The Prostaglandin Transporter Regulates Adipogenesis and ......esis and catabolism. The prostaglandin transporter (PGT) removes PGE 2 from the extracellular environment and thereby

Research Article

The Prostaglandin Transporter Regulates Adipogenesisand Aromatase Transcription

Kotha Subbaramaiah1, Clifford A. Hudis2, and Andrew J. Dannenberg1

AbstractCytochrome P450 aromatase, encoded by the CYP19 gene, catalyzes estrogen synthesis. In obese

postmenopausal women, increased estrogen synthesis in adipose tissue has been linked to hormone-

dependent breast carcinogenesis. Hence, it is important to elucidate the mechanisms that regulate CYP19

gene expression. Prostaglandin E2 (PGE2) stimulates the cyclic AMP (cAMP) ! protein kinase A (PKA) !cAMP responsive element binding protein (CREB) pathway leading to increased CYP19 transcription. The

prostaglandin transporter (PGT) removes PGE2 from the extracellular milieu and delivers it to the cytosol,

where it is inactivated. The main objective of this study was to determine whether PGT regulates CYP19

transcription. Silencing of PGT in preadipocytes increased PGE2 levels in the extracellular medium, thereby

stimulating the cAMP ! PKA pathway resulting in enhanced interaction between pCREB, p300, and the

CYP19 I.3/II promoter. A reciprocal decrease in the interaction between the CYP19 I.3/II promoter and

BRCA1, a repressor of CYP19 transcription, was observed. Overexpressing PGT reduced extracellular PGE2levels, suppressed the cAMP ! PKA pathway, enhanced the interaction between BRCA1 and p300, and

inhibited aromatase expression. We also compared the PGT ! aromatase axis in preadipocytes versus

adipocytes. Aromatase levels were markedly increased in preadipocytes versus adipocytes. This increase in

aromatase was explained, at least in part, by reduced PGT levels leading to enhanced PGE2! cAMP! PKA

signaling. In addition to regulating aromatase expression, PGT-mediated changes in extracellular PGE2levels were a determinant of adipocyte differentiation. Collectively, these results suggest that PGT

modulates adipogenesis and thereby PGE2-mediated activation of the cAMP ! PKA ! CREB pathway

leading to altered CYP19 transcription and aromatase activity. Cancer Prev Res; 4(2); 194–206. �2011 AACR.

Introduction

Obese postmenopausal women are at increased risk ofdeveloping hormone receptor–positive breast cancer (1).Approximately two thirds of patients with breast cancerhave tumors that express estrogen receptors and requireestrogen for tumor growth. After menopause, peripheralaromatization of androgen precursors in adipose tissue islargely responsible for estrogen production (2). Estrogensare synthesized from androgens in a reaction catalyzed bycytochrome P450 aromatase (aromatase), encoded by theCYP19 gene. Thus, the increased risk of hormone receptor–

positive breast cancer in obese postmenopausal women isbelieved to be attributable, in part, to elevated levels ofcirculating estradiol related to both increased adiposetissue and aromatase upregulation in adipose tissue (3–6).

Given the significance of estrogen synthesis in the patho-genesis of hormone-dependent breast cancer, intenseefforts have been made to elucidate the mechanisms thatregulate the transcription ofCYP19 (7). Several groups havecarried out studies pointing to the significance of prosta-glandin E2 (PGE2) as an inducer of aromatase. PGE2stimulates cyclic AMP (cAMP)-dependent signaling leadingto enhanced CYP19 transcription (8–10). COX catalyzesthe first step in the synthesis of PGE2 from arachidonic acid.Positive correlations have been detected between COX andaromatase expression in human breast cancer specimens(11–13). In mice that express a mammary-targeted COX-2transgene, increased PGE2 and aromatase levels wereobserved (14). Silencing of 15-hydroxyprostaglandin dehy-drogenase (15-PGDH), the key enzyme responsible forinactivating PGE2 upregulated aromatase (15). Recently,the tumor suppressor BRCA1 was found to negativelyregulate CYP19 expression (10, 16–18). This inhibitoryeffect of BRCA1 on aromatase expression was relieved byPGE2. Finally, two observational studies found that the use

Authors' Affiliations: 1Department of Medicine, Weill Cornell MedicalCollege; and 2Department of Medicine, Memorial Sloan-Kettering CancerCenter, New York

Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Kotha Subbaramaiah, Department of Medicine,Weill Cornell Medical College, 525 East 68th St., Rm. F-203A, New York,NY 10065. Phone: 212-746-4402; Fax: 212-746-4885. E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-10-0367

�2011 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 4(2) February 2011194

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of aspirin, an inhibitor of PGE2 production, was associatedwith a reduced risk of hormone receptor–positive breastcancer (19, 20).Levels of PGs depend on transport in addition to synth-

esis and catabolism. The prostaglandin transporter (PGT)removes PGE2 from the extracellular environment andthereby prevents its interaction with plasma membranereceptors (EP) for PGE2 (21). This transporter is a memberof the organic anion superfamily of transporting polypep-tides that contain 12-transmembrane spanning domains(22). PGT is rate limiting in the delivery of PGE2 tocytosolic 15-PGDH, which results in its oxidation andinactivation (23, 24). Although numerous studies haveshown the significance of enzymes involved in the synth-esis and catabolism of PGE2 as determinants of CYP19transcription and aromatase activity, the potential impor-tance of PGT in regulating aromatase expression isunknown. Hence, the primary objective of this studywas to evaluate whether changes in the expression ofPGT modulate CYP19 transcription. Preadipocytes containmuch higher levels of aromatase than mature adipocytesand were used as a model system (25). Here, we show thatPGT regulates adipogenesis and thereby the PGE2 ! cAMP! protein kinase A (PKA) ! cAMP responsive elementbinding protein (CREB) pathway leading, in turn, tochanges in CYP19 transcription and aromatase activity.Reciprocal changes in the interaction between BRCA1,p300, and the CYP19 I.3/II promoter contribute to thiseffect of PGT.

Materials and Methods

MaterialsMedium to grow visceral preadipocytes was purchased

from ScienCell Research Laboratories. Differentiating med-ium was obtained from ZenBio. FBS was purchased fromInvitrogen. Rabbit polyclonal antisera for human phos-pho-CREB (pCREB), CREB, p300, BRCA1, b-actin, andcontrol IgG were from Santa Cruz Biotechnology. Lowryprotein assay kits, horseradish peroxidase–conjugated sec-ondary antibody, glucose-6-phosphate, glycerol, pepstatin,leupeptin, glucose-6-phosphate dehydrogenase, and rote-none were from Sigma. cAMP enzyme immunoassay kitwas from Biomol. PKA activity assay kits were obtainedfrom Calbiochem. PGE2 and PGE2 enzyme immunoassayassay (EIA) kits were purchased from Cayman Chemicals.ECL Western blotting detection reagents were fromAmersham Biosciences. Nitrocellulose membranes werefrom Schleicher & Schuell. 1b-[3H]androstenedione and[32P]CTPwere from Perkin-Elmer Life Science. pSVbgal andplasmid DNA isolation kits were purchased from Promega.Luciferase assay reagents were from Analytical Lumines-cence. The PGT, BRCA1, aromatase, progesterone receptor(PR), aP2, and b-actin cDNAs were obtained from OpenBiosystems. The 18S rRNA cDNA was purchased fromAmbion. siRNAs [PGT, green fluorescent protein (GFP)]and RNeasy mini kits were purchased from Qiagen. Adi-pogenesis and chromatin immunoprecipitation (ChIP)

assay kits were purchased from Millipore. MuLV reversetranscriptase, RNase inhibitor, oligo-(dT)16, and SYBRgreen PCR master mix were obtained from Applied Bio-systems. Luciferase assay substrates and cell lysis bufferwere from BD Biosciences. The CYP19 I.3/II promoter-luciferase construct was kindly provided by Dr. S. Chen(City of Hope, Duarte, CA). The estrogen response element(ERE)-luciferase construct was from Panomics.

Cell culture and Oil Red O stainingHuman visceral preadipocytes were obtained from Scien-

Cell Research Laboratories. These primary cells were grownin preadipocyte medium containing 10% FBS. Preadipo-cytes were grown in differentiation medium for 48 hours toproduce mature adipocytes. Oil Red O staining of lipidvesicles was carried out and quantified to confirm thedifferentiation state of adipocytes. As detailed by the man-ufacturer (Millipore), the absorbance of extracted Oil RedO was measured in a plate reader at 490 nm.

PGE2 productionCells were plated in 6-well dishes and grown to 60%

confluence in growth medium. The amount of PGE2 in cellculture medium was measured by EIA. Levels of PGE2 werenormalized to protein concentrations.

Western blottingLysates were prepared by treating cells with lysis buffer

[150 mmol/L NaCl, 100 mmol/L Tris (pH 8.0), 1% Tween20, 50mmol/L diethyldithiocarbamate, 1mmol/L EDTA, 1mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL aproti-nin, 10 mg/mL trypsin inhibitor, and 10 mg/mL leupeptin].Lysates were sonicated for 20 seconds on ice and centri-fuged at 10,000 � g for 10 minutes to sediment theparticulate material. The protein concentration of thesupernatant was measured by the method of Lowry andcolleagues (26). SDS-PAGE was performed under reducingconditions according to the procedure of Laemmli (27).The resolved proteins were transferred onto nitrocellulosesheets as detailed by Towbin and colleagues (28). Thenitrocellulose membrane was then incubated with primaryantisera. Secondary antibody to IgG conjugated to horse-radish peroxidase was used. The blot was probed with theECLWestern blot detection system according to the instruc-tions of the manufacturer.

Northern blottingTotal RNA was prepared from cell monolayers, using an

RNAisolationkit fromQiagen.Tenmicrogramsof totalRNAper lane was electrophoresed in a formaldehyde-containing1% agarose gel and transferred to nylon-supported mem-branes. Aromatase, PGT, BRCA1, PR, aP2, b-actin, and 18SrRNA probes were labeled with [32P]CTP by random prim-ing. The blots were probed as described previously (14).

Real-time PCRTotal RNA was isolated using the RNeasy mini kit. One

microgram of RNA was reversed transcribed using murine

PGT Regulates Adipogenesis and Aromatase Transcription

www.aacrjournals.org Cancer Prev Res; 4(2) February 2011 195




leukemia virus reverse transcriptase and oligo-(dT)16primer. The resulting cDNA was then used for amplifica-tion. The volume of the PCR was 20 mL and contained 5 mLof cDNA with the following primers: for aromatase mRNA,the forward and reverse primers were 50-CACATCCTCAA-TACCAGGTCC-30 and 50-CAGAGATCCAGACTCG CATG-30; for BRCA1, the forward and reverse primers were 50-AGCCAGCCACAGGTACAGAG-30 and 50-AGTAGCCAG-GACAGTAGAAGGAC-30; for aP2, the forward and reverseprimers were 50-TGATGATCATGTTAGGTTTGGC and TGGAAACTTGTCTCCAGTGAA. Real-time PCR was performedusing 2x SYBR green PCR master mix on a 7900 HT real-time PCR system (Applied Biosystems) with b-actin (for-ward, 50-AGAAAATCTGGCACCACACC-30; reverse, 50-AGAGGCGTACAGGGATAGCA-30) serving as an endogen-ous normalization control. Relative fold induction wasdetermined using the CT (relative quantification) analysisprotocol.

cAMP levelsCells were plated at 5� 104 per well in 6-well dishes and

grown to 60% to 70% confluence before treatment.Amounts of cAMP were measured by EIA. Production ofcAMP was normalized to protein concentration.

PKA activityCells were plated at 5� 104 per well in 6-well dishes and

grown to 60% to 70% confluence before treatment. PKAactivity was measured according to the instructions of themanufacturer andwasnormalized toprotein concentration.

Aromatase activityTo determine aromatase activity, microsomes were pre-

pared from cell lysates by differential centrifugation usingestablishedmethods (14). To determine aromatase activity,microsomal proteinwas added to a 0.5mL reactionmixturecontaining 50mmol/L Tris-HCl (pH7.5), 5mmol/LMgCl2,5 mmol/L glucose-6-phosphate, 5 U glucose-6-phosphatedehydrogenase, 2 mmol/L rotenone, and 12.5 nmol/L 1b-[3H]androstenedione. Following preincubation for 3 min-utes, the reactionwas initiatedby the additionof 0.5mmol/LNADPH and allowed to run for up to several hours at 37�C.Adding 3 mL ice-cold chloroform and applying vigorousshaking and brief centrifugation terminated the reaction.The resulting aqueous layer was further extracted with 3 mLchloroform and treated with 0.5 mL 5% activated charcoal/0.5% dextran. Following centrifugation of the mixture, theradioactivity in the supernatant was counted. Aromataseactivity was quantified by measurement of the tritiatedwater released from 1b-[3H]androstenedione. The reactionwas also performed in the presence of a specific aromataseinhibitor, as a specificity control and without NADPH as abackground control. Aromatase activity was normalized toprotein concentration.

Transient transfectionsCells were seeded at a density of 5 � 104 per well in

6-well dishes and grown to approximately 50% confluence.

For each well, 2 mg of plasmid DNA were introduced intocells, using the Amaxa system. After 24 hours of incubation,themediumwas replaced with basalmedium. The activitiesof luciferase and b-galactosidase were measured in cellularextract. Preadipocytes overexpressing PGT or control vectorwere obtained by transfecting PGT expression vector orcontrol vector, using the Amaxa system. Following trans-fection, cells that could grow in puromycin (10 mg/mL)were selected and used.

ChiP assayChiP assay was performed with a kit according to the

manufacturer’s instructions. A total 2 � 106 cells werecross-linked in a 1% formaldehyde solution for 10 min-utes at 37�C. Cells were then lysed in 200 mL of SDS bufferand sonicated to generate 200- to 1,000-bp DNA frag-ments. After centrifugation, the cleared supernatant wasdiluted 10-fold with ChIP buffer and incubated with1.5 mg of the indicated antibody at 4�C. Immune com-plexes were precipitated, washed, and eluted as recom-mended. DNA-protein cross-links were reversed byheating at 65�C for 4 hours, and the DNA fragmentswere purified and dissolved in 50 mL of water. Tenmicroliters of each sample was used as a template forPCR amplification. CYP19 oligonucleotide sequences forPCR primers were as follows: forward, 50-AACCTGCT-GATGAAGTCACAA-30; reverse, 50-TCAGACATTTAGG-CAAGACT-30. This primer set encompasses the CYP19I.3/II promoter segment from nucleotide �302 to �38.PCR was performed at 94�C for 30 seconds, 60�C for 30seconds, and 72�C for 45 seconds for 30 cycles. ThePCR products generated from the ChIP template weresequenced, and the identity of the CYP19 promoter wasconfirmed. For real-time PCR analysis, ChIP-qPCR assaykits from Superarray Bioscience Corp. were used. Real-time PCR was performed as described previously.

StatisticsComparisons between groups were made by Student’s

t test. A difference between groups of P < 0.05 was con-sidered significant. All experiments were carried out aminimum of 3 times. Representative data are shown.

Results

PGT regulates aromatase expression in humanpreadipocytes

Initially, we determined the effect of silencing PGT onlevels of PGE2 in the cell culture medium. As shown inFigure 1A, silencing of PGT led to approximately a 1-foldincrease in PGE2 levels (Fig. 1A). This increase in PGE2 wasaccompanied by a corresponding increase in both aroma-tase expression and activity (Fig. 1B and C). To furtherinvestigate the significance of PGT as a determinant ofaromatase expression, it was overexpressed in preadipo-cytes. Overexpression of PGT led to reduced levels of PGE2in the cell culture medium, decreased aromatase expres-sion, and reduced aromatase activity (Fig. 1D–F).

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A B

C D

E F

Figure 1. PGT regulates aromatase expression. A–C, human preadipocytes were transfected with 2 mg of siRNAs to GFP (control) or PGT and allowed to growfor 36 hours prior to analysis. A, levels of PGE2 in the cell culture mediumwere determined by enzyme immunoassay. Means� SD are shown; n¼ 6. *, P < 0.01compared with control. Inset, Northern blotting was done using 10 mg total RNA per lane; the blot was probed for PGT and b-actin. B, total RNA was preparedfrom cells and levels of aromatase mRNA were determined by real-time PCR. Values were normalized to the expression levels of b-actin. Means � SD areshown; n ¼ 6. *, P < 0.01 compared with control. Inset, Northern blot analysis was done on 10 mg total RNA per lane and the blot was probed for aromataseand b-actin. C, aromatase activity was determined using microsomes prepared from cell lysates as in "Materials and Methods." Enzyme activity is expressedas fmol/mg protein/min. Means � SD are shown; n ¼ 6. *, P < 0.01 compared with control. D–F, PGT or control vector were overexpressed in humanpreadipocytes. D, levels of PGE2 in the cell culture medium were determined by enzyme immunoassay. Means� SD are shown; n¼ 6. *, P < 0.001 comparedwith control. Inset, Northern blot analysis was done using 10 mg of RNA per lane; the blot was probed for PGT and b-actin. E, total RNAwas prepared from cells,and levels of aromatase mRNA were determined by real-time PCR. Values were normalized to the expression levels of b-actin. Means� SD are shown; n¼ 6.*, P < 0.01 compared with control. Inset, Northern blot analysis was done using 10 mg of RNA per lane; the blot was probed for aromatase and b-actin.F, aromatase activity was determined using microsomes prepared from cell lysates as in "Materials and Methods." Enzyme activity is expressed asfmol/mg protein/min. Means � SD are shown; n ¼ 6. *, P < 0.01 compared with control.






Signal transduction pathway by which PGT regulatesCYP19 transcription

PGE2 is known to stimulate the cAMP ! PKA ! CREBpathway leading to induction of CYP19 gene expressionand increased aromatase activity (10). Hence, we nextevaluated the importance of PGT as a modulator of thissignal transduction pathway. Silencing PGT increasedlevels of cAMP and PKA activity (Fig. 2A and B). Conver-sely, overexpressing PGT led to a significant reduction incAMP levels and PKA activity (Fig. 2C and D). We nextevaluated whether changes in PGT expression modulatedaromatase promoter activity. As shown in Figure 2E, silen-cing PGT stimulated aromatase promoter activity. In con-trast, overexpressing PGT inhibited aromatase promoteractivity (Fig. 2F). ChIP assays were performed to evaluatewhether changes in the expression of PGT modulated thebinding of pCREB to the CYP19 promoter. Silencing PGTstimulated the phosphorylation of CREB and the recruit-ment of pCREB to the CYP19 promoter (Fig. 2G); over-expression of PGT inhibited the phosphorylation of pCREBand its recruitment to the CYP19 promoter (Fig. 2H).

Recently, PGE2 was found to suppress levels of BRCA1,which contributed, in turn, to enhanced CYP19 transcrip-tion and increased aromatase activity (10, 17). Therefore,we attempted to determine whether modulating PGT levelswould impact on BRCA1 levels. Silencing PGT suppressedBRCA1 levels (Fig. 3A), whereas overexpressing PGTinduced BRCA1 (Fig. 3B). ChIP assays were carried outto explore the effects of PGT on the interaction betweenBRCA1 and theCYP19 promoter. Silencing PGT suppressedthe interaction between BRCA1 and the CYP19 promoter(Fig. 3C), whereas overexpressing PGT stimulated the inter-action between BRCA1 and the CYP19 promoter (Fig. 3D).Because p300 is important for pCREB-dependent activa-tion of aromatase transcription, the interaction between theCYP19 promoter and p300 was also investigated. SilencingPGT increased the interaction between p300 and theCYP19promoter (Fig. 3E). In contrast, overexpressing PGTreduced the recruitment of p300 to the CYP19 promoter(Fig. 3F). To further understand the role of PGT in regulat-ing the transcription of aromatase, we also investigated theinteractions between BRCA1, p300, and pCREB underbasal conditions and following silencing or overexpressionof PGT. In control cells, immunoprecipitation experimentssuggested that p300 and BRCA1 were in a complex (Fig. 3Gand H). Following silencing of PGT, p300 and pCREB werein the complex but BRCA1 was not found (Fig. 3G). Incontrast, overexpression of PGT stimulated the interactionbetween p300 and BRCA1 but pCREB was not found in thecomplex (Fig. 3H).

Because aromatase activity can be rate limiting for thesynthesis of estradiol, we also evaluated the role of PGT as adeterminant of estrogen-dependent gene expression. ThePR, an estrogen target gene, is positively regulated by anERE (29, 30). As shown in Supplementary Figure 1, silen-cing PGT stimulated ERE-luciferase activity and inducedPR (Supplementary Fig. S1A and B). Overexpression ofPGT suppressed ERE-luciferase activity (Supplementary

Fig. S1C) and reduced levels of PR (SupplementaryFig. S1D). Collectively, these results indicate that PGTmodulates PGE2-mediated activation of the cAMP !PKA ! CREB pathway and thereby regulates aromatasetranscription and estrogen-dependent gene expression inpreadipocytes.

PGT regulates adipogenesis resulting in changes inaromatase expression

As mentioned earlier, aromatase expression is known tobe markedly higher in preadipocytes than in adipocytes(25). On the basis of our finding that PGT regulatesaromatase expression, we next investigated whether thedifference in aromatase levels in preadipocytes versus adi-pocytes could be explained at least, in part, by differences inPGT levels. Differentiating medium was used to converthuman preadipocytes to adipocytes. aP2 is a marker ofdifferentiated adipocytes. Higher levels of aP2 were foundin adipocytes than preadipocytes, confirming the differen-tiation state of the cells (Fig. 4A, inset). Levels of PGTmRNA were markedly increased in adipocytes comparedwith preadipocytes (Fig. 4A). Consistent with this finding,levels of PGE2 were approximately 4-fold lower in the cellculture medium of adipocytes than in preadipocytes(Fig. 4B). Given the evidence that PGE2 regulates the cAMP! PKA ! CREB signal transduction pathway leading tochanges in aromatase transcription, we next evaluated theimpact of differentiation on this axis. Reduced levels ofcAMP, PKA activity, pCREB, and aromatase were detectedin adipocytes versus preadipocytes (Fig. 4C–G). Levels ofPR, an estrogen-regulated gene, were also reduced in adi-pocytes (Fig. 4H). On the basis of our findings in preadi-pocytes that were engineered to overexpress PGT, we alsoinvestigated whether the transcription machinery that con-trols aromatase expression was altered in adipocytes versuspreadipocytes. Here, we show reduced binding of pCREB,increased BRCA1 expression and binding, and decreasedrecruitment of p300 to the CYP19 1.3/II promoter inadipocytes versus preadipocytes (Fig. 5A–D). The interac-tion between p300 and BRCA1 was increased, with areciprocal decrease in the interaction between p300 andpCREB in adipocytes versus preadipocytes (Fig. 5E). Takentogether, these results suggest that the higher levels of PGTin adipocytes contribute to the reduced expression ofaromatase in these cells compared with preadipocytes.

PGE2 suppresses adipocyte differentiation, the processby which preadipocytes become mature adipocytes (31,32). It is possible, therefore, that PGT regulates PGE2levels in the extracellular milieu and thereby adipogenesisand aromatase levels. Our finding that levels of PGT arereduced in preadipocytes versus adipocytes with a reci-procal increase in PGE2 levels in the cell culture mediumof preadipocytes (Fig. 4A and B) is consistent with thispossibility. The conversion of preadipocytes to adipocytesis associated with increased triglyceride levels in additionto elevated aP2 levels. To interrogate the potential impor-tance of PGT in regulating adipogenesis, siRNA was used.Silencing of PGT in preadipocytes markedly inhibited the

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Figure 2. PGT modulates thecAMP ! PKA ! aromatasepathway in preadipocytes. A andB, preadipocytes were transfectedwith 2 mg of siRNAs to GFP(control) or PGT and allowed togrow for 36 hours prior to analysis.C and D, control vector or PGTwere overexpressed. A and C,cellular levels of cAMP weredetermined. B and D, PKA activitywas determined. Means � SD areshown; n ¼ 6. *, P < 0.01compared with control. E,preadipocytes were transfectedwith 0.9 mg of CYP19 promoter-luciferase and 0.9 mg of controlsiRNA or PGT siRNA. All cells alsoreceived 0.2 mg of pSVbgal. Thirty-six hours after transfection,aromatase promoter activity wasmeasured. F, preadipocytes thatoverexpressed PGT or controlvector were transientlytransfected with 1.8 mg of CYP19promoter-luciferase and 0.2 mg ofpSVbgal. Following transfection,cells were harvested andaromatase promoter activity wasmeasured. E and F, luciferaseactivity was measured in celllysates, and the activitiesrepresent data that have beennormalized to b-galactosidaseactivity. Means � SD are shown;n ¼ 6. *, P < 0.01 compared withcontrol. G and H, ChIP assayswere performed. G, cells weretransfected with 2 mg of siRNAs toGFP (control) or PGT and allowedto grow for 36 hours prior toanalysis. H, control vector or PGTwere overexpressed inpreadipocytes and then subjectedto analysis. G and H, chromatinfragments wereimmunoprecipitated withantibodies against pCREB and theCYP19 I.3/II promoter wasamplified by real-time PCR. DNAsequencing was carried out, andthe PCR product was confirmed tobe the CYP19 1.3/II promoter,which was not detected whennormal IgG was used or antibodywas omitted fromimmunoprecipitation step (datanot shown). Means � SD areshown; n ¼ 3. *, P < 0.01. Insets(G and H), Western blotting wasperformed using 100 mg of proteinper lane and the blots were probedwith antibodies to pCREB andCREB.

BA

DC

FE

HG






ability of adipocyte differentiating medium to stimulatetriglyceride accumulation and induce aP2 expression(Fig. 6A and B). Differentiation of adipocytes also ledto reduced amounts of PGE2 in the cell culture medium,an effect that was blocked by silencing of PGT (Fig. 6C).

In this context, it was important to carry out additionalexperiments to further evaluate the importance of extra-cellular PGE2 in regulating adipocyte differentiation.Treatment of preadipocytes with exogenous PGE2 sup-pressed the ability of differentiating medium to stimulate

BA

DC

FE

HG

Figure 3. PGT modulates theinteractions of p300 with BRCA1and pCREB at the aromatasepromoter. A, C, E, and G,preadipocytes were transfectedwith 2 mg of siRNAs to GFP(control) or PGT. B, D, F, and H,control vector or PGT expressionvector were overexpressed; cellswere then subjected to analysis.A and B, total RNA was preparedfrom cells and levels of BRCA1mRNA were determined by real-time PCR. Values were normalizedto the expression levels of b-actin.Means � SD are shown; n ¼ 6.*, P < 0.01 compared with control.Insets, Northern blotting was doneon 10 mg RNA per lane and the blotwas probed for BRCA1 andb-actin. C–F, ChIP assays wereperformed. Chromatin fragmentswere immunoprecipitated withantibodies against BRCA1 (C, D)or p300 (E, F) and the CYP19 I.3/IIpromoter was amplified by real-time PCR. DNA sequencing wascarried out, and the PCR productwas confirmed to be the CYP191.3/II promoter. The CYP19 I.3/IIpromoter was not detected whennormal IgG was used or antibodywas omitted fromimmunoprecipitation step (datanot shown). Means � SD areshown; n¼ 3. *, P < 0.01. G and H,500 mg of cell lysate weresubjected to immunoprecipitationwith p300 antiserum and Westernblotting was performed for p300,BRCA1, and pCREB as indicated.These proteins were notimmunoprecipitated with controlIgG. Input is p300.

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triglyceride accumulation and aP2 expression, hallmarksof adipogenesis (Fig. 6D and E). Thus, either silencingPGT or treatment of preadipocytes with exogenous PGE2suppressed the ability of differentiating medium to sti-mulate adipogenesis. Treatment with PGE2 also sup-

pressed the increase in PGT levels associated withadipocyte differentiation (Fig. 6F). Taken together, ourdata suggest that PGT is a critical determinant of extra-cellular levels of PGE2 and thereby adipogenesis andaromatase expression.

Figure 4. Differentiation ofpreadipocytes induces PGT,leading to reduced aromataseexpression. A, total RNA wasprepared from cells and levels ofPGT mRNA were determined byreal-time PCR. Values werenormalized to the expressionlevels of b-actin. Inset, Northernblotting was done using 10 mg oftotal RNA per lane; the blot wasprobed for aP2 and b-actin. PA,preadipocytes; A, adipocytes.B, levels of PGE2 in the cell culturemedium were determined byenzyme immunoassay. C, cellularlevels of cAMP were determined.D, PKA activity was determined.E, Western blotting wasperformed using 100 mg of proteinper lane, and the blot was probedwith antibodies to pCREB andCREB. F, Northern blot analysiswas done using 10 mg of RNA perlane; the blot was probed foraromatase and b-actin.G, aromatase activity wasdetermined using microsomesprepared from cell lysates as in"Materials and Methods." Enzymeactivity is expressed as fmol/mgprotein/min. H, Northern blottingwas performed using 10 mg of totalRNA per lane. The blot wasprobed for the PR and 18S rRNA.A–D and G, means � SD areshown; n ¼ 6. *, P < 0.01compared with preadipocytes.

A B

C D

E F

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Discussion

Obese postmenopausal women are at increased risk ofdeveloping hormone receptor–positive breast cancer(33, 34). This increased risk has been attributed,in part, to elevated levels of circulating and tissue estro-gen related to both increased adipose tissue massand enhanced aromatase expression (3–6). Previousstudies indicate that PGE2 can stimulate CYP19 tran-scription resulting in elevated aromatase levels (8–10,14, 35, 36). The induction of aromatase by PGE2 seemsto be mediated, in part, by suppression of BRCA1, arepressor of CYP19 transcription (10, 17). Given the linkbetween estrogen synthesis and the development andprogression of hormone receptor–positive breast cancer(37, 38), we have attempted to further elucidate the

mechanisms that control the activation of CYP19 tran-scription by PGE2.

Extracellular PGE2 exerts its actions via cell surface Gprotein–coupled receptors (EP receptors) that activate anumber of signaling cascades (39). PGE2 via EP2 and EP4activates the cAMP ! PKA ! CREB pathway leading toenhanced CYP19 transcription and increased aromataseactivity (10). Extracellular PGE2 can be transported viaPGT into the cell, where it is inactivated by cytosolic 15-PGDH (24). Here, we show that changes in the levels ofPGT modulate extracellular levels of PGE2, which lead, inturn, to changes in CYP19 transcription and aromataseactivity. These findings highlight the importance of pros-taglandin transport into the cell as a determinant of localPGE2 levels and aromatase expression. Because changes inthe levels of PGT led to changes in aromatase activity, we

BA

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E

Figure 5. Differentiation ofpreadipocytes to adipocytesmodulates the composition of thetranscription complex at thearomatase promoter. A, chromatinfragments wereimmunoprecipitated withantibodies against pCREB and theCYP19 I.3/II promoter wasamplified by real-time PCR. B,total RNA was prepared from cellsand levels of BRCA1 mRNA weredetermined by real-time PCR.Values were normalized to theexpression levels of b-actin.Means � SD are shown; n ¼ 6.*, P < 0.01. C, ChIP assays wereperformed. Chromatin fragmentswere immunoprecipitated withantibodies against BRCA1, andthe CYP19 I.3/II promoter wasamplified by real-time PCR.D, ChIP assays were performed.Chromatin fragments wereimmunoprecipitated withantibodies against p300 and theCYP19 I.3/II promoter wasamplified by real-time PCR. A, C,and D, DNA sequencing wascarried out, and the PCR productwas confirmed to be the CYP191.3/II promoter. The CYP19 I.3/IIpromoter was not detected whennormal IgG was used or antibodywas omitted fromimmunoprecipitation step (datanot shown). Means � SD areshown; n ¼ 3. *, P < 0.01compared with preadipocytes.E, 500 mg of cell lysate weresubjected to immunoprecipitationwith p300 antiserum and Westernblotting was performed for p300,BRCA1, and pCREB as indicated.These proteins were notimmunoprecipitated with controlIgG. Input is p300.

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A B

C

D

F

E

Figure 6. PGT regulates adipogenesis. A–C, the bar labeled Vehicle represents human preadipocytes treated with preadipocyte medium containing vehicle for48 hours; the bar labeled Diff. Med. represents preadipocytes treated with differentiating medium for 48 hours; bars labeled Diff. Med.þControl siRNA or Diff.Med. þ PGT siRNA represent preadipocytes that were transfected with 2 mg of siRNAs to GFP (Control) or PGT before receiving differentiating medium for 48hours. A, top, total RNA was prepared from cells. The Northern blot was probed for PGT and b-actin. Bottom, cells were stained with Oil Red O and dye wasextracted. Absorbance of extracted dye was measured at 490 nm. Means � SD are shown; n ¼ 6. *. P < 0.01 compared with cells treated with controlsiRNA. B, total RNA was prepared from cells and levels of aP2 mRNA were determined by real-time PCR. Values were normalized to the expression levels ofb-actin. Means � SD are shown; n ¼ 6. *, P < 0.01 compared with control. C, levels of PGE2 in the cell culture medium were determined by enzymeimmunoassay. Means � SD are shown; n ¼ 6. *, P < 0.01 compared with cells treated with control siRNA. D–F, bars labeled Vehicle represent cells thatreceived preadipocyte medium containing vehicle for 48 hours; bars labeled Diff. Med. represent cells that received differentiating medium for 48 hours; barslabeled Diff. Med. þ PGE2 represent cells that received differentiating medium containing either 250 nmol/L or 500 nmol/L PGE2 for 48 hours. D, cells werestained with Oil Red O and dye was extracted. Absorbance of extracted dye was measured at 490 nm. Means � SD are shown; n ¼ 6. *. P < 0.01 comparedwith cells treated with differentiating medium alone. E, total RNA was prepared from cells and levels of aP2 mRNA were determined by real-time PCR.Values were normalized to the expression levels of b-actin. Means� SD are shown; n¼ 6. *, P < 0.01 compared with cells treated with differentiating medium.F, total RNA was prepared from cells. The Northern blot was probed for PGT and b-actin.






also investigated the role of PGT as a determinant ofestrogen-dependent gene expression. Silencing PGTincreased extracellular levels of PGE2, stimulated aromataseactivity, and induced the expression of the PR, a prototypicestrogen response gene. Consistent with these findings,overexpressing PGT led to reduced extracellular levels ofPGE2, decreased aromatase activity, and downregulationof PR.

Because PGE2 activates the cAMP ! PKA ! CREB path-way leading to enhanced CYP19 transcription, we investi-gated whether changes in the levels of PGT affected thissignal transduction pathway. Consistent with the change inextracellular PGE2 levels, silencing PGT induced cAMPlevels and PKA activity; overexpressing PGT led to reducedcAMP levels and PKA activity. Furthermore, silencing PGTstimulated the binding of pCREB to the CYP19 I.3/IIpromoter whereas binding of pCREB to the CYP19 I.3/IIpromoter was suppressed when PGT was overexpressed.The tumor suppressor BRCA1 plays a significant role inrepressing aromatase expression (10, 16–18, 40). BRCA1binds directly to the CYP19 1.3/II promoter region andsuppresses transcription (17). Agents including PGE2 thatstimulate cAMP signaling suppress BRCA1 levels resultingin enhanced CYP19 transcription (17, 41). We extend uponthese findings and show that PGT is a determinant ofBRCA1 levels. Silencing PGT suppressed levels of BRCA1and caused a decrease in its interaction with the CYP19 I.3/II promoter. In contrast, overexpression of PGT resulted inboth an increase in BRCA1 levels and an increase in itsinteraction with the CYP19 I.3/II promoter. To our knowl-edge, this is first time that changes in the expression of anytype of transporter have been linked to altered expressionof BRCA1. Our data also support the notion that changes inthe expression of BRCA1 in the stroma could be veryimportant for the development of hormone-dependentcancers.

The coactivator CBP/p300, which possesses histone acet-yltransferase activity (42), is also known to be importantfor PGE2-mediated activation of CYP19 transcription (10).Previously, PGE2 was found to stimulate the recruitment ofp300 to the CYP19 I.3/II promoter. In addition, the inter-action between p300 and pCREB was enhanced by treat-ment with PGE2. In the current study, we present evidencethat silencing PGT stimulated the recruitment of p300 tothe CYP19 I.3/II promoter. Importantly, the interactionbetween p300 and pCREB was enhanced whereas thatbetween p300 and BRCA1 was reduced by silencingPGT. In contrast, overexpressing PGT inhibited the recruit-ment of p300 to the CYP19 I.3/II promoter and stimulatedthe interaction between p300 and BRCA1.

Previously, levels of aromatase were reported to be sig-nificantly higher in preadipocytes than in adipocytes, afinding that we confirmed (25). PGE2 can suppress adipo-genesis, the process by which preadipocytes becomemature adipocytes (32). Importantly, we found that levelsof PGT were reduced in preadipocytes versus adipocyteswith a reciprocal increase in PGE2 levels in the cell culturemedium. Collectively, these findings suggested the

possibility that PGT may regulate adipogenesis leading,in turn, to changes in aromatase expression. To this end,we showed that silencing of PGT led to increased levels ofPGE2 in the cell culture medium and inhibition of adipo-cyte differentiation. The significance of this effect of PGT onPGE2 levels was highlighted by our finding that the treat-ment of preadipocytes with exogenous PGE2 also inhibitedadipocyte differentiation. Finally, we explored the possibi-lity that reduced levels of PGT in preadipocytes versusadipocytes could account for the significantly increasedlevels of aromatase in preadipocytes. The reduction inPGT levels in preadipocytes versus adipocytes led toenhanced PGE2 ! cAMP ! PKA signaling. Increasedbinding of pCREB, reduced binding of BRCA1, andincreased recruitment of p300 to the CYP19 1.3/II promo-ter were found in preadipocytes versus adipocytes. Thesefindings strongly suggest that the differentiation of prea-dipocytes to adipocytes leads to a change in the PGT !CYP19 axis, resulting in changes in aromatase levels. Nota-bly, ligands of PPARg stimulate adipocyte differentiation(43) and suppress aromatase expression (44). On the basis

Figure 7. Signal transduction pathway by which PGT regulates aromataseexpression. PGT, a transporter containing 12-transmembrane spanningdomains, mediates the rate-limiting step in PGE2 signal termination. Itcarries PGE2 from the extracellular environment across the plasmamembrane to the cytoplasm where 15-PGDH oxidizes it to 15-keto PGE2.Changes in amounts of PGT alter extracellular levels of PGE2, which exertsits effects by binding to EP receptors, G protein–coupled receptors. PGE2

via EP2 and EP4 activates the cAMP! PKA pathway resulting in enhancedinteraction between pCREB, p300, and aromatase promoter I.3/II. PGE2

also causes a decrease in amounts of BRCA1, a repressor of aromatasetranscription, and reduced interaction between BRCA1 and the aromatasepromoter I.3/II. The increased interaction between pCREB and p300 andthe reduced interaction between BRCA1 and p300 contribute to enhancedaromatase transcription and increased aromatase activity. Levels of PGTare reduced in preadipocytes versus adipocytes, resulting in increasedextracellular levels of PGE2. This increase leads, in turn, to activation of thecAMP ! PKA ! CREB pathway resulting in enhanced aromatasetranscription.

Subbaramaiah et al.





of our results, it will be worthwhile to determine whetherthese agents induce PGT and thereby suppress aromataselevels.Previous studies have focused on the importance of

enzymes involved in PGE2 synthesis and catabolism asdeterminants of aromatase expression (10–15). Thisstudy is the first to suggest that prostaglandin transportis a determinant of CYP19 transcription and aromataseexpression. Notably, the current findings could be impor-tant for understanding both normal physiology and dis-ease. For example, PGT expression is modulated inepithelial and stromal cells of the human endometriumduring the menstrual cycle (45). Our results suggest thatchanges in PGT levels will affect both prostaglandin levelsand estrogen synthesis in the local microenvironment,which could be important for normal menstrual function.Treatment with lipopolysaccharide (LPS) downregulatesPGT in both the lung and the liver (46), which could leadto increased aromatase levels and estrogen synthesis. Thispossibility should be explored, as estrogen antagonizessome of the proinflammatory effects of LPS (47). Levels ofPGT are reduced in a variety of tumors including lungcancer (48). Future studies are warranted to determinewhether reduced levels of PGT contribute to the elevatedlevels of aromatase found in some tumors (49). Given theimportance of changes in expression of PGT, an influxtransporter, on aromatase expression, it will also beworthwhile to determine whether multidrug resistance–

associated protein 4, an efflux transporter, plays a role inregulating aromatase expression. Taken together, our dataprovide evidence that PGT-mediated delivery of PGE2from the extracellular milieu to the cytoplasm regulatesadipocyte differentiation and the cAMP ! PKA ! CREBpathway leading to changes in the interaction betweenpCREB, BRCA1, p300, and the CYP19 I.3/II promoter(Fig. 7). Agents that induce PGT should enhance PGE2uptake and catabolism, suppress aromatase activity, andthereby reduce the risk of hormone receptor–positivebreast cancer, perhaps especially so among obese post-menopausal women.

Disclosure of Potential Conflicts of Interest

A.J. Dannenberg is a member of the Scientific Advisory Board ofTragara Pharmaceuticals, Inc., a company that is developing a selectiveCOX-2 inhibitor. The other authors disclosed no potential conflicts ofinterest.

Grant Support

This work was supported by the Breast Cancer Research Foundation andthe Botwinick-Wolfensohn Foundation (in memory of Mr. and Mrs. Ben-jamin Botwinick).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 22, 2010; revised December 6, 2010; acceptedDecember 14, 2010; published OnlineFirst January 6, 2011.

References1. Calle EE, Kaaks R. Overweight, obesity and cancer: epidemiological

evidence and proposed mechanisms. Nat Rev Cancer 2004;4:579–91.

2. Lorincz AM, Sukumar S. Molecular links between obesity and breastcancer. Endocr Relat Cancer 2006;13:279–92.

3. Key TJ, Appleby PN, Reeves GK, Roddam A, Dorgan JF, Longcope C,et al. Bodymass index, serum sex hormones, and breast cancer risk inpostmenopausal women. J Natl Cancer Inst 2003;95:1218–26.

4. Wake DJ, Strand M, Rask E, Westerbacka J, Livingstone DE, Soder-berg S, et al. Intra-adipose sex steroid metabolism and body fatdistribution in idiopathic human obesity. Clin Endocrinol 2007;66:440–6.

5. van Kruijsdijk RC, van derWall E, Visseren FL. Obesity and cancer: therole of dysfunctional adipose tissue. Cancer Epidemiol BiomarkersPrev 2009;18:2569–78.

6. Cleary MP, Grossmann ME. Minireview. Obesity and breast cancer:the estrogen connection. Endocrinology 2009;150:2537–42.

7. Bulun SE, Lin Z, Imir G, Amin S, Demura M, Yilmaz B, et al.Regulation of aromatase expression in estrogen-responsive breastand uterine disease: from bench to treatment. Pharmacol Rev2005;57:359–83.

8. Zhao Y, Agarwal VR, Mendelson CR, Simpson ER. Estrogen biosynth-esis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP,leading to activation of promoter II of the CYP19 (aromatase) gene.Endocrinology 1996;137:5739–42.

9. Zhao Y, Agarwal VR, Mendelson CR, Simpson ER. Transcriptionalregulation of CYP19 gene (aromatase) expression in adipose stromalcells in primary culture. J Steroid Biochem Mol Biol 1997;61:203–10.

10. Subbaramaiah K, Hudis C, Chang SH, Hla T, Dannenberg AJ. EP2 andEP4 receptors regulate aromatase expression in human adipocytesand breast cancer cells. Evidence of a BRCA1 and p300 exchange. JBiol Chem 2008;283:3433–44.

11. Brodie AM, Lu Q, Long BJ, Fulton A, Chen T, Macpherson N, et al.Aromatase and COX-2 expression in human breast cancers. J SteroidBiochem Mol Biol 2001;79:41–7.

12. Brueggemeier RW, Quinn AL, Parrett ML, Joarder FS, Harris RE,Robertson FM. Correlation of aromatase and cyclooxygenase geneexpression in human breast cancer specimens. Cancer Lett1999;140:27–35.

13. Irahara N, Miyoshi Y, Taguchi T, Tamaki Y, Noguchi S. Quantitativeanalysis of aromatase mRNA expression derived from various pro-moters (I.4, I.3, PII and I.7) and its association with expression of TNF-alpha, IL-6 and COX-2 mRNAs in human breast cancer. Int J Cancer2006;118:1915–21.

14. Subbaramaiah K, Howe LR, Port ER, Brogi E, Fishman J, Liu CH, et al.HER-2/neu status is a determinant of mammary aromatase activityin vivo: evidence for a cyclooxygenase-2-dependent mechanism.Cancer Res 2006;66:5504–11.

15. Wolf I, O’Kelly J, Rubinek T, Tong M, Nguyen A, Lin BT, et al. 15-hydroxyprostaglandin dehydrogenase is a tumor suppressor ofhuman breast cancer. Cancer Res 2006;66:7818–23.

16. Hu Y, Ghosh S, Amleh A, Yue W, Lu Y, Katz A, et al. Modulation ofaromatase expression by BRCA1: a possible link to tissue-specifictumor suppression. Oncogene 2005;24:8343–8.

17. Lu M, Chen D, Lin Z, Reierstad S, Trauernicht AM, Boyer TG, et al.BRCA1 negatively regulates the cancer-associated aromatase pro-moters I.3 and II in breast adipose fibroblasts and malignant epithelialcells. J Clin Endocrinol Metab 2006;91:4514–9.

18. Ghosh S, Lu Y, Katz A, Hu Y, Li R. Tumor suppressor BRCA1 inhibits abreast cancer-associated promoter of the aromatase gene (CYP19) inhuman adipose stromal cells. Am J Physiol Endocrinol Metab2007;292:E246–52.

19. Terry MB, Gammon MD, Zhang FF, Tawfik H, Teitelbaum SL, BrittonJA, et al. Association of frequency and duration of aspirin use and






hormone receptor status with breast cancer risk. JAMA 2004;291:2433–40.

20. Gierach GL, Lacey JV Jr, Schatzkin A, Leitzmann MF, Richesson D,Hollenbeck AR, et al. Nonsteroidal anti-inflammatory drugs and breastcancer risk in the National Institutes of Health-AARP Diet and HealthStudy. Breast Cancer Res 2008;10:R38.

21. Chang HY, Locker J, Lu R, Schuster VL. Failure of postnatal ductusarteriosus closure in prostaglandin transporter-deficient mice. Circu-lation 2010;121:529–36.

22. Schuster VL. Prostaglandin transport. Prostaglandins Other LipidMediat 2002;68–69:633–47.

23. Lu R, Kanai N, Bao Y, Schuster VL. Cloning, in vitro expression, andtissue distribution of a human prostaglandin transporter cDNA(hPGT).J Clin Invest 1996;98:1142–9.

24. Nomura T, Lu R, Pucci ML, Schuster VL. The two-step model ofprostaglandin signal termination: in vitro reconstitution with theprostaglandin transporter and prostaglandin 15 dehydrogenase.Mol Pharmacol 2004;65:973–8.

25. Dieudonne MN, Sammari A, Dos Santos E, Leneveu MC, Giudicelli Y,Pecquery R. Sex steroids and leptin regulate 11beta-hydroxysteroiddehydrogenase I and P450 aromatase expressions in humanpreadipocytes: Sex specificities. J Steroid Biochem Mol Biol 2006;99:189–96.

26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurementwith the Folin phenol reagent. J Biol Chem 1951;193:265–75.

27. Laemmli UK. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 1970;227:680–5.

28. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc Natl Acad Sci USA 1979;76:4350–4.

29. Read LD, Snider CE, Miller JS, Greene GL, Katzenellenbogen BS.Ligand-modulated regulation of progesterone receptor messengerribonucleic acid and protein in human breast cancer cell lines. MolEndocrinol 1988;2:263–71.

30. Savouret JF, Bailly A, Misrahi M, Rauch C, Redeuilh G, ChauchereauA, et al. Characterization of the hormone responsive element involvedin the regulation of the progesterone receptor gene. EMBO J1991;10:1875–83.

31. Yokota T, Meka CS, Medina KL, Igarashi H, Comp PC, Takahashi M,et al. Paracrine regulation of fat cell formation in bone marrow culturesvia adiponectin and prostaglandins. J Clin Invest 2002;109:1303–10.

32. Tsuboi H, Sugimoto Y, Kainoh T, Ichikawa A. Prostanoid EP4 receptoris involved in suppression of 3T3-L1 adipocyte differentiation. Bio-chem Biophys Res Commun 2004;322:1066–72.

33. Petrelli JM, Calle EE, Rodriguez C, ThunMJ. Bodymass index, height,and postmenopausal breast cancer mortality in a prospective cohortof US women. Cancer Causes Control 2002;13:325–32.

34. Feigelson HS, Patel AV, Teras LR, Gansler T, Thun MJ, et al. Adultweight gain and histopathologic characteristics of breast canceramong postmenopausal women. Cancer 2006;107:12–21.

35. Prosperi JR, Robertson FM. Cyclooxygenase-2 directly regulatesgene expression of P450 Cyp19 aromatase promoter regions pII,pI.3 and pI.7 and estradiol production in human breast tumor cells.Prostaglandins Other Lipid Mediat 2006;81:55–70.

36. Cai Z, Kwintkiewicz J, Young ME, Stocco C. Prostaglandin E2increases cyp19 expression in rat granulosa cells: implication ofGATA-4. Mol Cell Endocrinol 2007;263:181–9.

37. Strassmer-Weippl K, Goss PE. Prevention of breast cancer usingSERMs and aromatase inhibitors. J Mammary Gland Biol Neoplasia2003;8:5–18.

38. Goss PE, Ingle JN, Martino S, Robert NJ, Muss HB, Piccart MJ, et al. Arandomized trial of letrozole in postmenopausal women after fiveyears of tamoxifen therapy for early-stage breast cancer. N Engl JMed 2003;349:1793–802.

39. Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem2007;282:11613–7.

40. Chand AL, Simpson ER, Clyne CD. Aromatase expression isincreased in BRCA1 mutation carriers. BMC Cancer 2009;9:148.

41. Lu Y, Amleh A, Sun J, Jin X, McCullough SD, Baer R, et al. Ubiquitina-tion and proteasome-mediated degradation of BRCA1 and BARD1during steroidogenesis in human ovarian granulosa cells. Mol Endo-crinol 2007;21:651–63.

42. Das C, Lucia MS, Hansen KC, Tyler JK. CBP/p300-mediated acet-ylation of histone H3 on lysine 56. Nature 2009;459:113–7.

43. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out thebig picture. Cell 1996;87:377–89.

44. Rubin GL, Duong JH, Clyne CD, Speed CJ, Murata Y, Gong C, et al.Ligands for the peroxisomal proliferator-activated receptor gammaand the retinoid X receptor inhibit aromatase cytochrome P450(CYP19) expressionmediated by promoter II in human breast adipose.Endocrinology 2002;143:2863–71.

45. Kang J, Chapdelaine P, Parent J, Madore E, Laberge PY, Fortier MA.Expression of human prostaglandin transporter in the human endo-metrium across the menstrual cycle. J Clin Endocrinol Metab2005;90:2308–13.

46. Ivanov AI, Scheck AC, Romanovsky AA. Expression of genes con-trolling transport and catabolism of prostaglandin E2 in lipopolysac-charide fever. Am J Physiol Regul Integr Comp Physiol 2003;284:R698–706.

47. Murphy AJ, Guyre PM, Pioli PA. Estradiol suppresses NF-kappa Bactivation through coordinated regulation of let-7a and miR-125bin primary human macrophages. J Immunol 2010;184:5029–37.

48. Holla VR, BacklundMG, Yang P, Newman RA, DuBois RN. Regulationof prostaglandin transporters in colorectal neoplasia. Cancer Prev Res2008;1:93–9.

49. Niikawa H, Suzuki T, Miki Y, Suzuki S, Nagasaki S, Akahira J, et al.Intratumoral estrogens and estrogen receptors in human non-smallcell lung carcinoma. Clin Cancer Res 2008;14:4417–26.

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2011;4:194-206. Published OnlineFirst January 6, 2011.Cancer Prev Res Kotha Subbaramaiah, Clifford A. Hudis and Andrew J. Dannenberg

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