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Research Article Obesity Is Associated with Inflammation and Elevated Aromatase Expression in the Mouse Mammary Gland Kotha Subbaramaiah 1 , Louise R. Howe 2 , Priya Bhardwaj 1 , Baoheng Du 1 , Claudia Gravaghi 1 , Rhonda K. Yantiss 3 , Xi Kathy Zhou 4 , Victoria A. Blaho 3 , Timothy Hla 3 , Peiying Yang 5 , Levy Kopelovich 6 , Clifford A. Hudis 7 , and Andrew J. Dannenberg 1 Abstract Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese post- menopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)- mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflamma- tion, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-kB and increased levels of proinflammatory mediators (TNF-a, IL-1b, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal- vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflam- matory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-kB activity in macrophages leading to increased levels of TNF-a, IL-1b, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity ! inflammation ! aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis. Cancer Prev Res; 4(3); 329–46. Ó2011 AACR. Introduction More than 40,000 women in the United States die each year of metastatic breast cancer. Approximately two-thirds of breast cancer patients have tumors that express estrogen receptors. Obese postmenopausal women are at signifi- cantly increased risk of developing hormone receptor (HR)-positive breast cancer (1). Estrogens are synthesized from androgens in a reaction catalyzed by cytochrome P450 aromatase, encoded by the CYP19 gene (2). After menopause, peripheral aromatization of androgen precur- sors in adipose tissue is largely responsible for estrogen biosynthesis (3). The increased risk of HR-positive breast cancer in obese postmenopausal women has been attrib- uted, in part, to elevated levels of circulating estradiol related to both increased adipose tissue and elevated aro- matase expression in subcutaneous adipose tissue (4–7). Whether obesity alone leads to changes in aromatase expression in breast tissue is uncertain. Because of the significance of estrogen biosynthesis in the pathogenesis of HR-positive breast cancer, efforts have been made to elucidate the mechanisms that regulate the tran- scription of CYP19 (8). Multiple lines of evidence suggest a key role for proinflammatory mediators including TNF-a, IL-1b, and cyclooxygenase (Cox)-derived prostaglandin E 2 (PGE 2 ) in stimulating CYP19 transcription resulting in increased aromatase activity (9–17). Obesity causes inflam- mation and increased levels of proinflammatory mediators in adipose tissue (6, 18–20). In humans, a correlation has been observed between increased body mass index and elevated levels of aromatase in subcutaneous fat (7). This Authors' Affiliations: Departments of 1 Medicine, 2 Cell and Developmental Biology, 3 Pathology and Laboratory Medicine, and 4 Public Health, Weill Cornell Medical College, New York; 5 Department of General Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 6 Divi- sion of Cancer Prevention, National Cancer Institute, Bethesda, Maryland; and 7 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York Note: Supplementary material for this article is available at Cancer Pre- vention Research Online (http://cancerprevres.aacrjournals.org/). Corresponding Authors: Andrew J. Dannenberg, Department of Medi- cine and Weill Cornell Cancer Center, 525 East 68th St., Room F-206, New York, NY 10065. Phone: 212-746-4403; Fax: 212-746-4885. E-mail: [email protected]; or Kotha Subbaramaiah, Department of Med- icine and Weill Cornell Cancer Center, 525 East 68th St., Room F-203, New York, NY 10065. Phone: 212-746-4402; Fax: 212-746-4885. E-mail: [email protected] doi: 10.1158/1940-6207.CAPR-10-0381 Ó2011 American Association for Cancer Research. Cancer Prevention Research www.aacrjournals.org 329 for Cancer Research. on July 14, 2018. © 2011 American Association cancerpreventionresearch.aacrjournals.org Downloaded from

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Research Article

Obesity Is Associated with Inflammation and Elevated AromataseExpression in the Mouse Mammary Gland

Kotha Subbaramaiah1, Louise R. Howe2, Priya Bhardwaj1, Baoheng Du1, Claudia Gravaghi1,Rhonda K. Yantiss3, Xi Kathy Zhou4, Victoria A. Blaho3, Timothy Hla3, Peiying Yang5,Levy Kopelovich6, Clifford A. Hudis7, and Andrew J. Dannenberg1

AbstractElevated circulating estrogen levels are associated with increased risk of breast cancer in obese post-

menopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-

mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting

estrogens are then secreted into the systemic circulation. The potential links between obesity, inflamma-

tion, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed

necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary

glands and visceral fat. The presence of CLS was associated with activation of NF-kB and increased levels of

proinflammatory mediators (TNF-a, IL-1b, Cox-2), which were paralleled by elevated levels of aromatase

expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-

vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflam-

matory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes.

Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-kB activity in

macrophages leading to increased levels of TNF-a, IL-1b, and Cox-2, each of which contributed to the

induction of aromatase in preadipocytes. The discovery of the obesity! inflammation! aromatase axis in

the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms

underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women,

the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their

generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor

prognosis. Cancer Prev Res; 4(3); 329–46. �2011 AACR.

Introduction

More than 40,000 women in the United States die eachyear of metastatic breast cancer. Approximately two-thirdsof breast cancer patients have tumors that express estrogenreceptors. Obese postmenopausal women are at signifi-cantly increased risk of developing hormone receptor

(HR)-positive breast cancer (1). Estrogens are synthesizedfrom androgens in a reaction catalyzed by cytochromeP450 aromatase, encoded by the CYP19 gene (2). Aftermenopause, peripheral aromatization of androgen precur-sors in adipose tissue is largely responsible for estrogenbiosynthesis (3). The increased risk of HR-positive breastcancer in obese postmenopausal women has been attrib-uted, in part, to elevated levels of circulating estradiolrelated to both increased adipose tissue and elevated aro-matase expression in subcutaneous adipose tissue (4–7).Whether obesity alone leads to changes in aromataseexpression in breast tissue is uncertain.

Because of the significance of estrogen biosynthesis in thepathogenesis of HR-positive breast cancer, efforts have beenmade to elucidate the mechanisms that regulate the tran-scription of CYP19 (8). Multiple lines of evidence suggest akey role for proinflammatory mediators including TNF-a,IL-1b, and cyclooxygenase (Cox)-derived prostaglandin E2(PGE2) in stimulating CYP19 transcription resulting inincreased aromatase activity (9–17). Obesity causes inflam-mation and increased levels of proinflammatory mediatorsin adipose tissue (6, 18–20). In humans, a correlation hasbeen observed between increased body mass index andelevated levels of aromatase in subcutaneous fat (7). This

Authors' Affiliations: Departments of 1Medicine, 2Cell andDevelopmentalBiology, 3Pathology and Laboratory Medicine, and 4Public Health, WeillCornell Medical College, NewYork; 5Department of General Oncology, TheUniversity of Texas M.D. Anderson Cancer Center, Houston, Texas; 6Divi-sion of Cancer Prevention, National Cancer Institute, Bethesda, Maryland;and 7Department of Medicine, Memorial Sloan-Kettering Cancer Center,New York

Note: Supplementary material for this article is available at Cancer Pre-vention Research Online (http://cancerprevres.aacrjournals.org/).

Corresponding Authors: Andrew J. Dannenberg, Department of Medi-cine andWeill Cornell Cancer Center, 525 East 68th St., Room F-206, NewYork, NY 10065. Phone: 212-746-4403; Fax: 212-746-4885. E-mail:[email protected]; or Kotha Subbaramaiah, Department of Med-icine andWeill Cornell Cancer Center, 525 East 68th St., Room F-203, NewYork, NY 10065. Phone: 212-746-4402; Fax: 212-746-4885. E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-10-0381

�2011 American Association for Cancer Research.

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constellation of findings suggested the possibility that obe-sity-related inflammation is causally linked to increasedaromatase expression. Although studies have shown thatobesity causes inflammation in both visceral and subcuta-neous fat (20), it is unknownwhether similar changes occurin the mammary gland.

In this study, we had several objectives. Our first goal wasto determine whether obesity led to inflammation in themammary gland in addition to visceral fat. Second it wasimportant to establish whether obesity was associated withincreased levels of both proinflammatory mediators andaromatase in the mammary gland and visceral fat. Ourthird objective was to develop a cellular model that couldexplain the link between obesity, inflammation, andincreased aromatase expression. Here we show that obesitycaused inflammation in the mammary glands (MG) andvisceral fat of mice. Increased levels of aromatase mRNAand activity paralleled the elevated levels of TNF-a, IL-1b,and Cox-2 in the mammary gland and visceral fat of obesemice. Finally, we present evidence that activation of macro-phages, a component of the inflammatory cell infiltrate inadipose and mammary tissues of obese mice, led toincreased production of proinflammatorymediators whichcontributed, in turn, to the induction of aromatase. Col-lectively, these results provide potential insights into whyobese postmenopausal women are at increased risk ofdeveloping HR-positive breast cancer. Moreover, overex-pression of aromatase in the adipose tissue of obesemice offers a possible explanation for why the recom-mended doses of aromatase inhibitor are less effectivein the treatment of HR-positive breast cancer in obeseversus lean women (21). The discovery of the obesity !inflammation ! aromatase axis provides the basis fordeveloping mechanism-based strategies to reduce the riskof HR-positive breast cancer in this growing segment of thepopulation.

Materials and Methods

MaterialsMedium to grow visceral preadipocytes was purchased

from ScienCell Research Laboratories. FBS was purchasedfrom Invitrogen. Neutralizing antibodies and enzymeimmunoassay (EIA) kits for TNF-a and IL-1b were pur-chased from R&D systems. Antibodies to phospho-p65,p65, histone H3, Cox-2, and b-actin were from Santa CruzBiotechnology. Lowry protein assay kits, lipopolysacchar-ide, butylated hydroxytoluene (BHT), IFNg , horseradishperoxidase–conjugated secondary antibody, collagenasetype 1, bovine serum albumin (BSA), glucose-6-phosphate,glycerol, pepstatin, leupeptin, glucose-6-phosphate dehy-drogenase, and rotenone were from Sigma. BAY11-7082was purchased from InvivoGen. Celecoxib was obtainedfrom LKT Laboratories. Saturated fatty acids were obtainedfrom Nu-CheK Prep. ECL Western blotting detectionreagents were from Amersham Biosciences. Nitrocellulosemembranes were from Schleicher & Schuell. 1b-[3H]-androstenedione and [32P]dCTP were from Perkin-Elmer

Life Science. pSVbgal, electrophoretic mobility gel shift,and plasmid DNA isolation kits were from Promega. TheF4/80 and aP2 cDNAs were obtained from Open Biosys-tems. The 18S rRNA cDNA was purchased from Ambion.RNeasy mini kits were purchased from Qiagen. MuLVreverse transcriptase, RNase inhibitor, oligo (dT)16, andSYBR green PCR master mix were obtained from AppliedBiosystems. Real-time PCR primers were synthesized bySigma-Genosys. Luciferase assay substrates and cell lysisbuffer were from BD Biosciences. PGE2, PGE2-d4, andPGE2 EIA kits were purchased from Cayman Chemicals.Protein assay kits were purchased from Bio-Rad. Chroma-tin immunoprecipitation (ChIP) assay kits were purchasedfromMillipore. Control siRNA and siRNAs for p65, TNF-a,IL-1b, and Cox-2 were purchased from Thermo Scientific.

Animal modelsBoth dietary and genetic models of obesity were used. In

the dietary model, at 5 weeks of age, ovary intact andovariectomized (OVX) C57BL/6J female mice (JacksonLaboratories) were randomized (n ¼ 10/group) to receiveeither low fat (LF) or high fat (HF) diets. The LF(D12450Bi) and HF (D12492i) diets contain 10 kcal%fat and 60 kcal% fat, respectively (Research Diets) and arecommonly used in studies of obesity (22). Mice were fedthese diets ad libitum for 10 weeks before being sacrificed.In the genetic model, female ob/ob and control C57BL/6Jmice were obtained at 8 weeks of age (Jackson Labora-tories) and fed PicoLab Rodent Diet 20, #5053 (W.F. Fisher& Son) ad libitum for 3 weeks prior to sacrifice. Followingsacrifice, MG and visceral fat were snap frozen in liquidnitrogen and stored at �80�C for molecular analysis orformalin fixed for histological and immunohistochemicalanalyses. In experiments to separate the adipose and stro-mal-vascular fractions (SVF) of themammary gland, tissueswere directly subjected to cell fractionation. The animalprotocol was approved by the Institutional Animal Careand Use Committee at Weill Cornell Medical College.

Light microscopy and immunohistochemistryFour-micron thick sections were prepared from forma-

lin-fixed, paraffin-embedded mammary gland tissue andvisceral fat and stained with hematoxylin and eosin. Thetotal number of inflammatory foci per section was quanti-fied by a pathologist (RKY) and the amount of tissuepresent on each slide was recorded, to determine thenumber of inflammatory foci per cm2 of tissue.

Immunohistochemical stains against F4/80 were per-formed using standard techniques. Briefly, 4-mm forma-lin-fixed, paraffin-embedded tissue sections of mammarygland were deparaffinized and rehydrated prior to antigenretrieval in citrate buffer, pH 6.0 (Dako). Endogenousperoxidase activity was quenched with 0.3% H2O2, andnonspecific binding blocked with 0.5% Tween-20 in 5%BSA prior to overnight incubation with rat anti-mouse F4/80 primary antibody (Serotec). Horseradish peroxidase-labeled goat anti-rat IgG (Jackson ImmunoResearch) andNovaRed substrate (Vector Labs) were used for detection.

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Sections were counterstained with hematoxylin (VectorLabs).

Separation of stromal-vascular and adipocytefractionsTo fractionate mammary gland tissue into SVF and adi-

pocyte fractions, we utilized a modified version of an exist-ing protocol (23). Mammary gland tissue was minced intosmall pieces and placed in sterile plastic tubes with Krebs-Ringer buffer containing 25 mmol/L NaHCO3, 11 mmol/Lglucose, 25 mmol/L Hepes (pH 7.4), 2% BSA, and 1.5 mg/mL collagenase type I. The ratio between adipose tissuemass and incubation solution was 1:4 (w/v). The tissuesuspension was incubated at 37�C with gentle shaking for45 to 60 minutes. Once digestion was completed, sampleswere passed through a sterile 250-mm nylon mesh (VWR).The suspension was centrifuged at 200 � g for 10 minutes;the pelleted cells were collected as SVF and the floatingcells were considered the adipocyte-enriched fraction.The adipocytes were washed twice with Krebs-Ringer-bicar-bonate-Hepes-BSAbuffer and centrifugedas before. TheSVFwas resuspended in erythrocyte lysis buffer consisting of0.154 mol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/LEDTA, and incubated at room temperature for 10 minutes.The erythrocyte-depleted SVF was centrifuged at 400 � gfor 5 minutes, the pellet was resuspended and washed4 times in Krebs-Ringer-bicarbonate-Hepes-BSA bufferand centrifuged at 400 � g for 5 minutes. After washing,the SVF and adipocyte fractions were subjected to analysisor cultured.

Tissue cultureHuman visceral preadipocytes (ScienCell) were grown in

preadipocyte medium containing 10% FBS. 3T3-L1 cells(ZenBio) were grown in DMEM supplemented with 10%calf bovine serum. THP-1 cells (ATCC) were maintained inRPMI-1640 medium (Invitrogen) supplemented with 10%FBS. These cells were treated with phorbol 12-myristate 13-acetate (10 ng/mL) overnight to differentiate them intomacrophages. Human monocytes (Astarte Biologics) wereactivated with IFNg (15 ng/mL) and lipopolysaccharide(15 ng/mL) in RPMI-1640 medium for 4 days. THP-1 cellsand bloodmonocyte–derived macrophages were then trea-ted with saturated fatty acids. To prepare conditionedmedium (CM), these cells were treated with saturated fattyacids for 12 hours in medium comprised of RPMI-1640and preadipocyte medium at a 1:1 ratio. Following treat-ment with fatty acids, the medium was removed and cellswere washed thrice with PBS to remove fatty acids. Subse-quently, fresh medium was added for 24 hours. This CMwas then collected and centrifuged at 4,000 rpm for 30minutes to remove cell debris. CM was then used to treatpreadipocytes.

Immunoblot analysisLysates were prepared by treating cells with lysis buffer

(150 mmol/L NaCl, 100 mmol/L Tris, pH 8.0, 1% Tween20, 50 mmol/L diethyldithiocarbamate, 1 mmol/L EDTA,

1 mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL apro-tinin, 10 mg/mL trypsin inhibitor, and 10 mg/mL leupep-tin). Lysates were sonicated for 20 seconds on ice andcentrifuged at 10,000 � g for 10 minutes to sedimentthe particulate material. The protein concentration of thesupernatant was measured by the method of Lowry andcolleagues (24). SDS-PAGE was done under reducing con-ditions on 10% polyacrylamide gels. The resolved proteinswere transferred onto nitrocellulose sheets and then incu-bated with primary antisera. Antibodies to phospho-p65,p65, histone H3, Cox-2, and b-actin were used. Secondaryantibody to IgG conjugated to horseradish peroxidase wasused. The blot was probed with the ECL Western blotdetection system.

Northern blottingTotal RNA was prepared from SVF and adipocytes

derived from the mammary gland using an RNA isolationkit. Ten micrograms of total RNA/lane were electrophor-esed in a formaldehyde-containing 1% agarose gel andtransferred to nylon-supported membranes. F4/80, aP2and 18S rRNA probes were labeled with [32P]dCTPby random priming. The blots were probed as describedpreviously (25).

Quantitative real-time PCRTotal RNA was isolated using the RNeasy mini kit. For

tissue analyses, poly A RNA was prepared with an OligotexmRNA mini kit (Qiagen). Poly A RNA was reversed tran-scribed using murine leukemia virus reverse transcriptaseand oligo (dT)16 primer. The resulting cDNAwas then usedfor amplification using primers listed in SupplementaryTable S1. Glyceraldehyde 3 phosphate dehydrogenase(GAPDH) and b-actin were used as endogenous normal-ization controls for tissue and cell culture analyses, respec-tively. Real-time PCR was performed using 2x SYBR greenPCR master mix on a 7500 Real-time PCR system (AppliedBiosystems). Relative fold induction was determined usingthe ddCT (relative quantification) analysis protocol.

Transient transfectionsNF-kB-luciferase (Panomics) and pSV-bgal were trans-

fected into THP-1 cells using the Amaxa system. After 24hours of incubation, the medium was replaced with basalmedium. The activities of luciferase and b-galactosidasewere measured in cellular extracts. For siRNA transfections,THP-1 cells were plated at 60% confluence and transfectedwith nontargeting siRNA or siRNA targeting TNF-a, IL-1b,Cox-2, and p65 using Dharmafect 4 for 36 hours. Subse-quently, the cells were treated with vehicle or saturated fattyacids.

ChIP assay and qPCRChIP assays were performed with a kit according to the

manufacturer’s instructions. A total of 3.5 � 106 cells werecross-linked in a 1% formaldehyde solution for 10 minutesat 37�C. Cells were then lysed in 200 mL of SDS buffer andsonicated to generate 200 to 1,000-bp DNA fragments.

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After centrifugation, the cleared supernatant was diluted10-fold with ChIP buffer and incubated with 1.5 mg of theindicated antibody at 4�C. Immune complexes were pre-cipitated, washed, and eluted as recommended. DNA-pro-tein cross-links were reversed by heating at 65�C for 4hours, and the DNA fragments were purified and dissolvedin 50 mL of water. A total of 10 mL of each sample was usedas a template for PCR amplification. The following PCRprimers were used: TNF-a promoter, forward 50-GATCCG-GAGGAGATTCCTTGA-30 and reverse 50-ACACTCCAGG-CACTTAAGGGTCCCGACTCAAGTA-30 (�255 to �655);IL-1b promoter, forward 50-CGTGGGAAAATCCAGTATTT-TAATG-30 and reverse 50CAAATGTATCACCATGC-AAATATGC-30 (�490 to �190), and Cox-2 promoterforward 50-AAAGCTATGTATGTATGTGCTGCAT-30 andreverse 50-AACCGAGAGAACCTTCCTTTTTAT-30 (�7 to�621). PCR was performed at 94�C for 30 seconds,60�C for 30 seconds, and 72�C for 45 seconds for 30cycles. The PCR products generated from the ChIP templatewere sequenced, and the identity of TNF-a, IL-1b, and Cox-2 promoters was confirmed. Real-time PCR was performedas described above.

Electrophoretic mobility shift assayNuclear extracts were prepared from mouse MGs using

an electrophoretic mobility shift assay (EMSA) kit. Forbinding studies, oligonucleotides containing NF-kB sites(Active Motif) were used. The complementary oligonucleo-tides were annealed in 20 mmol/L Tris (pH 7.6), 50 mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L dithiothreitol.The annealed oligonucleotide was phosphorylated at the 50

end with [g-32P]ATP and T4 polynucleotide kinase. Thebinding reaction was performed by incubating 5 mg ofnuclear protein in 20 mmol/L HEPES (pH 7.9), 10%glycerol, 300 mg of bovine serum albumin, and 1 mg ofpoly(dI�dC) in a final volume of 10 mL for 10 minutes at25�C. The labeled oligonucleotides were added to thereaction mixture and allowed to incubate for an additional20minutes at 25�C. The samples were electrophoresed on a4% nondenaturing polyacrylamide gel. The gel was thendried and subjected to autoradiography at �80�C.

Aromatase activityTo determine aromatase activity, microsomes were pre-

pared from cell lysates and tissues by differential centrifu-gation using established methods (12). Aromatase activitywas quantified by measurement of the tritiated waterreleased from 1b-[3H]-androstenedione (26). The reactionwas also performed in the presence of letrozole, a specificaromatase inhibitor, as a specificity control and withoutNADPH as a background control. Aromatase activity wasnormalized to protein concentration.

PGE2 analysisLevels of PGE2 in the mammary gland were determined

using modified versions of previously established methods(27, 28). Approximately 30 mg of frozen tissue was groundto a fine powder with a liquid nitrogen-cooled mortar.

Aliquots of 10 mL of internal standard (100 ng/mL), 300 mLof methanol containing 0.25% BHT, and 5 mL of formicacid were added to the pulverized tissue. Samples were thensonicated for 3minutes at 0�Cwith an Ultrasonic Processor(Misonix). Following centrifugation, an aliquot (300 mL) ofsupernatant was mixed with 1.7 mL water and subjected tosolid phase extraction. The solution was then applied to anOasis cartridge (3 cc, 60 mg; Waters Corp) preconditionedwith 2 mL methanol and 2 mL 0.1% formic acid. PGs wereeluted with 1.5 mL of methanol followed by 1.5 mL ethylacetate after the cartridge was washed with 2 mL of 0.1%formic acid. The eluate was then evaporated to drynessunder a stream of nitrogen. Samples were then reconsti-tuted in 100 mL of methanol/0.1% formic acid (50:50, v:v),before liquid chromatography/tandem mass spectroscopicanalysis. Protein concentration was determined by themethod of Bradford according to the manufacturer’sinstructions (Bio-Rad).

Liquid chromatography—mass spectrometry (LC/MS/MS) analyses were performed using a Tandem QuadrupoleMass Spectrometer (Agilent) equipped with an Agilent1200 binary pump HPLC system using a modified versionof the method of Yang and colleagues (28). PGs werechromatographically separated using a Gemini 3-mm C6-phenyl 4.6 � 100-mm analytical column (Phenomenex).The mobile phase consisted of 0.1% formic acid in water(mobile phase A) and 0.1% formic acid in acetonitrile(mobile phase B). The chromatographic baseline resolu-tion for the PGs of interest was achieved using a lineargradient that went from 20% to 40%mobile phase B for 12minutes and then from 40% to 70% for 8minutes. This wasthen increased to 90% mobile phase B over the next 2minutes and kept at this level for an additional 3 minutes.The flow rate was 0.8 mL/minute with a column tempera-ture of 40�C. The mass spectrometer was operated innegative electrospray ionization mode with capillary vol-tage of 2.9 kV, a cone gas flow rate of 10 L/minute, and ashear gas flow rate of 12 mL/minute at 350�C. PGs weredetected and quantified using multiple reaction modemonitoring of the transitions m/z 351 ! 271 for PGE2and m/z 355 ! 275 for PGE2-d4. The results are expressedas nanograms of PGE2 per mg of protein. For measure-ments of PGE2 in cell culture media, EIA kits were used.

Enzyme immunoassayTNF-a and IL-1b levels were quantified in cell culture

media using EIA kits.

StatisticsData generated in this study include mouse weights, the

number of inflammatory foci, tissue TNF-a, IL-1b, Cox-2,and aromatase mRNA levels and aromatase activity. Ingeneral, endpoints that conform to normality assumption,such as the mouse weight data, were summarized in termsof mean � SD and compared across multiple groups usingANOVA. Pair-wise comparison of mouse weights were thencarried out using Tukey’s test to identify pairs of groupswith significant difference in weights and adjusted for

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multiple comparisons. Endpoints that are usually not nor-mally distributed were summarized in terms of median andrange and with box-plots graphically. Differences in theseendpoints were examined across multiple groups using thenonparametric Kruskal–Wallis test. The Wilcoxon rank-sum test was then used for pair-wise comparisons. P valueswere adjusted for multiple comparisons using the conser-vative Bonferroni method. To examine the magnitudeof difference between 2 experimental groups in differentfractions of mammary gland tissue, linear mixed-effectsmodels were used to take into account both the withinand between mouse variation. Data were log-transformedwhere appropriate to ensure that the underlyingnormality assumption was satisfied. For in vitro studies,comparisons between groups were made using Student’st test. A difference between groups of P < 0.05 was con-sidered significant.

Results

Obesity causes inflammation and elevated aromataseexpression in the MGs and visceral fat of mice

Initially, we investigated the effects of dietary fat andovarian function on weight gain in female mice. Ovaryintact or OVX mice were fed LF or HF diets for 10 weeks.Both ovariectomy and feeding a HF diet led to weight gain(Fig. 1A). Thus, mice that were both fed a HF diet andsubjected to ovariectomy gained the most weight. Pre-viously, inflammatory lesions known as crown-like struc-tures (CLS) have been identified in the adipose tissue ofobese mice and humans (18, 19, 29). Hence, we nextinvestigated whether CLS (Fig. 1B) were present in theMGs and visceral fat of obese mice. A marked increasein the number of these inflammatory lesions was found inthe mammary gland and visceral fat of mice that gained the

Figure 1. Diet-induced obesity causes inflammation in the mammary gland and visceral fat. Ovary intact or OVX female mice (n ¼ 10/group) were fedeither a LF or HF diet for 10 weeks. A, weight of mice in the diet-induced obesity experiment, shown as mean � SD. A significant difference in averageweights was observed across groups (P < 0.001, ANOVA). In particular, OVX mice fed an HF diet had a significantly higher average weight compared withthe other groups (Padj < 0.001, Tukey's test). B, hematoxylin and eosin-stained slides were evaluated for the presence of inflammatory foci containingmacrophages that surrounded necrotic adipocytes (arrow, 200�). C and D, box-plots of the number of inflammatory foci in MGs and visceral fat of micein the different treatment groups. Significant differences were observed across the 4 experimental groups for both tissue types (P < 0.001, Kruskal–Wallistest). In pair-wise comparisons, OVX mice fed the HF diet showed significantly greater number of inflammatory foci in mammary gland compared withthose in the LF diet groups (Padj ¼ 0.01, Wilcoxon rank-sum test, P values were adjusted for multiple comparisons with Bonferroni method), and in visceral fatcompared with those in the other groups (Padj � 0.01). E, inflammatory foci contain F4/80-positive macrophages that formed a typical crown aroundadipocytes (200�). F, box-plots of PGE2 levels in MGs of mice in the different treatment groups. Significant differences were observed across the 4experimental groups (P < 0.001, Kruskal–Wallis test). In pair-wise comparisons, OVX mice fed the HF diet showed significantly greater PGE2 levels comparedwith those in the LF diet groups (Padj ¼ 0.01, Wilcoxon rank-sum test, P values were adjusted for multiple comparisons with Bonferroni method).

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most weight (Fig. 1C and D). To determine whether macro-phages were present in these inflammatory lesions, immu-nohistochemistry was performed to assess F4/80, amacrophage marker. These studies revealed that F4/80-positive cells were abundant in the CLS (Fig. 1E). BecausePGE2 levels are increased in a variety of inflammatoryconditions, levels of PGE2 were determined in the MGsof mice in each of the treatment groups. Consistent withthe histological evidence of inflammation, obesity wasassociated with elevated levels of PGE2 (Fig. 1F).

Real-time PCR was used to quantify levels of proin-flammatory mediators in mammary gland and visceralfat. Obesity-related inflammation was associated withelevated levels of TNF-a, IL-1b, and Cox-2 in both themammary gland and visceral fat (Fig. 2). Importantly,each of these proinflammatory molecules is a knowninducer of CYP19 transcription and aromatase activity(8–17). Accordingly, we next quantified aromatasemRNA levels and activity in the MGs and visceral fat ofthe different treatment groups. Remarkably, the changesin aromatase expression and activity paralleled thechanges in levels of TNF-a, IL-1b, and Cox-2 mRNAs inboth the mammary gland (Fig. 2D and E) and visceral fat(Fig. 2I and J). Taken together, these data suggest thatdiet-induced obesity causes inflammation in the mam-mary gland and visceral fat resulting in increased aroma-tase expression and activity.

To confirm that the aforementioned findings were notunique to the diet-induced obesity model, ob/ob mice wereused as a second model of obesity. Ob/ob mice are leptin-deficient and have been widely used in studies of obesity.At 11 weeks of age, the ob/ob mice weighed 54.3 � 2.2 gmwhereas lean wild-type mice weighed 19.2 � 0.8 gm (P <0.001). Consistent with the findings in the diet-inducedmodel of obesity, significant increases in the number ofinflammatory foci and levels of proinflammatory media-tors (TNF-a, IL-1b, Cox-2) were observed in the MGs andvisceral fat of ob/ob vs. wild-type mice (Table 1). Impor-tantly, levels of aromatase mRNA and activity were alsosignificantly increased in both the MGs and visceral fat ofob/ob mice (Table 1). Once again, the elevated levels ofaromatase paralleled the increased amounts of proinflam-matory mediators.

The SVF of the mammary gland is a source ofproinflammatory mediators that induce aromatase

Previous studies have suggested that obesity-relatedinflammation is associated with increased levels of

Figure 2. Diet-induced obesity is associated with increased levels of proinflammatory mediators and aromatase in the mammary gland and visceral fat.Ovary intact or OVX femalemice (n¼ 10/group) were fed either a LF or HF diet for 10weeks. Real-time PCRwas carried out on RNA isolated from themammarygland (n ¼ 10/group) and visceral fat (n ¼ 10/group) of mice in each of the four groups. box-plots of TNF-a, IL-1b, and Cox-2 mRNA expression in MGs (A–C)and visceral fat (F–H) are shown. Significant differences were observed across the 4 experimental groups for each proinflammatory mediator (P < 0.005).In pair-wise comparisons, OVX mice fed a HF diet showed significantly higher expression of the 3 genes in mammary gland and visceral fat comparedwith mice in the LF or LFþOVX groups (Padj� 0.02). Box-plots of relative aromatasemRNA levels and activity in MGs (D, E) and visceral fat (I, J) of mice in eachof the 4 treatment groups are shown. Significant differences were observed across the 4 experimental groups for aromatase expression and activity(P < 0.001). In pair-wise comparisons, OVX mice fed a HF diet had significantly higher levels of aromatase activity (femtomoles/mg protein/hour) and mRNAexpression in mammary gland and visceral fat compared with those in the other groups (Padj � 0.05).

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proinflammatory mediators in the SVF of abdominal fat(30). Hence, our next goal was to evaluate whether obesityled to increased levels of proinflammatory mediators andaromatase in the SVF or adipocyte fractions of the mam-mary gland. We focused on the diet-induced obesitymodel which includes mice subjected to OVX becauseof the link between obesity and increased risk of HR-positive breast cancer in the postmenopausal state. Inaddition, weight gain was more modest in this modelcompared with the ob/ob model in which the mice weremorbidly obese. We compared LF versus HF þ OVXbecause mice in these 2 groups exhibited the greatestdifferences in both weight and inflammation (Fig. 1).SVF and adipocyte fractions of the mammary gland wereseparated. To confirm that the separation of SVF andadipocyte fractions of the mammary gland was adequate,we performed analyses of F4/80 and aP2, markers ofmacrophages and adipocytes, respectively. F4/80 wasexpressed in the SVF but not in the adipocyte fractionisolated from the mammary gland (Fig. 3A). Conversely,aP2 was found in the adipocyte fraction but not in theSVF. Interestingly, obesity (HF þ OVX vs. LF) was asso-ciated with markedly increased levels of TNF-a, IL-1b, andCox-2 mRNAs in the SVF but not in the adipocyte fraction(Fig. 3B). Increased levels of aromatase mRNA and activ-ity were observed in both SVF and adipocyte fractions inobese mice (Fig. 3C). However, the magnitude of increasewas much greater in the adipocyte fraction compared withthe SVF. Because aromatase is a rate-limiting enzyme forestrogen biosynthesis, we also quantified levels of theprogesterone receptor (PR) and pS2, prototypic estrogen-inducible genes. Obesity was associated with increasedexpression of both PR and pS2 (Fig. 3D).Collectively, the aforementioned findings suggest that

obesity causes inflammation that is characterized bymacrophage-enriched CLS in the mammary gland. Ele-vated levels of proinflammatory mediators were foundin the SVF; these proinflammatory mediators are likelyto induce CYP19 transcription and aromatase expression

together with aromatase activity in other cell type(s)including adipocytes via a paracrinemechanism. Therefore,a series of experiments was performed to evaluate thispossibility. Initially, SVF isolated from the MG of micein the HFþOVX versus LF groups was cultured to quantifythe production of proinflammatory mediators. SVF derivedfrom the HF þ OVX group produced markedly increasedlevels of TNF-a, IL-1b, and PGE2 compared with SVF fromthe LF group (Fig. 4A–C). Consistent with the differencein PGE2 production, higher levels of Cox-2 protein werefound in SVF of the HF þ OVX versus LF group (Fig. 4D).Preadipocytes express higher levels of aromatase thanmature adipocytes and are often used as a model cellsystem in studies of aromatase (31). Next experimentswere carried out to determine if cross-talk between macro-phages and preadipocytes can explain the increased aro-matase levels found in the adipocyte fraction of the MG ofobese versus lean mice. Notably, treatment with CM-derived from the SVF of the HF þ OVX versus LF groupled to higher levels of aromatase mRNA and activity inmouse preadipocyte 3T3-LI cells (Fig. 4E). Experimentswere then carried out to evaluate the importance of eachof the SVF-derived inflammatory mediators (TNF-a, IL-1b, and PGE2) for inducing aromatase. Antibodies wereused to neutralize TNF-a and IL-1b in CM-derived fromthe HF þ OVX mammary gland SVF. Neutralizing eitherTNF-a (Fig. 4F) or IL-1b (Fig. 4G) attenuated CM-mediated induction of aromatase in 3T3-L1 cells. Inaddition, treatment of macrophage-enriched SVF withcelecoxib, a selective Cox-2 inhibitor, blocked the releaseof PGE2 into the CM (Fig. 4H). Following Cox-2 inhibi-tion, the ability of SVF-derived CM to induce aromatasemRNA and aromatase activity in 3T3-L1 cells was mark-edly attenuated (Fig. 4I). Taken together, these datasuggest that SVF isolated from the MG of obese versuslean mice (HFþOVX versus LF) produces increased levelsof TNF-a, IL-1b, and Cox-2-derived PGE2, each of whichcontributes, in turn, to the induction of aromatase inpreadipocytes.

Table 1. Significantly greater inflammation, proinflammatory molecule levels, and aromatase expressionwere found in both the MG and visceral fat (VF) of ob/ob compared with wild-type mice.

Endpoint MG VF

Wild-type ob/ob P Wild-type ob/ob P

Inflammatory foci 0.8 (0.0, 2.6) 13.8 (8.5, 20.4) <0.001 0 (0.0,10.6) 64.5 (25.5, 202) <0.001Relative TNF-a expression 0.9 (0.6, 4.9) 4.9 (0.7, 9.7) 0.007 1.0 (0.5, 2.8) 5.3 (0.6, 8.7) 0.02Relative IL-1b expression 1.0 (0.4, 2.3) 2.9 (0.3, 9.7) 0.02 1.1 (0.03, 6.0) 5.7 (1.1, 7.9) 0.005Relative Cox-2 expression 1.0 (0.4, 2.5) 3.8 (1.2, 5.7) 0.001 1.0 (0.4, 3.7) 2.2 (0.6, 4.8) 0.02Relative aromatase expression 1.2 (0.2, 5.6) 6.1 (0.04, 7.6) 0.007 0.9 (0.4, 2.7) 1.8 (0.8, 6.8) 0.009Aromatase activity 90 (68,112) 210 (146, 278) <0.001 98 (67, 154) 272 (177, 355) <0.001

NOTE: Inflammatory foci, number of inflammatory foci per cm2 of tissue; real-time PCR was used to quantify relative TNF-a, IL-1b,Cox-2, and aromatase transcript levels; aromatase activity, femtomoles/mg protein/hour. Values are summarized in median (range),P values are based on Wilcoxon rank-sum test, n ¼ 10/gp.

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Figure 3.Diet-inducedobesity is associatedwith elevated levels of proinflammatorymediators andaromatase indifferent compartmentsof themammarygland.Adipocyte (Adipo) and SVF were prepared fromMGs of ovary intact mice fed a low fat (LF) diet or mice that were subjected to ovariectomy (OVX) and fed an HFdiet for 10weeks. A, RNA fromSVF and adipocyte fractionswas isolated from3MGs and subjected to northern blotting. Blotswere probed for F4/80 and aP2 asindicated. B-D, Real-time PCRwas used to quantifymRNA levels for TNF-a, IL-1b, Cox-2, aromatase, PR and pS2. B, box-plots of the TNF-a, IL-1b, and Cox-2mRNA levels in the SVF and the adipocyte fractions of MGs (n ¼ 10/group) are shown. In comparison to lean mice (LF), obese mice (HF þ OVX) showedsignificantly higher expression levels of all 3 proinflammatory mediators in the SVF but not in the adipocyte fraction of the mammary gland (P < 0.001). C, box-plots of relative aromatase mRNA levels and aromatase activity in the SVF and adipocyte fractions of MGs (n ¼ 10/group) are shown. Aromatase activity isexpressedas femtomoles/mgprotein/hour.D, relativeprogesterone receptor (PR) andpS2mRNAexpression in theSVFandadipocyte fractionsof themammarygland (n ¼ 10/group) are shown.

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Figure 4. Proinflammatory mediators derived from the SVF of the mammary gland of obese mice induce aromatase in preadipocytes. SVF cells derived fromthe mammary gland of mice in the LF and HF þOVX groups were grown overnight in DMEM. Conditioned medium (CM) was then collected and analyzedor used to treat 3T3-LI preadipocytes. Levels of TNF-a (A), IL-1b (B), andPGE2 (C) inCMweredeterminedby enzyme immunoassay.D,Cox-2protein abundancewasdeterminedby immunoblotting ofwhole-cell lysates.b-actinwasused as a loading control. E, 3T3-L1 cellswere treatedwithCMderived from themammarygland SVF of LF or HF þ OVX mice for 24 hours. Relative aromatase mRNA levels and aromatase activity were then determined. F and G, neutralizing eitherTNF-a (F) or IL-1b (G) suppressed the ability of CM derived from HF þ OVX SVF to induce aromatase in 3T3-L1 cells. In both F and G, the bar labeledControl represents 3T3-L1 cells that received DMEMmedium alone; the bar labeled CM represents 3T3-L1 cells that received CMderived fromHFþOVX SVF.In addition, CM fromHFþOVXSVFwas incubatedwith control IgG, TNF-a IgG, or IL-1b IgG overnight at 4�C to neutralize TNF-a (F) and IL-1b (G), respectively.3T3-L1 cells were then treated as indicated for 24 hours prior to measurements of relative aromatase mRNA levels and aromatase activity. H, SVF cellsderived from the mammary gland of mice in the HF þ OVX group were grown overnight. The cells were then treated with fresh DMEM containing vehicle(HFþOVX) or 5mmol/L celecoxib (HFþOVXþ celecoxib) for 6 hours.CMwascollected andanalyzed forPGE2 levels (H) or used to treat 3T3-LI preadipocytes (I).I, inhibiting Cox-2 in SVF cells derived from HF þ OVX mammary gland attenuated the ability of CM to induce aromatase in 3T3-L1 cells. The bar labeledControl represents 3T3-L1 cells that received DMEM medium; the bar labeled CM represents 3T3-L1 cells that received CM derived from HF þ OVX SVF;the bar labeled CM þ celecoxib represents 3T3-L1 cells that received CM derived from HF þ OVX SVF treated with 5 mmol/L celecoxib. 3T3-L1 cells weretreated as indicated for 24 hours prior to measurements of relative aromatase mRNA levels and aromatase activity. Aromatase activity is expressed asfemtomoles/mg protein/minute. Columns, means (n ¼ 3); bars, SD. *, P < 0.05.

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Figure 5. Treatment ofmacrophages with saturated fattyacids causes dose-dependentinduction of proinflammatorymediators. THP-1 cells weretreated with the indicatedconcentrations of saturated fattyacid (LA, MA, PA, and SA) for 24hours. Real-time PCR was used toquantify TNF-a, IL-1b, and Cox-2mRNAs (A, C, E). Levels of TNF-aprotein, IL-1b protein and PGE2 inthe culture medium weredetermined by enzymeimmunoassay (B, D, G). Cox-2protein abundance wasdetermined by immunoblotting ofwhole-cell lysates. b-actin wasused as a loading control (F).Columns, means (n¼ 6); bars, SD.*, P < 0.05.

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Saturated fatty acids stimulate the production ofproinflammatory mediators by macrophages leadingto increased aromatase levelsLipolysis is increased in obesity (32). Moreover, satu-

rated fatty acids released from adipocytes can activatemacrophages resulting in an inflammatory response(30). Hence, our next goal was to determine whether theelevated levels of proinflammatory mediators in the SVF ofthe mammary gland of obese versus lean mice might beexplained by the effects of saturated fatty acids on macro-phages. Experiments were carried out to determine ifsaturated fatty acids stimulated the production of proin-flammatory molecules by macrophages leading, in turn, toelevated aromatase expression in preadipocytes. Saturatedfatty acids ranging in chain length from C12 to C18including lauric acid (LA), myristic acid (MA), palmiticacid (PA), and stearic acid (SA) were used. Treatment witheach of these saturated fatty acids caused dose-dependentinduction of TNF-a, IL-1b, and Cox-2 in THP-1 cells, a cellline with properties of human monocyte–derived macro-phages (Fig. 5). Induction of Cox-2 mRNA by saturatedfatty acids was associated with a corresponding increase inCox-2 protein and PGE2 production (Fig. 5F and G).Saturated fatty acids also induced proinflammatory med-

iators in human blood monocyte–derived macrophages(Supplementary Fig. 1). Treatment with CM derived fromeither saturated fatty acid–treated THP-1 cells or humanblood monocyte–derived macrophages induced aromatasemRNA and activity in human preadipocytes (Fig. 6). Neu-tralizing either TNF-a or IL-1b attenuated CM-mediatedinduction of aromatase in preadipocytes (Fig. 7). Use ofsiRNA to silence either TNF-a or IL-1b in THP-1 cells led tosimilar suppressive effects (Supplementary Fig. 2). In addi-tion, either silencing of Cox-2 or treatment of THP-1 cellswith celecoxib blocked saturated fatty acid–mediatedinduction of PGE2 release into the CM (Fig. 7 and Supple-mentary Fig. 2). Following Cox-2 inhibition, the ability ofmacrophage-derived CM to induce aromatase mRNA oraromatase activity in preadipocytes was attenuated. Takentogether, these data suggest that saturated fatty acid–mediated induction of TNF-a, IL-1b, and Cox-2 in macro-phages contributes to the induction of aromatase inpreadipocytes.

Activation of NF-kB contributes to increasedaromatase levels in obesity

NF-kB plays a significant role in regulating the expres-sion of proinflammatory mediators in macrophages (33).

Figure 6. CM from saturated fattyacid–treated macrophagesinduces aromatase inpreadipocytes. THP-1 cells orhuman blood monocyte–derivedmacrophages were treated with 0to 10 mmol/L LA, MA, PA, or SA asdetailed in the Materials andMethods section to generate CM.CM was then used to treatpreadipocytes for 24 hours. A andB, preadipocytes treated with CMderived from THP-1 cells. C and D,preadipocytes treated with CMderived from human bloodmonocyte–derived macrophages.Aromatase mRNA (A, C) andactivity levels (femtomoles/mgprotein/minute; B, D) weredetermined. Columns, means(n ¼ 6); bars, SD. *, P < 0.05.

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Hence, experiments were performed to investigate thepotential role of this transcription factor in saturated fattyacid–mediated induction of TNF-a, IL-1b, and Cox-2 inmacrophages. Transient transfections were performed withan NF-kB-luciferase reporter construct. Saturated fatty acidsinduced NF-kB-luciferase activity in THP-1 cells (Fig. 8A).EMSA indicated that saturated fatty acids stimulated bind-ing of nuclear protein to a 32P-labeled NF-kB consensussequence (Fig. 8B). This bindingwas abrogatedwhen a large

excess of cold probe was used (data not shown). Supershiftassays indicated that p65, a component of NF-kB, waspresent in the binding complex (Fig. 8C). Consistent withthe activation of NF-kB, saturated fatty acids stimulated thephosphorylation of p65 and its translocation from cytosolto nucleus (Fig. 8D and E). To further evaluate the potentialrole of NF-kB in regulating the expression of TNF-a, IL-1bandCox-2 inmacrophages, it was important to determine ifsaturated fatty acids stimulated the binding of p65 to the

Figure 7. Proinflammatory mediators in CM of SA-treated macrophages induce aromatase in preadipocytes. A–D, THP-1 cells were treated with vehicleor 10 mmol/L SA as detailed in theMaterials andMethods section to generate CM. As indicated, CM from SA-treated cells was then incubated with neutralizingantibodies (Ab) to TNF-a, IL-1b, or control IgG overnight at 4�C. E–G, THP-1 cells were treated with vehicle, SA or SA and 5 mmol/L celecoxib for 12 hours.Following treatment, the medium was removed and cells were washed. Subsequently, fresh medium was added for 24 hours to generate CM. E, celecoxibsuppressed SA-mediated induction of PGE2 production. A–D, F and G, preadipocytes were treated with THP-1 cell-derived CM for 24 hours prior tomeasurements of aromatase mRNA and activity. Aromatase mRNA (A, C, F) and activity levels (B, D, G) were determined in preadipocytes. Activity isexpressed as femtomoles/mg protein/minute. Columns, means (n ¼ 6); bars, SD. *, P < 0.05.

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promoters of each of these proinflammatory genes. ChIPassays were carried out. Each of the saturated fatty acidsstimulated phospho-p65 binding to the promoters of theproinflammatory genes (Fig. 8F).

To further evaluate the importance of p65 in regulatingthe production of both proinflammatory mediators andthe induction of aromatase, THP-1 cells were treated withsmall interfering RNA to p65 (Fig. 9A). Silencing of p65

Figure 8. Saturated fatty acids activate NF-kB in THP-1 cells. A, cells were transiently transfected with NF-kB-luciferase and pSV-bgal constructs.Cells were then treated with 10 mmol/L LA, MA, PA, or SA for 24 hours. Activities represent data that have been normalized to b-galactosidase activity.Columns, mean (n¼ 6); bar, SD. B and C, 10 mg of nuclear protein was incubated with a 32P-labeled oligonucleotide containing NF-kB binding sites. B, lanes 1to 4 represent binding with nuclear protein from cells treated with the indicated concentration of SA for 1 hour. C, lanes 1 to 4 represent binding with nuclearprotein from cells treated with vehicle (lane 1) or 10 mmol/L SA (lanes 2–4) for 1 hour. Lanes 2 to 4 represent nuclear protein incubated with normal IgG(lane 2) or antibodies to p65 (2 mL, lane 3; 1 mL, lane 4). B and C, the protein-DNA complexes that formed were separated on a 4% polyacrylamide gel. D, cellswere treated as indicated with 0 to 10 mmol/L LA, MA, PA, or SA for 30minutes. The abundance of phospho-p65 and p65 protein in cell lysates was determinedby immunoblotting. E, cells were treated with vehicle (control) or 10 mmol/L SA for 30 minutes. The abundance of phospho-p65 was determined byimmunoblotting in cytosolic (C) and nuclear (N) preparations. b-actin and histone H3 represent cytosolic and nuclear markers, respectively. F, cells weretreated with vehicle (Control) or 10 mmol/L LA, MA, PA, and SA for 3 hours. ChIP assays were performed. Chromatin fragments were immunoprecipitated withantibodies against phospho-p65 and the TNF-a, IL-1b, andCox-2 promoters were amplified by real-time PCR. DNA sequencing was carried out, and the PCRproducts were confirmed to be correct promoters. These promoters were not detected when normal IgG was used or when antibody was omitted from theimmunoprecipitation step (data not shown). Means � SD are shown; n ¼ 3.

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inhibited the production of proinflammatory mediators(TNF-a, IL-1b, PGE2) in response to treatment with satu-rated fatty acids (Fig. 9, B-D). Silencing of p65 in THP-1cells also suppressed CM-mediated induction of aromataseexpression and activity in preadipocytes (Fig. 9E and F). Wealso evaluated the effects of BAY11-7082, a pharmacolo-gical inhibitor of NF-kB. Treatment of THP-1 cells withBAY11-7082 suppressed both SA-mediated activation ofNF-kB and the production of increased amounts of TNF-a,IL-1b, and PGE2 (Supplementary Fig. 3). Following NF-kBinhibition, the ability ofmacrophage-derived CM to inducearomatase expression and activity was suppressed (Supple-mentary Fig. 3, D and E).

Because of the important role that NF-kB was found toplay in regulating proinflammatory mediator productionand aromatase expression in vitro, complementary studieswere carried out on tissues from the diet-induced modelof obesity. EMSAs were performed using nuclear extracts

isolated from mammary gland tissue in the differenttreatment groups. Consistent with the elevated levels ofTNF-a, IL-1b, and Cox-2 in MGs of obese mice (Fig. 2),NF-kB binding activity was increased in this group(Fig. 10A). Once again, p65 was found in the bindingcomplex (Fig. 10B). As shown previously, higher levels ofTNF-a, IL-1b, and Cox-2 message were found in the SVFversus adipocyte fraction of MGs derived from obese mice(Fig. 3B). Notably, higher levels of NF-kB binding activitywere also found in the SVF versus adipocyte fractionsprepared from the MGs of obese mice (Fig. 10C). Con-sistent with the in vitro findings (Fig. 8), supershift assaysindicated that p65 was present in the binding complex(Fig. 10D). Taken together, our findings suggest that NF-kB plays a key role in regulating the production ofproinflammatory mediators in macrophages leading, inturn, to the induction of aromatase in the adipocytefraction of the mammary gland.

Figure 9. Silencing of p65 inhibits saturated fatty acid (FA)–mediated induction of proinflammatory mediators and aromatase. A, THP-1 cells were transfectedwith control siRNA or siRNA to p65. The abundance of p65 protein was determined by immunoblotting in cell lysates. b-actin was used as a loading control.B–D, THP-1 cells were untreated or transfected with control siRNA or p65 siRNA. Subsequently, cells were treated with 0 or 10 mmol/L LA, MA, PA, orSA as detailed in the Materials and Methods sections to generate CM. Enzyme immunoassays were used to quantify levels of TNF-a (B), IL-1b (C), andPGE2 (D) in the CM. E and F, preadipocytes were treated with THP-1 cell-derived CM for 24 hours prior to measurements of aromatase mRNA andactivity. Activity is expressed as femtomoles/mg protein/minute. Columns, means (n ¼ 6); bars, SD. *, P < 0.05.

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Discussion

Obese postmenopausal women are at increased risk ofdeveloping HR-positive breast cancer compared with leanpostmenopausal women. This increased risk has beenattributed, in part, to elevated levels of circulating estrogensthat reflect both increased adipose tissue mass andenhanced aromatase expression in subcutaneous fat(4–7). Given the link between estrogen biosynthesis andthe development and progression of HR-positive breastcancer, we sought to determine whether obesity-inducedinflammation led to increased aromatase expression in themammary gland in addition to adipose tissue.Previous studies of adipose tissue indicate that obesity is

associated with adipocyte death leading to the accumula-tion of macrophages. The macrophages are aggregated inCLS that form around individual necrotic adipocytes in thevisceral and subcutaneous fat of obese humans and mice(18–20, 29, 34). Here we observed crowns of macrophagessurrounding adipocytes in both the mammary gland andvisceral fat of obese mice. To our knowledge, this is the firstreport of CLS in the mammary gland. Consistent with thehistological evidence of CLS, representing foci of inflam-mation, increased levels of TNF-a, IL-1b, and Cox-2 werefound in the mammary gland and visceral fat in bothdietary and genetic models of obesity. The increased levelsof proinflammatory mediators occurred in the macro-

phage-enriched SVF of the mammary gland. These findingsare consistent with prior evidence that macrophages are amajor source of proinflammatory mediators in adiposetissue in obese mice and humans (20, 34).

Proinflammatory mediators including TNF-a, IL-1b, andCox-2-derived PGE2 are known inducers of CYP19 tran-scription resulting in increased aromatase activity (10, 13,14, 16, 17, 26, 35). In this study, elevated levels of proin-flammatory mediators were paralleled by increased levelsof aromatase in the mammary gland and visceral fat ofobese mice suggesting a causal relationship. In support ofthis possible mechanism, SVF isolated from the MG ofobese mice produced increased levels of TNF-a, IL-1b, andCox-2-derived PGE2 compared with SVF from lean mice.Importantly, each of these proinflammatory moleculescontributed to the ability of SVF-derived CM to inducearomatase in preadipocytes. Notably, both TNF-a and IL-1b induce Cox-2 and PGE2 production (36). It’s possible,therefore, that the ability of these proinflammatory cyto-kines to stimulate PGE2 synthesis contributes to the induc-tion of aromatase (16).

Macrophage-derived cytokines have been suggested tostimulate lipolysis in adipocytes leading to increasedrelease of saturated fatty acids (37). These saturated fattyacids activate TLR4 signaling in macrophages and therebystimulate the production of proinflammatory mediators(38, 39). Therefore, in obesity, a paracrine loop involving

Figure 10.NF-kB is activated in the SVF of the mammary gland of obese mice. A–D, 10 mg of nuclear protein was incubated with a 32P-labeled oligonucleotidecontaining NF-kB binding sites. A, binding of nuclear protein from unfractionated MGs of 17 mice in the indicated treatment groups. B, lanes 1 to 7represent binding of nuclear protein from MGs in the HF þ OVX group. Lane 1, binding reaction without nuclear protein; lane 2, binding of nuclearprotein to labeled oligonucleotide; lanes 3 to 5, nuclear protein incubated with normal IgG (lane 3) or phospho-p65 antibody (2mL, lane 4; 1 mL, lane 5);lanes 6 and 7, nuclear protein was incubated with labeled oligonucleotide and a 10� excess (lane 6) or 50� excess of cold probe (lane 7). C, binding ofnuclear protein isolated from the adipocyte or SVF fractions of the mammary gland of 5 mice in the HF þ OVX group. D, binding of nuclear proteinfrom SVF isolated from a mammary gland in the HF þ OVX group. Lane 1, binding of nuclear protein to labeled oligonucleotide; lane 2, nuclear proteinwas incubated with labeled oligonucleotide and a 50� excess of cold probe; lanes 3 and 4, nuclear protein incubated with normal IgG (lane 3) or 2mLof phospho-p65 antibody (lane 4). In A–D, the protein-DNA complexes that formed were separated on a 4% polyacrylamide gel.

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macrophages and adipocytes may amplify the inflamma-tory state leading to increased aromatase expression(40; Fig. 11). We present evidence that saturated fatty acidtreatment of macrophages led to increased productionof TNF-a, IL-1b, and Cox-2, each of which contributedto enhanced aromatase expression in preadipocytes.Neutralizing antibodies, small interfering RNAs and selec-tive pharmacological inhibitors were used to establish acausal link between the overproduction of each of theseproinflammatory mediators by macrophages and theinduction of aromatase in preadipocytes. To evaluatewhether the obesity-related increase in aromatase activitywas functionally significant, levels of PR and pS2, 2 estro-gen receptor target genes, were determined (41). Increasedlevels of PR and pS2 were found in the adipocyte fractionof the MGs of obese mice. Collectively, these findingsare consistent with the concept that obesity caused inflam-mation in the mammary gland, which resulted in increasedexpression of aromatase that was followed by increasedestrogen biosynthesis, resulting in changes in gene expres-sion. Therefore, our data suggest that inflammation-relatedincreases in aromatase expression in addition to increasedadipose mass may contribute to the increased risk ofHR-positive breast cancer in obese postmenopausalwomen. Given the link that we have established betweenobesity, CLS and increased aromatase expression, it ispossible that CLS will prove to be a biomarker of breastcancer risk.

We present evidence that obesity led to activation of NF-kB in the mammary gland. This finding can explain theobserved increase in proinflammatory gene expression andthe resulting increase in aromatase expression. In support ofthis conclusion, silencing of p65 or use of a pharmacolo-gical inhibitor of NF-kB blocked saturated fatty acid–mediated induction of proinflammatory mediators inmacrophages and thereby suppressed CM-mediated induc-tion of aromatase in preadipocytes. Previously, tumor cellNF-kB was suggested to play a role in breast carcinogenesisincluding in models of HR-negative breast cancer (42). Thecurrent results suggest that stromalNF-kB plays a key role inregulating aromatase expression via a paracrinemechanism(Fig. 11). Hence, NF-kBmay play a role in bothHR-positiveand HR-negative breast cancer. Future studies will berequired to determine whether single agents or combina-tions of agents that inhibit the activation of NF-kB willsuppress the increased levels of aromatase found in themammary gland and visceral fat of obese mice. Interven-tions that disrupt the obesity! inflammation! aromataseaxis may prove useful in reducing the risk of HR-positivebreast cancer in postmenopausal women.

In the dietary model of obesity, we found that a HF dietcausedmore severe inflammation, greater proinflammatorycytokine expression and increased aromatase levels in themammary gland and visceral fat of OVX compared withovary intact mice. Rodent OVX has been used as a model ofhuman menopause because it provides the means to studythe metabolic consequences that arise through the loss ofovarian function (43). Studies have consistently shown thatOVX causes expansion of adipose tissue, insulin resistance,and inflammation (43). These effects, which are associatedwith increased breast cancer risk in postmenopausalwomen, can be reversed by systemic administration ofestradiol (44, 45). Consequently, it appears that circulatingestradiol plays a critical role in preventing obesity andinflammation. However, obesity in postmenopausalwomen and its attendant increase in breast cancer risk areassociated with an increase in serum estradiol, although theactual concentrations are much lower than that found inpremenopausal women (46). It is possible, therefore, thatvery high estradiol concentrations can be formed in thebreast tissue of obese postmenopausal women and that thisis responsible for the increased risk of HR-positive breastcancer rather than circulating estradiol. If so, circulatingestradiol in postmenopausal women might only be a bio-marker for breast cancer risk and not the primary mediatorof the increased risk. The answers to these questions couldhave profound implications for the chemoprevention ofHR-positive breast cancer in postmenopausal women.

Our data also raise important questions concerning therelationship between obesity and the progression of breastcancer. Numerous studies have shown that obesity isassociated with poor prognosis including an increased riskof distantmetastasis and cancer-related death (47, 48). Thisis true for both premenopausal and postmenopausal breastcancer patients. A variety of mechanisms have been sug-gested to explain the relationship between obesity and poor

Figure 11. Paracrine interactions between adipocytes and macrophagescan explain the elevated levels of aromatase in the mammary gland andvisceral fat of obese mice. In obesity, lipolysis is increased resulting inincreased concentrations of free fatty acids. Saturated fatty acids triggerthe activation of NF-kB in macrophages resulting in enhanced productionof proinflammatory mediators (PGE2, TNF-a, IL-1b). Each of theseproinflammatory mediators contributes to the induction of aromatase inpreadipocytes and adipocytes.

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prognosis. For example, increased levels of estrogens, insu-lin or proinflammatory mediators can potentially drivetumor progression (6, 49). Here, we present the firstevidence that obesity leads to activation of NF-kB,increased levels of proinflammatory mediators and ele-vated aromatase expression in the mammary gland. In adifferent context, each of these effects has been linked totumor progression. Based on the current results, additionalstudies are warranted to further interrogate the mechan-isms underlying the link between obesity and breast cancerprogression. It’s possible that agents that suppress theobesity ! inflammation axis will have a role in inhibitingtumor progression. Finally, we note that the efficacy ofanastrozole, an aromatase inhibitor, in the adjuvant treat-ment of HR-positive breast cancer is reduced in obesecompared to lean postmenopausal women (21). It hasbeen theorized that a standard dose of anastrozole isinsufficient to completely suppress the elevated estrogenlevels resulting from obesity. Our finding that obesitycauses elevated aromatase expression in adipose tissueprovides a mechanism that may contribute to the reducedefficacy of an aromatase inhibitor in obese women givenstandard therapeutic doses. Future studies are warranted to

determine the dose of aromatase inhibitor that is requiredto completely suppress tissue estrogen biosynthesis inobese women.

Disclosure of Potential Conflicts of Interest

Andrew 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.

Acknowledgments

We thank Dr. Ian Blair for his thoughtful reading of the manuscript andhelpful discussion and Claire Kent for her excellent technical assistance.

Grant Support

This work was supported by NIH R25CA105012, NCI N01-CN-43302,the Breast Cancer Research Foundation, the Botwinick-Wolfensohn Foun-dation (inmemory ofMr. andMrs. Benjamin Botwinick), andNewChapter,Inc.

Received December 15, 2010; accepted January 12, 2011; publishedonline March 3, 2011.

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