheral macrophages and granulocyteslementary Fig. S14B), as reported previously (36, 37).
cytopexiste

ant (P > 0.05).

acrjournals.org


ssion

his current study we provide compelling evidence that0 is a SphK1 inhibitor and a potent radiosensitizer ofn prostate cancer both in vitro and in vivo.eral lines of evidence suggest that FTY720 has an anti-rative potential in prostate cancer cells. FTY720 wasusly reported to induce apoptosis in DU145 cells4). Here we show for the first time that FTY720 is ainducer of apoptosis in hormone-refractory, meta-

prostate cancer PC-3 cells. FTY720-induced apoptosis-3 cells was dose dependent and was preceded bycant upregulation of caspase 3 and 7 activity (Fig. 1).720 has been previously reported to inhibit SphK1(35). In our study we show for the first time that

0 has a capacity to inhibit SphK1 enzymatic activityo (Fig. 2) and that this inhibition is not related to ation in SphK1 expression (Supplementary Fig. S1A).inhibition seems to be a crucial step in FTY720-

ed prostate cancer apoptosis, because SphK1 over-ssion exerts a cytoprotective effect (Fig. 2). This
rotection is partial, however, which indicates thence of parallel, SphK1-independent mechanisms of
5. FTY720 radiosensitizes PC-3 andcells by SphK1 inhibition. PC-3 (A) and(B) cells were pretreated for 1 hourmol/L FTY720 and exposed to 5 Gy

ation. Cells were incubated for 24 hours,hK1 activity (top), S1P (middle), ande content (bottom) were measured.s, mean of three independentents performed in triplicate; bars, SE..05; **, P < 0.01; ***, P < 0.001; ns, not




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0-induced prostate cancer apoptosis. We verified theement of several previously suggested mechanisms of

ber of distant metastases (lungs, liver, pancreas, mesenteric and kidney) p0.01; ***, P < 0.001; ns, not significant (P > 0.05).

0-induced cancer cell apoptosis in our system. Inst to La Montagne et al. (24), knockdown of S1P re-

action(26), w



s showed that FTY720-induced cell death was S1Por independent, suggesting an intracellular mode of

mal. Columns, points, means of 8 animals; bars, SE. *, P < 0.05;

6. FTY720 radiosensitizes human fluorescent PC-3 tumors established in nude mice. Three weeks after tumor implantation, mice were randomizedr groups and subjected to daily i.p. injections of 2.5 mg/kg FTY720 (total dose 35 mg/kg), 4 Gy radiotherapy every 3 days (total dose 20 Gy),ination of these treatments, or a sham treatment by i.p. injections. A, volume of s.c. tumor measured with a caliper at weekly intervals. B, tumor massed primary green fluorescent protein (GFP)-labeled tumor (top) and representative fluorescent primary prostate tumors at the time of autopsyoups treated with sham (Cont), FTY720, γ-irradiation (Irr), and a combination of FTY720 and γ-irradiation (bottom). Red arrows, primary orthotopicmors. C, SphK1 activity (top), and S1P (middle) and ceramide (bottom) levels were measured in tissue extracts obtained from primary tumors.

(Supplementary Fig. S2). Similarly to Azuma et al.e found that FTY720 induced a downregulation of

Cancer Research



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hosphorylation (Supplementary Fig. S3). This effectphK1 dependent, however, because it could be mim-by SphK1 inhibition and restored by SphK1 overex-on. Finally, our data show that FTY720-mediatedide accumulation was not responsible for FTY720-d apoptosis because pretreatment of prostate cancerith inhibitors of sphingomyelinases and de novo cer-synthesis pathway did not abrogate FTY720-inducedsis (Supplementary Fig. S4). Overall our data suggestK1-mediated S1PR-independent mechanism of pros-ancer cell apoptosis induced by FTY720.ingolipids have previously been shown to play a rolestate cancer radioresistance. Radioresistant prostater cell lines are deficient in the generation of pro-otic ceramide and sphingosine, and the addition orsed production of these lipids is radiosensitizing (3,). In contrast to completely radioresistant, androgen-ive LNCaP prostate cancer cells, both DU145 andormone-refractory metastatic prostate cancer cells re-to γ-irradiation in a dose-dependent manner. Withmind, one of the major aims of our study was to

igate possible therapeutic dose intensification. Inst to several DNA-damaging therapies that were pre-y shown to downregulate SphK1 activity (13, 40, 41),e report that in both PC-3 and DU145 metastaticte cancer cell lines γ-irradiation–induced apoptosisot associated with SphK1 inhibition (Fig. 3) or anse in intracellular ceramide (Fig. 5). Interestingly incells, irradiation even induced a slight activationK1 (but not through transcription), an effect ob-in several cancer cell lines treated with DNA-

ing agents (13, 42). We therefore conclude that intatic prostate cancer cells γ-irradiation does not reg-the SphK1/S1P pathway, which suggests that its ex-manipulation may provide benefit in terms of doseification.have shown that SK1 inhibition leads to a promo-f the effects of cytotoxic chemotherapy on cell lines4, 16). Although metastatic prostate cancer cells arelly responsive to γ-irradiation, sustained SphK1 ac-may provide an “escape from apoptosis” for theseWe hypothesized that SphK1 inhibition may, in ad-to its effects on chemotherapy, serve as potentialensitizer. Here we show for the first time that0 can sensitize PC-3, DU145, LNCaP-C4-2B, PC-3M,2Rv1 prostate cancer cells to irradiation by apoptosision in a dose-dependent fashion (Fig. 4, and Supple-ry Figs. S5, S6, and S13). Our data suggest that0-mediated radiosensitization is mediated by SphK1tion (Fig. 5) and is similar to the effects that can beed by the SphK inhibitor SKI-II or SphK1 siRNA, Supplementary Fig. S7). Conversely, the radiosensi-n can be blocked by SphK1 overexpression (Supple-ry Fig. S10) or extracellular S1P (Supplementary11). Interestingly, although FTY720 induced only aincrease in ceramide (Fig. 5), it induced a significant
ent increase in intracellular sphingosine (Supplemen-ig. S8A) acting in synergy with γ-irradiation. As
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usly reported (3), both sphingosine and ceramidect as radiosensitizers (Supplementary Fig. S8B), buting the generation of intracellular ceramide hasfect on PC-3 cell survival (Supplementary Fig. S9),ming that its low intracellular levels are not suffi-to induce prostate cancer cell death. Our data showTY720-induced radiosensitization is caspase inde-nt (Supplementary Fig. S12), suggesting that the de-n of prosurvival signaling (e.g., Akt, SphK1/S1P) maye major mechanism of FTY720-induced apoptosis/ensitization.s implies that although γ-irradiation can inducete cancer cell death, its effect can be potentiateddownregulation of SphK1 activity. These data corre-ith our previous findings on SphK1 downregulationtiating the effects of docetaxel chemotherapy in pros-ancer (14, 16). These results are also in line with ourus findings in which SphK1 inhibition by dimethyl-osine was shown to sensitize completely radioresis-ndrogen-sensitive LNCaP prostate cancer cells todiation (3).efficacy of FTY720 in animal cancer models includingte cancer was shown previously (29, 43). Here we showe first time that FTY720 can radiosensitize hormone-tory metastatic human prostate cancer tumors estab-in nude mice. Our in vivo data show that treatmentTY720 alone decreased the size of both s.c. and ortho-tumors (Fig. 6). Furthermore, cotreatment with FTY720d in a further reduction in both orthotopic and s.c. tu-treated with γ-irradiation. In our models the efficacy of0 was correlated to a decrease in tumor SphK1 activity1P levels (Fig. 6C).the selected dose (2.5 mg/kg/day), treatment with0 alone had only a moderate effect on primary tumorh (Fig. 6). In contrast to previous data showing theof FTY720 to block cell invasion in prostate cancer

dimensional cell cultures (28), in our model treatmentTY720 alone did not influence the number of distanttases. Surprisingly γ-irradiation alone led to a signifi-eduction in the number of distant metastases, whichrther enhanced by cotreatment with FTY720, suggest-synergetic mechanism of action between FTY720 anddiation.accordance with previous studies, prolonged treat-with 2.5 mg/kg/day FTY720 led to a moderate lym-nia (Supplementary Fig. S14B). The major reportedlication of FTY720 is a transient decrease in therate due to a “first dose” effect, and no toxic effectseen observed in healthy volunteers treated for 7cutive days with 5 mg/day FTY720 (44, 45). At thedose (2.5 mg/kg/day) we did not observe any toxic-gross abnormalities (like weight loss or i.p. inflam-n) in animals treated with FTY720. It is worththat until recently no clinically tested SphK1 inhi-were available. Conversely, recent data from phaseals show clinical safety and significant potential of
TY720 (fingolimod) in multiple sclerosis patients0).



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sitizes hormone-resistant prostate cancer to docetaxel. Int Jncer 2009;125:2728–36.iba K. FTY720, a new class of immunomodulator, inhibits lympho-

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Support

Royal Society (grant P24841 to D. Pchejetski), The Prostate Cancer(grant 110630 to D. Pchejetski), Institut National du Cancer (O. Cuvillier),ational de la Recherche Scientifique (O. Cuvillier) Institut National de lade la Recherche Médicale and Ministère de la Santé (Interface Program,lier), and Association pour la Recherche sur le Cancer (O. Cuvillier).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 04/26/2010; revised 07/22/2010; accepted 08/09/2010; publishedirst 10/19/2010.

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2010;70:8651-8661. Published OnlineFirst October 19, 2010.Cancer Res Dmitri Pchejetski, Torsten Bohler, Leyre Brizuela, et al. Radiotherapy by Inhibition of Sphingosine Kinase-1FTY720 (Fingolimod) Sensitizes Prostate Cancer Cells to

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