Estradiol Induces Export of Sphingosine 1-Phosphate fromBreast Cancer Cells via ABCC1 and ABCG2*

Received for publication, September 8, 2009, and in revised form, January 28, 2010 Published, JBC Papers in Press, January 28, 2010, DOI 10.1074/jbc.M109.064162

Kazuaki Takabe‡§, Roger H. Kim‡§, Jeremy C. Allegood§¶, Poulami Mitra§¶, Subramaniam Ramachandran‡§,Masayuki Nagahashi‡§1, Kuzhuvelil B. Harikumar§¶, Nitai C. Hait§¶, Sheldon Milstien§¶, and Sarah Spiegel§¶2

From the ‡Division of Surgical Oncology, Department of Surgery, ¶Department of Biochemistry and Molecular Biology, and§Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298

Sphingosine 1-phosphate (S1P), a potent sphingolipid medi-ator produced by sphingosine kinase isoenzymes (SphK1 andSphK2), regulates diverse cellular processes important forbreast cancer progression acting in an autocrine and/or para-crine manner. Here we show that SphK1, but not SphK2,increased S1P export fromMCF-7 cells.Whereas for both estra-diol (E2) and epidermal growth factor-activated SphK1 and pro-duction of S1P, only E2 stimulated rapid release of S1P anddihy-dro-S1P from MCF-7 cells. E2-induced S1P and dihydro-S1Pexport required estrogen receptor-�, not GPR30, and was sup-pressed either by pharmacological inhibitors or gene silencingofABCC1 (multidrug resistant protein 1) orABCG2 (breast can-cer resistance protein). Inhibiting these transporters alsoblocked E2-induced activation of ERK1/2, indicating that E2activates ERK via downstream signaling of S1P. Taken together,our findings suggest that E2-induced export of S1Pmediated byABCC1 and ABCG2 transporters and consequent activation ofS1P receptors may contribute to nongenomic signaling of E2important for breast cancer pathophysiology.

Sphingosine 1-phosphate (S1P)3 is a potent sphingolipidmetabolite that regulates a number of biological processes crit-ical for breast cancer such as cell growth and survival, move-ment and invasion, angiogenesis, and immunity (1–3). Most ofthe biological effects of S1P are mediated by five specific Gprotein-coupled receptors located on the cell surface (desig-nated S1P1–5) (2, 4). S1P is produced in cells by two closelyrelated sphingosine kinase (SphK) isoenzymes, SphK1 andSphK2, which have distinct localizations and functions (5).SphK1 has been implicated in breast cancer progression,whereas the role of SphK2 still remains unclear (1). Enforcedexpression of SphK1 in estrogen receptor (ER)-positive MCF-7human breast cancer cells significantly increased cell growth in

response to 17�-estradiol (E2) (6), enhanced resistance to anti-cancer drugs (7, 8), and produced larger xenograft tumors withhigher microvessel density in an E2-dependent manner (9).SphK1 is overexpressed in human breast cancer and has beenassociated with tumor angiogenesis and resistance to radiationand chemotherapy (10, 11). Moreover, microarray analyses of1,269 breast tumor samples revealed a worse outcome forpatients with high SphK1 expression (12).SphK1 activity is enhanced by growth factors important for

breast cancer progression, such as epidermal growth factor(EGF) (7) and E2 (6). The physiological actions of E2 mainlyresult from regulation of transcription by estrogen receptorsER-� and ER-� (13). Because ERs are overexpressed in�70% ofbreast cancers, anti-estrogen hormonal therapies alone are aseffective as any other chemotherapeutic drugs for breast cancertreatment (14). E2 also elicits a variety of rapid nongenomicevents mediated by either ER located at the plasma membrane(15), or the seven-transmembrane receptor, GPR30 (16–18).Some of these rapid actions of E2 in breast cancer cells mayresult from stimulation of SphK1, and “inside-out” signaling ofS1P leading to transactivation of EGF receptors (19). Yet, themechanism by which S1P is released from breast cancers isunknown. How S1P generated inside cancer cells is exported tothe outside is an important issue to resolve as it does not spon-taneously traverse the lipid bilayer due to its polar head group.In this study,we show that SphK1, but not SphK2, is responsiblefor production of the S1P that is released from MCF-7 breastcancer cells. Moreover, E2, but not EGF, induces S1P releasealthough both activate SphK1 and generate S1P inside the cell.Furthermore, ABCG2 and ABCC1 are involved in E2-mediatedexport of S1P raising the possibility that secretion of S1P byABC transporters may be a contributing factor to the poorprognosis of patients overexpressing these drug-resistantproteins.

EXPERIMENTAL PROCEDURES

Reagents—S1P andMK571 were from Biomol Research Labo-ratory (Plymouth Meeting, PA). D-erythro-[3-3H]Sphingosine(23 Ci/mmol) and [�-32P]ATP (3000 Ci/mmol) were fromPerkinElmer Life Sciences. E2 and ICI 182,780 were from Sigma.FumitremorginC (FTC)was purchased fromAlexis Biochemicals(San Diego, CA) and GPR30 from Calbiochem/EMD Biosciences(San Diego, CA). Anti-tubulin and anti-ERK2 antibodies werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit poly-clonal antibodies against SphK1 and SphK2were described previ-ously (20, 21). Anti-lamin A/C, anti-ER-�, anti-ABCC1, anti-

* This work was supported, in whole or in part, by National Institutes of HealthGrants R37GM043880 and R01CA61774 (to S. S.), Virginia CommonwealthUniversity Grant BIRCWH K12HD055881, and Susan G. Komen for the CureCareer Catalyst Research Grant KG090510 (to K. T.).

1 Supported by the SUMITOMO Life Social Welfare Services Foundation.2 To whom correspondence should be addressed: Dept. of Biochemistry and

Molecular Biology, Virginia Commonwealth University School of Medicine,Richmond, VA 23298-0614. Tel.: 804-828-9330; Fax: 804-828-8999; E-mail:sspiegel@vcu.edu.

3 The abbreviations used are: S1P, sphingosine 1-phosphate; EGF, epidermalgrowth factor; ER, estrogen receptor; ERK, extracellular signal-regulatedkinase; E2, 17�-estradiol; FTC, fumitremorgin C; LC-ESI-MS/MS, liquid chro-matography electrospray ionization tandem mass spectrometry; siRNA,small interfering RNA; SphK, sphingosine kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10477–10486, April 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ABCG2, and anti-phospho-ERK1/2(Thr202/Tyr204) antibodies were fromCell Signaling (Beverly, MA); anti-phospho-SphK1 (Ser225) was fromExalpha (Maynard, MA); anti-ABCC1 was from Alexis Biochemi-cals (San Diego, CA); and anti-ABCG2 was from Novus Biologicals(Littleton, CO). Secondary anti-bodies were obtained from JacksonImmunoResearch (West Grove,PA). The internal standard mixturefor mass spectrometry containingC17-sphingosine, C17-sphinga-nine, C17-S1P, and C17-dihydro-S1P was obtained fromAvanti PolarLipids (Alabaster, AL).Cell Culture and Transfection—

MCF-7 and MDA-MB-231 humanbreast carcinomacells were obtainedfrom American Type Culture Col-lection (Manassas, VA). MCF-7cells were grown in phenol red-freeimproved minimal essential me-dium (supplemented with 0.25%glucose and 10% fetal bovine serumas described (22)). For down-regula-

FIGURE 1. Expression of SphK1 but not SphK2 increases S1P secretion. MCF-7 cells were transfected with vector, V5-SphK1, or V5-SphK2. A and C, equalamounts of cell lysate proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-V5 antibody. Blots werethen stripped and re-probed with anti-tubulin antibody to demonstrate equal loading. B and D, SphK1 and SphK2 activities in lysates were determined withisoenzyme-specific assays. E and F, vector (open bars), V5-SphK1 (black bars), or V5-SphK2 (gray bars)-transfected MCF-7 cells were incubated for 10 min with [3H]sphin-gosine (1.5 �M, 0.45 �Ci) in serum-free medium, washed extensively, fresh medium was added, and labeled lipids were extracted differentially at the indicated timesfrom medium (F and H) and cells (E and G) into aqueous phases containing [3H]S1P (E and F) and organic phases (G and H) and quantified by scintillation counting asdescribed under “Experimental Procedures.” Data are the mean � S.D. of duplicate determinations. Cellular [3H]S1P is expressed as picomole/million cells and [3H]S1Pin the medium is expressed as picomole/ml secreted by one million cells. *, p � 0.05 compared with vector. Similar results were obtained in two additionalexperiments.

FIGURE 2. Down-regulation of SphK1 but not SphK2 decreases S1P secretion. MCF-7 cells were transfectedwith control siRNA or siRNA targeted to SphK1 or SphK2, as indicated. Equal amounts of lysate or nuclearproteins were immunoblotted with anti-SphK1 (A) or anti-SphK2 (C). Blots were stripped and reprobed withanti-tubulin (A) or lamin (C) to confirm equal loading. B and D, RNA was isolated from duplicate cultures andSphK1 and SphK2 mRNA determined by quantitative real time PCR and normalized to levels of glyceraldehyde-3-phosphate dehydrogenase mRNA. E and F, siControl (open bars), siSphK1 (black bars), or siSphK2 (gray bars)transfected MCF-7 cells were incubated for 10 min with [3H]sphingosine (1.5 �M, 0.45 �Ci) in serum-freemedium, washed extensively, fresh medium was added, and [3H]S1P in cells (E) or secreted into the medium (F)was measured at the indicated times. Data are the mean � S.D. of duplicate determinations. Similar resultswere obtained in two additional experiments. *, p � 0.05 compared with siControl.

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tion with small interfering RNAs (siRNA), MCF-7 cells weretransfected with ON-TARGET plus SMARTpool siRNAs tar-geted to SphK1, SphK2, ABCC1, or ABCG2 or control siRNA(Dharmacon, Lafayette, CO) according to the manufacturer’sinstructions.Quantitative PCR—Total RNA was isolated with TRIzol re-

agent (Invitrogen). RNA was reverse transcribed withSuperScript II (Invitrogen). For real-time PCR, pre-mixedprimer-probe sets were purchased from Applied Biosystems(Foster City, CA) and cDNA was amplified with the ABI Prism7900HT as described (23).Western Blotting—MCF-7 cells were lysed and proteins were

detected by immunoblotting, as described previously (22).SphK Assays—SphK1 and SphK2 activities were determined

exactly as described previously (24). Activity is expressed aspicomole of S1P formed/min/mg of protein.Measurement of Cellular [3H]S1P and Its Release—S1P

release was determined as previously described (25). Brieflycells were labeled for 10 min at 37 °C with [3H]sphingosine (1.5�M, 0.4 �Ci), washed with ice-cold medium, and incubated at37 °C for the indicated times. Lipids were extracted from themedium by addition of chloroform, methanol, and 3 N NaOH(1:1:0.1, v/v/v), followed by vigorous mixing and phase separa-tion. The basic aqueous phase contained S1P, devoid of sphin-

gosine, whereas the majority ofphospholipids were in the organicphase. Cells were washed with coldphosphate-buffered saline, scrapedwith methanol, sonicated on ice,chloroform added, and vortexed. 1M NaCl and 3 N NaOH were addedtomake the final ratios of methanol,chloroform, 1 M NaCl, 3 M NaOH(1:1:1:0.1) (v/v/v/v). Radioactivity in100-�l aliquots of aqueous andorganic phases was determinedby scintillation counting. Cellular[3H]S1Pwas expressed as picomole/million cells based on the specificactivity of [3H]sphingosine. [3H]S1Pin the medium was expressed aspicomole/ml secreted by one mil-lion cells.Measurement of Mass Levels of

Released S1P and Dihydro-S1P byLiquidChromatography-ElectrosprayIonization Tandem Mass Spectrome-try (LC-ESI-MS/MS)—MCF-7 cells(2.5 � 105/well) cultured for 24 h in6-well plates were washed twicewith 2 ml of phosphate-bufferedsaline, and treated as described inthe figure legends. 1-ml aliquots ofmediumwere centrifuged at 2000�g for 10 min. 150 �l of phospha-tase inhibitor mixture containingsodium orthovanadate (2 mM),sodium pyrophosphate (4 mM), and

sodium fluoride (100 mM) were added to 850 �l of supernatantin 13 � 100-mm borosilicate tubes with Teflon-lined caps andmixed with CH3OH (5 ml) and CHCl3 (2.5 ml) and the internalstandard mixture (500 pmol of each sphingolipid in 20 �l ofethanol). After sonication for 30 s, the single-phasemixturewasincubated at 48 °C overnight. 1 MKOH inCH3OH (0.75ml) wasadded followed by incubation in a shaking water bath for 2 h at37 °C to hydrolyze glycerolipids. Extracts were neutralized withglacial acetic acid, 75% transferred to a new tube, and reducedto dryness with a SpeedVac. Dried residues were reconstitutedin 0.3 ml of mobile phase for LC-ESI-MS/MS analysis and cen-trifuged at 16,000 � g for 30 s to remove particulates beforetransfer to autoinjector vials.For LC-ESI-MS/MS analyses, a Shimadzu LC-20 AD binary

pump system coupled to a SIL-20AC autoinjector and DGU20A3degasser interfaced with an ABI 4000 QTrap mass spectrometer(Applied Biosystems) operating in a triple quadrupole mode wasused.Q1andQ3were set topassmolecularlydistinctiveprecursorand product ions (m/z 380.4 to 264.4 for S1P and m/z 382.4 to266.4 for dihydro-S1P), using N2 to collisionally induce dissocia-tions in Q2. Lipids were separated with a binary solvent system ata flow rate of 1.5 ml/min using a Supelco 2.1� 50-mmDiscoveryC18 column (Sigma). Prior to injection of the sample, the columnwas equilibrated for 0.5minwith a solventmixture of 80% solvent

FIGURE 3. Effect of E2 and EGF on SphK1 and S1P formation and secretion. A, MCF-7 cells were stimulatedwith E2 for the indicated times. B, MCF-7 cells were transfected with control siRNA or siRNA targeted to SphK1and stimulated without or with E2. C, MCF-7 cells were stimulated with EGF for the indicated times. A–C, celllysates were prepared, and equal amounts of protein were separated by SDS-PAGE and analyzed by immuno-blotting with anti-phospho-SphK1 antibody. Blots were stripped and re-probed with anti-tubulin to ensureequal loading and transfer. The asterisks indicate nonspecific immunostained bands and the arrowheads indi-cate SphK1. D, MCF-7 cells were stimulated without or with E2 for the indicated times. Cells were lysed andSphK1 activity (circles) was measured with sphingosine added as Triton X-100 mixed micelles and SphK2activity (triangles) with sphingosine added as a bovine serum albumin complex in the presence of 1 M KCl. *, p �0.05 for SphK1 activity at all time points compared with unstimulated (t � 0). E and F, MCF-7 cells were serumstarved overnight, prelabled with [3H]sphingosine (1.5 �M, 0.45 �Ci), and washed extensively, fresh mediumwas added and then stimulated without (vehicle) or with EGF (50 ng/ml) or E2 (10 nM) for 5 min, and the [3H]S1Pin cells (E) or secreted into the medium (F) determined. Data are expressed as mean � S.D. Similar results wereobtained in two independent experiments. *, p � 0.05.

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A (CH3OH/H2O/HCOOH, 58:41:1, v/v/v, containing 5 mM

ammonium formate) and 20% solvent B (CH3OH/HCOOH, 99:1,v/v, containing 5 mM ammonium formate). After injection, theA/B ratiowasmaintained at 80:20 for 0.8min, followed by a lineargradient to 100% B over 1.2 min, held at 100% B for 1.4 min, fol-lowed by a 0.3-min gradient return to 80:20A/B. The columnwasre-equilibrated with 80:20 A/B for 0.5 min before each injection.S1P and dihydro-S1P in the medium was expressed as pico-mole/ml secreted by onemillion cells.ERK1/2 Activation—MCF-7 cells were cultured in 6-well

plates in phenol red-free improved minimal essential/Ham’sF-12 medium (1:1) containing 10% fetal bovine serum. The fol-lowing day cells werewashed once in phosphate-buffered salineand then cultured in fresh phenol red-free, serum-freemediumwith glucose for 3 days as described (26) to minimize basallevels of ERK1/2 phosphorylation.Statistical Analysis—Experiments were repeated at least

three times with consistent results. Statistical differencesbetween groups were determined with unpaired Student’s ttest. p � 0.05 was considered significant.

RESULTS

SphK1, but Not SphK2, Is Involved in Export of S1P byMCF-7Cells—In ER positive MCF-7 breast cancer cells, E2 stimulatesproduction of S1P that has been implicated in tumorigenesis by

activation of its receptors presenton the same or neighboring cells (6,7, 9, 19, 20, 27). However, it is notclear how breast cancer cells secreteS1P or which of the two sphingosinekinase isozymes expressed areinvolved. To address these ques-tions, we utilized a recently devel-oped differential extraction assayto examine [3H]S1P export fromMCF-7 cells overexpressing SphK1or SphK2 (25). In agreement withprevious studies (7, 20), transienttransfection of SphK1 or SphK2caused large increases in isoen-zyme-specific enzymatic activity(Fig. 1, B and D), accompanied byprotein expression with the ex-pected molecular masses (Fig. 1, Aand C). Expression of both SphK1and SphK2 increased intracellular[3H]S1P formation in MCF-7 cellspre-labeled with [3H]sphingosine(Fig. 1E). However, there was only asignificant increase in export of[3H]S1P fromMCF-7 cells expressingSphK1, but not from those expressingSphK2 (Fig. 1F). SphK1 or SphK2expression did not affect 3H-labeledsphingolipids in the cells (Fig. 1G) oruptake of sphingosine from themedium (Fig. 1H).To investigate the involvement of

endogenous SphK1 and SphK2, the expression of each isoformin MCF-7 cells was down-regulated. Endogenous SphK1 andSphK2mRNA levels were reduced by 90 and 75%, respectively,by transfection with siRNA targeted to SphK1 or SphK2, com-paredwith scrambled siRNA (Fig. 2,B andD). The specificity ofthese siRNAs was demonstrated by their inability to inhibitexpression of the other isoform of SphK (Fig. 2, B and D).ReducedmRNA levels of SphK1 and SphK2 were accompaniedby markedly reduced protein expression as determined byWestern blotting with isotype-specific antibodies (Fig. 2,A andC). Although reduction of expression of either SphK1 or SphK2significantly decreased intracellular [3H]S1P formation (Fig.2E), only down-regulation of SphK1, not SphK2, significantlydecreased export of S1P compared with control siRNA trans-fectants (Fig. 2, E and F).E2, but Not EGF, Induces Export of S1P—E2 and EGF have

been shown to stimulate SphK1 activity in MCF-7 cells leadingto increased intracellular S1P (7, 19, 20, 27). In agreement, bothE2 andEGF rapidly activated SphK1 as determined by enhancedphosphorylation on Ser225 (Fig. 3,A–C), an event important forits activation (28). As expected, down-regulation of SphK1eliminated the phosphorylated SphK1 immunoreactive band(Fig. 3B). Consistent with a previous study (19), E2 stimulatedthe enzymatic activity of SphK1 determined with an isoen-zyme-specific assay and had almost no effect on SphK2 activity

FIGURE 4. E2 but not EGF stimulates release of S1P and dihydro-S1P from MCF-7 cells. MCF-7 cells werestimulated with vehicle (open circles) or 20 nM E2 (filled squares) for the indicated times (A and C), for 5 min withthe indicated concentrations of E2 or EGF (B), or with 10 ng/ml of EGF (filled triangles) for the indicated times (Cand D). S1P (A–C) and dihydro-S1P (D) released into the medium were measured by LC-ESI-MS/MS. Data areexpressed as picomole of phosphorylated sphingoid base/ml and are mean � S.D. Levels of S1P and dihydro-S1P in the cells at zero time (7.4 � 1 and 2.8 � 0.2 pmol/ml, respectively) were subtracted from all values. *, p �0.01 compared with vehicle. Similar results were obtained in three independent experiments.

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(Fig. 3D). E2 and EGF increased formation of intracellular[3H]S1P inMCF-7 cells to similar extentswithin 5min (Fig. 3E).Interestingly, however, only E2 significantly increased export ofS1P (Fig. 3F).It was important to confirm these results by determination of

mass levels of S1P produced from endogenous pools of sphin-gosine and released into the medium. Analysis of S1P releasedfrom cells presents a challenge due to the low amounts relativeto the large volume of medium. For this purpose, we developeda sensitive and quantitative mass spectrometric method capa-ble of measuring femtomole amounts of S1P. E2 induced a sig-nificant increase in S1P released from MCF cells, peakingwithin 5 min (Fig. 4A), and was dose-dependent, reaching amaximum at a concentration of 10–20 nM (Fig. 4B). Thisincrease in S1P as well as the levels of S1P present in themedium at the beginning of the experiment were not due to thepresence of fragments of cell membranes as centrifugation ofthe medium at 100,000 � g for 1 h did not alter these values.In agreement with the [3H]S1P secretion results (Fig. 3F),

stimulation with EGF at any concentration examined did notsignificantly increase export of S1P, throughout a time courseof 15 min (Fig. 4C). Interestingly, LC-ESI-MS/MS analysisrevealed that export of dihydro-S1Pwas also enhanced by treat-ment with E2 but not by EGF (Fig. 4D).

Previous studies showed that transfection with catalyticallyinactive SphK1 suppressed secretion of S1P after E2 stimulation(19), suggesting that SphK1 activation is necessary forincreased S1P production and release. In agreement, E2increased intracellular levels of S1P that was dependent on theexpression of SphK1 (Fig. 5A). Depletion of SphK1 also abol-ished the elevated release of S1P (Fig. 5B) and dihydro-S1P (Fig.5C) in response to E2. In contrast, down-regulation of SphK2did not eliminate the E2-stimulated release of S1P (Fig. 5B) anddihydro-S1P (Fig. 5C). Moreover, although ectopic expressionof SphK1 or SphK2 increased release of S1P into the medium,only expression of SphK1 but not SphK2 further enhanced theE2-stimulated release of S1P (Fig. 5D). These results suggestthat SphK1 ismainly responsible for formation of S1P and dihy-dro-S1P secreted in response to E2.ER-�, but Not GPR30, Is Required for E2-mediated Rapid

Release of S1P—Nongenomic responses to E2 are mediated byeither ER located at the plasma membrane (15, 29) or by a Gprotein-coupled receptor, GPR30 (17, 26). To clarify whetherER-� and/or GPR30 are responsible for E2-mediated S1Pexport, we first utilized MDA-MB-231 breast cancer cells thatlack both ER-� andGPR30 (18, 30). In agreement with previousstudies (6), E2 did not activate SphK1 in MDA-MB-231 cells asdetermined by Western blotting with anti-phospho-Ser225

(data not shown). In contrast to MCF-7 cells, which expressboth ER-� and GPR30, E2 did not stimulate export of S1P fromMDA-MB-231 cells (Fig. 6A).Moreover, whenER-�was down-regulated inMCF-7 cells by prolonged treatment with the pureestrogen antagonist ICI 182,780 (Fig. 6C), the ability of E2 tostimulate S1P exportwas completely abolished (Fig. 6B). UnlikeE2, neither ICI 182,780, which is also a GPR30 agonist (17), norG1, a selective GPR30 agonist (31), significantly increased S1P(Fig. 6D) or dihydro-S1P (Fig. 6E) export from MCF-7 cells

determined by LC-ESI-MS/MS. Hence, only ER-� is necessaryfor E2-induced S1P export and not GPR30.ABCC1 and ABCG2Mediate E2-induced Export of S1P—Re-

cent studies suggest that members of the ABC transporter fam-ilymight be involved in release of S1P from several types of cells(32–35). Three ABC transporters, ABCB1 (also known as mul-tidrug resistant gene 1 (MDR1)) or p-glycoprotein, ABCC1(also known as multidrug resistance protein 1 (MRP1)), andABCG2 (also known as breast cancer resistance protein),appear to account for 80–90% of multidrug-resistant human

FIGURE 5. SphK1 is important for E2-mediated release of S1P and dihy-dro-S1P from MCF-7 cells. A, MCF-7 cells were transfected with siControl orsiSphK1 and treated with vehicle (open bars) or 10 nM E2 (black bars) for 5 min.Lipids were extracted from 106 cells and S1P was determined by LC-ESI-MS/MS. *, p � 0.01 compared with vehicle. B, MCF-7 cells were transfected withsiControl, siSphK1, or siSphK2 and treated with vehicle (open bars) or 10 nM E2(black bars) for 5 min. S1P (B) and dihydro-S1P (DHS1P) (C) released into themedium was determined by LC-ESI-MS/MS. Levels at zero time were sub-tracted and data expressed as mean � S.D. *, p � 0.01 compared with vehicle.Similar results were obtained in two additional experiments. D, MCF-7 cellswere transfected with vector, SphK1, or SphK2 and treated with vehicle (openbars) or with 10 nM E2 (black bars) for 5 min. S1P released into the medium wasdetermined by LC-ESI-MS/MS. Levels at zero time were subtracted and dataexpressed as mean � S.D. *, p � 0.05 compared with unstimulated vector; #,p � 0.05 compared with E2-stimulated vector. Similar results were obtained intwo additional experiments.

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tumor cells (36). In agreement with previous reports (36, 37),MCF-7 cells have high expression levels of ABCG2, lessABCC1, and no ABCB1 (Fig. 7A). To examine the involvementof these transporters in S1P and dihydro-S1P export fromMCF-7 cells, their expression was down-regulated by genesilencing. Transfection with siRNA targeted to ABCC1 orABCG2 reduced endogenous ABCC1 and ABCG2 mRNA lev-els by more than 60 and 90%, respectively, compared withscrambled control siRNA (Fig. 7, B andC) and also reduced thecorresponding protein expression (Fig. 7, D and E). Interest-ingly, down-regulation of ABCC1 increased ABCG2 mRNAwith a concomitant increase inABCG2protein (Fig. 7,B andE),and conversely, decreasing ABCG2 mRNA resulted inincreased ABCC1 mRNA and protein (Fig. 7, C and D).Although themagnitude of the response to E2 by cells transfectedwith control siRNAwas similar to that of the naïve cells (Fig. 4A),down-regulation of ABCC1 significantly decreased E2-inducedS1P release, and down-regulation of ABCG2 completely abro-gated it (Fig. 7F). The greater susceptibility to depletion ofABCG2might bedue to its higher expression andgreater down-regulationcompared with the decrease in expression of ABCC1 (Fig. 7).

Down-regulation of either ABCC1 or ABCG2 also decreasedE2-induced dihydro-S1P release (Fig. 7G).To further substantiate the involvement of ABCC1 and

ABCG2 in S1P and dihydro-S1P export from MCF-7 cells, theeffects of MK571 or FTC, pharmacological inhibitors ofABCC1 and ABCG2, respectively (32, 36, 38), were also exam-ined. E2-stimulated release of S1P and dihydro-S1P fromMCF-7 cells was significantly reduced by preincubation withMK571 or FTC (Fig. 8, A and B). Moreover, a combination ofboth inhibitors totally abrogated E2-induced release of S1P (Fig.8, A and B).E2-induced S1P Secreted via ABCC1 or ABCG2 Activates

ERK1/2—Oneof themostwell knownnongenomic effects of E2is ERK1/2 activation (reviewed in Refs. 15, 18, and 26). Previousstudies have suggested that activation of SphK1 and productionof S1P might be involved in E2-induced ERK1/2 activation (6,19). If so, prevention of S1P release should negate this responseto E2. In agreement with previous studies (6, 18, 19, 26, 39),treatment of MCF-7 cells with E2 rapidly activated ERK1/2, asdemonstrated with phosphospecific antibodies, with maximalresponse obtained at 10 min, which returned to basal levels

FIGURE 6. ER-� is necessary for E2-mediated release of S1P and dihydro-S1P. A, MDA-MB-231 cells were stimulated with vehicle (open circles) or 10 nM E2(filled squares) for the indicated times and S1P released into the medium was measured by LC-ESI-MS/MS. Levels of S1P in the medium of MDA-MB-231 cells atzero time (0.6 � 0.1 pmol/ml) were subtracted from all values. B, MCF-7 cells were pretreated for 18 h without or with 10 �M ICI 182,780 and then stimulatedwith 10 nM E2 and S1P release was determined at the indicated times by mass spectrometry. Levels of S1P in the medium of MCF-7 cells at zero time (8.8 � 0.7pmol/ml) was subtracted and data expressed as mean � S.D. C, equal amounts of proteins from lysates of duplicate cultures of MDA-MB-231 and MCF-7 cellstreated as in A and B, respectively, were separated by SDS-PAGE and analyzed by immunoblotting with anti-ER-� antibody. Blots were stripped and reprobedwith anti-tubulin to confirm equal loading. D and E, MCF-7 cells were treated with vehicle, 10 nM E2, 10 �M ICI 182,780, or 10 nM G1 for 5 min. S1P (D) anddihydro-S1P (E) released into the medium were determined by LC-ESI-MS/MS. Levels at zero time were subtracted and data are expressed as mean � S.D. *, p �0.01 compared with vehicle. Similar results were obtained in two additional experiments.

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within 30 min (data not shown). Inhibition of either ABCC1 orABCG2 using MK571 or FTC, respectively, drastically reducedactivation of ERK1/2 induced by E2 (Fig. 8C), consistent withthe ability of these inhibitors to reduce export of S1P (Fig. 8A)and dihydro-S1P (Fig. 8B). As expected, ERK1/2 activation byEGF, which does not enhance S1P secretion from MCF7 cells,was not inhibited by inhibition of ABCC1 or ABCG2 (Fig. 8C).Moreover, down-regulation of ABCG2 markedly reduced E2-but not EGF-mediated ERK1/2 activation (Fig. 8D).

DISCUSSION

Numerous studies have suggested that S1P is important inbreast cancer, acting in an autocrine and/or paracrine manner(1, 11, 40). In this study, we have shown that SphK1, not SphK2,is necessary for release of S1P from MCF-7 ER-positive breastcancer cells. This is probably due to the distinct subcellularlocalization of the two kinase isoforms. SphK1 is primarily cyto-solic and rapidly translocated to the plasma membrane uponstimulation, where produced S1Pmay be readily available to betransported outside the cells (41, 42). In contrast, SphK2 hasbeen shown to be localized to the nucleus of MCF-7 cells, asdetermined by confocal microscopy and Western blotting (22,

43, 44). Surprisingly, although bothE2 and EGF stimulated SphK1 inMCF-7 cells, only E2 rapidly stimu-lated export of S1P and dihydro-S1P. This suggests that activation ofSphK1 and production of S1P at theplasma membrane is not sufficientfor its release and indicates thatactive transport might also beinvolved. Although our initialefforts to examine secretion of S1Putilized labeled sphingosine, thismethod has the caveat that itrequires treatment of cells withexogenous sphingosine and doesnot provide any information onsecretion of endogenously pro-duced S1P. However, consistenttrends were obtained when secre-tion of mass levels of S1P was deter-mined by a sensitive LC-ESI-MS/MS method that we developed.Moreover, we were able to demon-strate for the first time that MCF-7cells also export dihydro-S1P,whichis also a ligand for all of the S1Preceptors. E2-stimulated release ofboth S1P and dihydro-S1P was de-pendent on SphK1.Studies from several laboratories,

including ours, have suggested theinvolvement of ABC transporters inS1P export from mast cells (32),platelets (33), endothelial cells (34),astrocytes (35), thyroid follicularcell (45), fibroblasts (46), and eryth-

rocytes (47). Similarly, using pharmacological and molecularapproaches, we demonstrated that ABCC1 and ABCG2 areinvolved in E2-mediated transport of S1P and dihydro-S1P outof MCF-7 cells. Yet in MCF7 cells, ABCG2 predominates,which might be due to its greater expression in these cells. It isstill not completely understoodwhether these two transportersoperate serially rather than in parallel and whether both arenecessary or whether one is sufficient.Overexpression ofABCC1 andABCG2 appear to account for

a large fraction of multidrug-resistant breast tumors (48, 49). Itis tempting to speculate that enhanced expression of theseABCtransporters is involved in multidrug resistance not only due toincreased drug efflux from the cell, but also due to export of S1Pand dihydro-S1P, because by binding to S1P receptors they canplay significant roles in regulating angiogenesis,metastasis, andbreast cancer progression. Hence, export of S1P by ABC trans-porters may be a contributing factor to the poor prognosis ofpatients who overexpress these multidrug-resistant proteins.Interestingly, MCF7 cells constitutively export relatively highlevels of S1P into the culture medium compared with MDA-MB-231 cells that are not dependent on ABCC1 and ABCG2transporter activity or expression and are not affected by

FIGURE 7. Down-regulation of ABCC1 or ABCG2 decreases E2-mediated release of S1P or dihydro-S1P.A, RNA was isolated from MCF-7 cells and mRNA of ABCB1, ABCC1, and ABCG2 determined by quantitative realtime reverse transcription-PCR and normalized to cyclophilin mRNA. B–E, MCF-7 cells were transfected withcontrol siRNA (open bars) or siRNA targeted to ABCC1 (black bars) or ABCG2 (gray bars), as indicated. Expressionof ABCC1 and ABCG2 was determined by quantitative real-time reverse transcription-PCR and normalized toglyceraldehyde-3-phosphate dehydrogenase mRNA. D and E, equal amounts of cell lysate proteins from dupli-cate cultures were immunoblotted with anti-ABCC1 (D) or anti-ABCG2 (E). Blots were stripped and reprobedwith anti-clathrin heavy chain (CHC) or anti-tubulin, as indicated, to confirm equal loading. F and G, MCF-7 cellswere transfected with siControl, siABCC1, or siABCG2 and stimulated with vehicle or E2 (10 nM) for the indicatedtimes. S1P (F) and dihydro-S1P (G) released into the medium was determined by LC-ESI-MS/MS. Levels of S1Pand dihydro-S1P at zero time (7 � 0.7 and 1.5 � 0.3 pmol/ml, respectively) were subtracted and data expressedas mean � S.D. All values at 5 min were statistically significant (p � 0.01) compared with siControl treated withE2 at 5 min. Similar results were obtained in two additional experiments.

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decreased temperature nor did theysignificantly increase with time.ABCC1 and ABCG2 transporters

have been shown to play a majorrole in resistance of breast cancer tosome important chemotherapyagents, such as doxorubicin (49).ABCG2 is often expressed in cancerstem cell populations and couldhelp them survive cytotoxic or tar-geted therapies leading to tumorregrowth or relapse (38, 49). A fas-cinating connection is that S1P,which is secreted by ABCG2, hasalso been shown to be important forstem cell growth, survival, mainte-nance of pluripotentiality (50), andhoming to sites of action (51). Ofnote, it was recently shown thatexpression of SphK1 is linked withresistance of MCF-7 cells to tamox-ifen (52), suggesting that targetingthe SphK1/S1P axis might be a use-ful approach to overcome anti-es-trogen resistance in human breastcancer.E2 stimulates mitogenesis of

MCF-7 cells in the absence of anyother growth factors. In the classicalor genomic mechanism, E2 mole-cules diffuse into the cell and bind tothe ER, which exists as two differentforms, ER-� and ER-�. The bindingof E2 to either of its receptorsinduces a conformational change,resulting in interactions with thetranscriptional co-regulators andregulatory DNA sequences of targetgenes. The resulting changes in lev-els of the proteins they encodeunderlie many overall physiologicalresponses following E2 exposure.However, there are rapid biochemi-cal and physiological responses thatoccur within minutes after E2administration that cannot beaccounted for by changes in geneexpression mediated by nuclearERs. There is still controversyregarding the rapid nongenomicresponses to E2 and the receptorsthat mediate these events. Oneschool of thought is that plasmamembrane-localized ER-� initiatesE2-dependent rapid signaling cas-cades (29). On the other hand, it hasbeen suggested that GPR30 is themembrane receptor that mediates

FIGURE 8. Pharmacological inhibitors of ABCC1 or ABCG2 decrease E2-mediated release of S1P and dihy-dro-S1P and ERK1/2 activation. MCF-7 cells were preincubated for 2 h without or with 20 �M MK571 or 20 �M

FTC, or both, followed by stimulation with vehicle or E2 (10 nM) for the indicated times (A and B), for 10 min with10 nM E2, or with 10 ng/ml EGF (C). S1P (A) and dihydro-S1P (B) released was determined by LC-ESI-MS/MS.Levels of S1P and dihydro-S1P in the cells at zero time (7 � 0.6 and 2.9 � 0.2 pmol/ml, respectively) weresubtracted and data expressed as mean � S.D. All values at 5 and 15 min were statistically significant (p � 0.05)compared with control cells treated with E2. Similar results were obtained in two additional experiments.C, equal amounts of proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-phos-pho-ERK1/2 antibody. D, MCF-7 cells were transfected with siControl or siABCG2 and stimulated with vehicle,E2 (20 nM), or EGF (10 ng/ml) for 10 min, and ERK1/2 activation was determined by immunoblotting withanti-phospho-ERK1/2 antibody. Blots were stripped and reprobed with anti-total ERK antibodies to confirmequal loading and transfer.

FIGURE 9. Scheme highlighting the importance of ABC transporters and S1P/dihydro-S1P export fromthe cell in nongenomic effects of E2. See text for more details. For simplicity, many other known signalingpathways downstream of ER are not shown. Binding of E2 to ER-� and not GPR30 stimulates release of S1P (anddihydro-S1P, not shown here) via ABC transporters, ABCC1 and ABCG2. This S1P in turn binds to and activatesS1P receptors to stimulate ERK1/2 leading to downstream signaling events important for breast cancer prolif-eration, progression, and invasion.

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some of the nongenomic signaling of E2 (16, 18, 26). Becausethese E2 receptors have different pharmacologies, it was possi-ble to determine which was involved in E2-stimulated export ofS1P and dihydro-S1P. Interestingly, neither ICI 182,780, aknown antagonist of ER-� and an agonist for GPR30 (17), norG1, a specific GPR30 agonist (31), had significant effects on S1Por dihydro-S1P export, in contrast to E2. In accordance, down-regulation of ER-� abolished E2-mediated release of S1P anddihydro-S1P. Importantly, suppression of export of S1P anddihydro-S1P by inhibitors of ABCC1 and ABCG2 markedlydecreased activation of ERK1/2 in response to E2. Our resultssupport the notion that activation of SphK1 by E2, export of S1Pvia ABC transporters, and consequent activation of S1P recep-tors (Fig. 9) may contribute to nongenomic signaling of E2important for breast cancer pathophysiology. Furthermore, theS1P receptors may be the G protein-coupled receptors thatmediate rapid nongenomic responses to E2 rather than GPR30,which has been shown to be present in endoplasmic reticulum,not the plasma membrane (17, 53). Our work, taken togetherwith the criss-cross model for E2 signaling proposed bySukocheva et al. (19), suggests that E2 stimulates SphK1,increasing production of S1P in the vicinity of the ABC trans-porters, ABCC1 and ABCG2, at the plasma membrane, whichfacilitate its export (Fig. 8). This S1P in turn binds to and acti-vates S1P receptors, to stimulate ERK1/2 and increase EGFRtransactivation in a matrix metalloprotease-dependent man-ner. Finally, the demonstration that ER-� expression is neces-sary for S1P and dihydro-S1P export from breast cancer cellsmay, at least in part, explain why ER-positive breast cancershows reduced responses to modern combined chemotherapycompared with ER-negative tumors, as found in a randomizedclinical trial involving more than 6500 advanced breast cancerpatients (54).

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E2 and ABC Transporters in S1P Export

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Milstien and Sarah SpiegelRamachandran, Masayuki Nagahashi, Kuzhuvelil B. Harikumar, Nitai C. Hait, Sheldon

Kazuaki Takabe, Roger H. Kim, Jeremy C. Allegood, Poulami Mitra, SubramaniamABCC1 and ABCG2

Estradiol Induces Export of Sphingosine 1-Phosphate from Breast Cancer Cells via

doi: 10.1074/jbc.M109.064162 originally published online January 28, 20102010, 285:10477-10486.J. Biol. Chem. 

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