Progestin effects on cell proliferation pathways in the postmenopausal mammary gland

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1 Wood et l. Brest Cner Reserh 213, 15:R62 RESEARCH ARTICLE Open Aess Progestin effets on ell prolifertion pthwys in the postmenopusl mmmry glnd Chrles E Wood 1, Dniel Brnstetter 2, Allison P Jo 3, J Mrk Cline 1, Thoms C Register 1, Kthy Rohrh 2, Li-Y Hung 2, Hermin Borgerink 1 nd Willim C Dougll 3* Astrt Introdution: Menopusl hormone therpies vry widely in their effets on rest ner risk, nd the mehnisms underlying these differenes re unler. The primry gols of this study were to hrterize the mmmry glnd trnsriptionl profile of estrogen + progestin therpy in omprison with estrogen-lone or tiolone nd investigte pthwys of ell prolifertion in postmenopusl primte model. Methods: Ovrietomized femle ynomolgus mque monkeys were rndomized into the following groups: pleo (), orl onjugted equine estrogens (), with medroxyprogesterone ette (MPA) ( + MPA), nd tiolone given t low or high dose (Lo or Hi Ti). All study tretment doses represented humn linil dose equivlents nd were dministered in the diet over period of 2 yers. Results: Tretment with + MPA hd the gretest effet on glol mrna profiles nd mrkers of mmmry glnd prolifertion ompred to or tiolone tretment. Chnges in the trnsriptionl ptterns resulting from the ddition of MPA to were relted to inresed growth ftors nd deresed estrogen reeptor (ER) signling. Speifi genes indued y + MPA tretment inluded key memers of proltin reeptor (PRLR)/signl trnsduer nd tivtor of trnsription 5 (STAT5), epiderml growth ftor reeptor (EGFR), nd reeptor tivtor of nuler ftor kpp B (RANK)/reeptor tivtor of nuler ftor kpp B lignd (RANKL) pthwys tht were highly ssoited with rest tissue prolifertion. In ontrst, tiolone did not ffet rest tissue prolifertion ut did eliit mixed pttern of ER gonist tivity. lusion: Our findings indite tht estrogen + progestin therpy results in distint moleulr profile ompred to estrogen-lone or tiolone therpy, inluding upregultion of key growth ftor trgets ssoited with mmmry rinogenesis in mouse models. These hnges my ontriute to the promotionl effets of estrogen + progestin therpy on rest ner risk. Keywords: Postmenopusl hormone therpy, Estrogen, Progestin, Tiolone, Brest ner, Gene expression, RANKL, Denosum Introdution Different types of postmenopusl hormone therpy (HT) hve een widely used for more thn 6 yers to llevite symptoms of menopuse nd prevent ssoited onditions suh s osteoporosis. The primry form of HT during muh of this time hs een estrogen-lone therpy (ET) [1]. In the mid-199s, the linil use of estrogen plus progestin therpy (EPT) egn fter severl studies demonstrted tht progestins opposed the dverse * Correspondene: dougllw@mgen.om 3 Therpeuti Innovtion Unit (TIU), Amgen In, Settle, WA 98119, USA Full list of uthor informtion is ville t the end of the rtile effets of ET on endometril ner risk [2]. These findings helped spur rpid inrese in EPT presriptions from less thn two million in 1995 to 24 million in 21 [1]. At thetime,themostommontypeofhtusedintheus ws onjugted equine estrogens () with or without the progestin medroxyprogesterone ette (MPA), whih together ounted for more thn 6% of totl HT presriptions [1]. Use of HT fell drmtilly in 22 fter the relese of the primry results from the Women s Helth Inititive (WHI) Estrogen + Progestin Tril [1,3]. In this lrge rndomized linil tril, postmenopusl women reeiving 213 Wood et l.; liensee BioMed Centrl Ltd. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 2 of 16 EPT ( + MPA) tretment hd signifintly higher inidene of invsive rest ner ompred with those tking the pleo [3,4]. Susequent reports noted inresed rest ner mortlity for women tking EPT [5] nd deresed rest ner inidene following disontinution of EPT [6]. These results supported prior epidemiologi studies [7,8] ut differed from the sister WHI Estrogen-Alone Tril, in whih lone did not inrese the inidene of invsive rest ner mong women with prior hysteretomy [9,1]. Colletively, these studies onfirmed tht the promotionl effets of EPT on ertin types of rest ner were greter thn those seen with estrogen lone. A prominent lterntive to trditionl menopusl HTs used outside the US is tiolone. This unique steroidl ompound is onverted in tissue-seletive mnner to estrogeni, progestogeni, nd ndrogeni metolites. Estrogeni metolites hve een shown to redue menopusl symptoms nd frture risk [11,12], wheres the progestogeni Δ-4 isomer metolite (seletively produed in the uterus) hs een shown to provide endometril protetion from estrogeni effets [13,14]. However, the effets of tiolone therpy on rest ner risk re ontroversil. In rndomized linil tril of older postmenopusl women, tiolone therpy resulted in signifintly lower rest ner risk [12], wheres in seprte linil tril, rest ner survivors hd inresed risk of reurrene following tiolone tretment [15]. Despite numerous prelinil nd linil studies, the mehnisti tions of different HTs on rest tissue hve not een lerly defined. In mouse models, ovrin hormones ontriute signifintly to mmmry glnd rinogenesis [16]; however, differenes etween mouse nd humn mmmry glnd ntomy, development, nd/ or hormonl ontrol of prolifertion my limit the trnsltionl relevne of trgets identified in the mouse for estrogen nd progestogen funtion. Mques re nthropoid primtes with high overll geneti oding sequene identity to humns, inluding key genes relted to rest ner suseptiility [17]. Prior work hs shown lose similrities etween mque nd humn mmmry glnd iology, inluding responses to exogenous estrogen nd progestogen therpies, sex steroid reeptor expression, nd ge-relted hyperplsti nd neoplsti lesions [18]. This inludes study on ged rhesus mques nd suggests lifetime inidene of mmmry glnd ner t out 6% [19], similr to lower-risk humn popultions. Previous studies in this model hve lso shown tht the ddition of progestin to n estrogen inreses mmmry glnd prolifertion nd density eyond tht seen with estrogen lone, in support of the lter WHI findings relted to rest ner risk [2]. The primry im of this study ws to ssess the effets of long-term tretment with, + MPA, or tiolone on the trnsriptionl profiles nd signling pthwys of the norml postmenopusl primte mmmry glnd. Our min gols were to identify speifi trgets ssoited with hormone-indued rest prolifertion nd evlute their reltions with ndidte pthwys known to drive mmmry rinogenesis in mouse tumor models. Methods Study design nd tretments This study utilized rhived tissue smples from prllelrm design experiment in whih 149 ovrietomized dult femle ynomolgus mques (M fsiulris) with men estimted ge of six to eight yers were rndomized to reeive one of the following five tretments for two yers: pleo (ontrol; n = 31); t.42 mg/kg (; n = 28); + MPA t.167 mg/kg ( + MPA; n = 29); tiolone t.5 mg/kg (Lo Ti; n = 3); nd tiolone t.2 mg/kg (Hi Ti; n = 31). Dose equivlents pproximted stndrd HT doses of (.625 mg/dy) nd MPA (2.5 mg/dy) in postmenopusl women, nd tiolone doses were designed to pproximte.75 mg/dy (low) nd 3. mg/dy (high) doses in women. Serum onentrtions of estrogens, MPA, nd tiolone metolites were similr to those reported in women tking omprle therpies [21]. Tretments were dministered in the ontrol diet with sein plus ltlumin s the protein soure nd mronutrient omposition sed on typil humn diet in the USA [22]. Animls were housed in soil groups of five nimls eh nd fed 6 kl/kg (plus 1% extr to ount for wste) twie dily, with drug tretments split etween the two feedings. Dily doses were sled to 1,8 kl of diet (the estimted dily intke for US womn) to ount for differenes in metoli rtes etween monkeys nd humn sujets. All nimls were onsidered multiprous sed on historil dt from the originl reeding olony nd uterine histology. Histology outomes were desried previously [21]; no mmmry glnd tumors were deteted. All proedures involving mques in this study were onduted in ompline with Stte nd Federl lws nd stndrds of the US Deprtment of Helth nd Humn Servies nd were pproved y the Wke Forest University Animl Cre nd Use Committee. The filities nd lortory niml progrm of Wke Forest University re fully redited y the Assoition for the Assessment nd Aredittion of Lortory Animl Cre. Gene mirorry nlyses Mmmry glnd tissues were olleted during neropsy t the end of the two-yer tretment period [21], nd designted portions were snp-frozen in liquid nitrogen nd stored t 7 C for gene expression nlyses.totl mmmry glnd RNA ws extrted from frozen smples using Tri Regent (Moleulr Reserh Center, Cininnti, OH, USA), purified using RNesy Mini kit (QIAGEN, Vleni, CA, USA), nd quntitted using NnoDrop ND-1 UV vis spetrophotometer (NnoDrop, Wilmington, DE,

3 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 3 of 16 USA). Nulei id inttness nd qulity were onfirmed using n Agilent 21 Bionlyzer (Agilent Tehnologies, Wilmington, DE, USA). Biotinylted RNA smples were prepred ording to the stndrd Enzo Biorry protool (Enzo Life Sienes, Frmingdle, NY, USA) nd hyridized using the stndrd Affymetrix (Snt Clr, CA, USA) protool for eukryoti smples. Biotinylted RNA from eh smple ws hyridized to Affymetrix GeneChip Rhesus Mque Genome Arrys (Snt Clr, CA, USA), wshed nd stined in n Affymetrix GeneChip Fluidis Sttion (Snt Clr, CA, USA), nd snned with n Affymetrix GeneChip Snner 3 (Snt Clr, CA, USA). Intensity dt were extrted from snned imges nd heked for qulity using Affymetrix GeneChip Operting Softwre nd Expression sole (MAS5 lgorithm; Snt Clr, CA, USA). Mirorry ssys were performed t Bekmn Coulter Genomis (Morrisville, NC, USA). Four rndomly seleted smples per group were run on mirorry from ontrol,, + MPA, nd Hi Ti groups. Mirorry dt nlyses were performed using the GeneSifter softwre progrm (Perkin Elmer/Geospiz, Settle, WA, USA). Intensity dt were RMA-normlized, onverted to log 2 sle, sreened for heterogeneity mong smples nd groups, nd evluted using supervised nlysis of vrine (ANOVA) nd pirwise omprisons etween tretments. Prinipl omponents nlysis (PCA), pttern nvigtion, luster nlysis, het mpping, nd KEGG pthwy nlyses were performed on filtered dt susets, s desried in the Results setion. Differenes in gene numers ltered y eh tretment were ompred using either Fisher s ext test or hi-squre test. Euliden distnes (representing the numeri differene etween tretment vetors) were lulted s prt of hierrhil lustering dendrogrms using verge linkge. Pthwys relted to ell prolifertion were evluted using z-sores generted in KEGG nlyses; z-sore more thn 2. ws onsidered signifint overrepresenttion of genes in prtiulr pthwy. All P vlues were orreted when possile for multiple omprisons using the Benjmini nd Hoherg method (P dj ), whih derives flse disoveryrteestimtefromtherwp-vlues [23]. Representtion of differentilly expressed genes within speifi funtionl tegories ws evluted using Ingenuity Pthwy Anlysis softwre v6 (Ingenuity Systems, Redwood City, CA, USA) using Fisher s ext test with Benjmini nd Hoherg orretion nd expressed s -log 1 of the P vlue for gene numers within eh tretment group. Mirorry dt re pulily ville on the NCBI Gene Expression Omnius dtse (ession numer GSE27228). Quntittive gene expression Expression of genes ssoited with prolifertion, epithelil density, growth ftor signling, oestrogen reeptor (ER) expression nd tivity, estrogen metolism, nd reeptor tivtor of nuler ftor kpp-b (RANK)/RANK lignd (RANKL) pthwy tivity were mesured in mmmry glnd smples using quntittive rel-time reverse trnsriptse polymerse hin retion (qpcr). Mquespeifi qpcr primer-proe sets for internl ontrol genes (glyerldehyde-3-phosphte dehydrogense (GAPDH); et-tin (ACTB)) were generted through the Applied Biosystems Tqmn Assy-y-Design servie (Foster City, CA, USA). Soures for ll trget primer/proe sets re given in Additionl file 1: Tle S1. All proes spnned n exon-exon juntion to eliminte genomi DNA mplifition. qpcr retions (2 μl volume) were performed on n Applied Biosystems 75 Fst Rel-Time PCR system (Foster City, CA, USA) using stndrd Tqmn regents nd thermoyling protool [24]. Reltive expression ws determined using the ΔΔCt method. The Ct vlues for the ontrol genes GAPDH nd ACTB were verged for use in internl lirtion, nd premenopusl rest tissue RNA ws referene for plte-to-plte prllel lirtion. Clultions were performed using Applied Biosystems Reltive Quntifition 75 Softwre v2..1 (Foster City, CA, USA). Immunohistohemistry Immunohistohemil (IHC) stining ws performed on fixed prffin-emedded mmmry glnd setions. Slides were deprffinized, rehydrted in wter, prepred y het-indued epitope retrievl using Div Deloker (Biore Medil, ord, CA, USA) nd Deloking Chmer Plus (Biore Medil, ord, CA, USA) t het nd pressure yles of 125 C for 3 nd 1 seonds. Slides were grdully ooled y repling the retrievl solution with deionized wter nd rinsed twie in wsh uffer (Dko Wsh Buffer, DkoCytomtion Crpinteri, CA; five minutes) efore loding on Dko Autostiner (Dko North Ameri In, Crpinteri, CA). Setions were loked for endogenous peroxidses nd nonspeifi inding of stining regents y sequentilly inuting with 3% hydrogen peroxidse (hydrogen peroxidse lok, Thermo Sientifi Wlthm, MA; 1 minutes), Avidin (Vetor Ls, Burlingme, CA; 15 minutes), Biotin (Vetor Ls, Burlingme, CA; 15 minutes), nd TNB (Perkin Elmer; 2 minutes). Tris-NCl-loking uffer ws removed nd repled with nti-humn RANK (N-2B1.1 or N-1H8.1; Amgen, Settle, WA) or RANKL (M366; Amgen, Settle, WA) mouse monolonl ntiodies or isotype-mthed ontrol mouse IgG (BD Phrmingen, Sn Jose, CA) t onentrtions of 5 μg/ml for nti-rank nd 1 μg/ml for nti-rankl for 6 minutes. A iotinylted, got ntimouse IgG (Vetor Ls, Burlingme, CA) seondry ntiody in 1% norml humn serum Tris-NCl-loking uffer ws pplied t onentrtion of 7.5 μg/ml followed y 3 minute inution. Slides were sequentilly inuted

4 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 4 of 16 with streptvidin-horserdish peroxidse (SA-HRP; Perkin Elmer, Wlthm, MA; 3 minutes) t 1:15 dilution in TNB, tyrmide signl mplifition TSA (Perkin Elmer, Wlthm, MA; five minutes) t 1:1 dilution in mplifition diluent (Perkin Elmer, Wlthm, MA), nd then SA-HRP (Perkin Elmer, Wlthm, MA; 3 minutes) t 1:15 dilution in TNB. Slides were then inuted with diminoenzidine hromogen (Dko, Crpinteri, CA; five minutes), ounterstined with hemtoxylin (Dko, Crpinteri, CA; 3 seonds), llowed to turnlueintpwterfortwo minutes efore dehydrting with sending onentrtions of ethnol, lered with xylene, nd mounted. The intensity of IHC stining ws sored on semiquntittive sle ( = sent, 1 = wek, 2 = moderte, 3 = intense), linded to tretment group y ord-ertified pthologist. Inidene ws sored s positive IHC signl (ny intensity). Immunostining of slides for Ki-67 ntigen ws desried previously [21]. For dul leling experiments, the following modifitions to the ove proedure were performed. Antigen retrievl ws performed using Div AR regent (Biore, ord, CA; DV24G1) t 9 C overnight in the Deloking Chmer. Setions were loked s desried ove, inuted with either ntiprogesterone reeptor PGR (Dko, Crpinteri, CA; M3569 t 1:4; 4 minutes) or nti-ki-67 (Epitomis, Burlingme, CA; t 1:4; 6 minutes), deteted with Dko Mouse or Rit Envision + Systems (Dko, Crpinteri, CA, 3 minutes), nd visulized y Dko DAB+ (Dko, Crpinteri, CA, 1 minutes). Antiody stining from the first PR/Ki-67 IHC inution/stining were loked y rinsing setions in distilled wter, eluting, nd inuting the slides in Div AR regent (Biore, ord, CA) t 98 C for 1 minutes. Slides were wshed nd loked s desried ove; followed y inution with either nti- RANKL (M366 t 2 μg/ml), nti-rank (N-1H8.1 t 5 μg/ml), or mouse IgG1 isotype ontrol (5 μg/ml) for 6 minutes. Seondry ntiody inution ws performed s desried ove, followed y inution in streptvidin lkline phosphtse, tyrmide mplifition nd repet of strepvidin lkline phosphtse. Slides were then inuted with permnent red hromogen (Dko, Crpinteri, CA; 2 minutes), ounterstined with Myer s hemtoxylin, wshed nd queous mounted. Histologi imges were photogrphed using Nikon Elipse E6 mirosope with Nikon (Tokyo, Jpn) DXM12 digitl mer. The resulting imges were white-lned using Adoe Photoshop CS softwre (Sn Jose, CA); no dditionl imge modifitions were employed. Sttistil nlyses Dt were nlyzed using the SAS sttistil pkge v9 (SAS Institute, Cry, NC, USA). All dt were evluted for norml distriution nd homogeneity of vrines mong groups. Gene expression dt were evluted using nonprmetri Kruskl-Wllis test followed y two-sided Wiloxon rnk sum pirwise nlysis nd reported s fold-hnge of ontrol with 9% onfidene intervl. One ontrol group niml died during the ourse of the study, reduing the numer of ontrol nimls to 3. All pirwise P vlues were djusted for the numer of pirwise tests using Bonferroni orretion. Pre-plnned pirwise tests inluded eh tretment group versus ontrol nd + MPA versus nd Hi Ti groups. Intensity of RANK/RANKL immunostining ws evluted using one-wy ANOVA with Bonferroni s post test. To evlute the orreltion of gene or protein expression with IHC, dt were log trnsformed nd liner regression nlysis ws performed. A two-tiled signifine level of.5 ws used for ll omprisons. Results EPT eliits distint effets on glol gene expression profiles Glol mmmry glnd expression profiles were evluted y mirorry nlysis. A totl of 52,865 rry proe sets were deteted t qulity sore of more thn 2.. Of these, proes for 1,534 different genes were signifintly ltered t fold hnge (FC) more thn 1.5 nd n djusted ANOVA P <.5. HTs resulted in distint effets on mmmry glnd gene expression. Overll trnsriptionl effets were gretest for + MPA nd lowest for Hi Ti. For exmple, + MPA resulted in greter numer of signifintly ltered genes versus ontrols (n = 145) ompred with (n = 437) nd Hi Ti (n = 6) (P <.1) (Figure 1). Among these genes, PCA nd hetmp nlysis showed modest overlp in trnsriptionl profiles for nd + MPA nd profile for Hi Ti most similr to the ontrol group (Figure 1, 1). Funtionl nlysis of signifintly ltered genes showed overrepresenttion of severl tegories relted to ell prolifertion nd ner risk (Figure 1d). The most highly represented funtionl gene tegory ws ner. Within this lss, greter numer of differentilly regulted genes ws seen for + MPA (n = 497, P <1-34 )ompredwith (n = 166, P =1-14 ) nd Hi Ti (n = 2, P =.1). Other funtionl tegories showing greter overrepresenttion in the + MPA group inluded ellulr movement, signling, nd intertion (Figure 1d). EPT inreses ell prolifertion nd growth ftor signling mrkers Similr to glol profiles, quntittive expression of speifi mrkers for epithelil prolifertion (ntigen identified y monolonl ntiody Ki-67 (MKI67)) nd density (kertin 19 (KRT19)) ws highest for + MPA, intermedite for, nd lowest for tiolone groups ompred with pleo (Figure 2). Tretment effets relted to ell prolifertion inluded the Jk/Stt nd ErB pthwys, whih

5 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 5 of MPA Hi Ti 125 Component 2 +MPA Hi Ti Component 1 +MPA Hi Ti 35 -Log (P- vlue) Cner Cellulr Growth nd Prolifertion Cell Deth Cell Cyle Cellulr Movement Cell-to-ell Signling nd Intertion +MPA Hi Ti Hi Ti +MPA d Figure 1 Glol trnsriptionl ptterns in the rest tissue following tretment with ontrol (pleo), onjugted equine estrogens (), + medroxyprogesterone ette (MPA), nd high-dose tiolone (Hi Ti). () Venn digrm showing greter numers of genes ltered y + MPA ompred with nd Hi Ti. () prinipl omponent nlysis nd () hetmp of differentilly regulted genes showing greter overlp etween nd Hi Ti groups nd the + MPA nd groups. (d) Funtionl tegories of signifintly ltered trnsripts showing overrepresenttion of genes involved in ner nd severl relted lsses. mrna profiles were determined y mirorry nlysis. All digrms orrespond to signifintly ltered trnsripts with fold-hnge of more thn 1.5 versus ontrol group in t lest one group, djusted nlysis of vrine (ANOVA) P <.5, nd qulity more thn 2. were overrepresented mong upregulted genes, nd the trnsforming growth ftor (TGF)-et pthwy, whih ws signifintly overrepresented mong downregulted genes. On mirorry nlysis, Jk/Stt nd ErB-relted genes relted primrily to signl trnsduer nd tivtor of trnsription 5 (STAT5) nd epiderml growth ftor reeptor (EGFR) signling pthwys, respetively. On qpcr, expression of key moleules within these pthwys ws highest for + MPA tretment, showing similr overll expression ptterns to tht seen for MKI67 (Figure 2, 2) nd high orreltion for individul mrkers, prtiulrly STAT5A nd mphiregulin (AREG) (R >.7, P <1-2 for oth) [see Additionl file 2: Figures S1, S1]. In ontrst, no signifint tretment effets were seen on qpcr for individul mrkers of TGF-et pthwy tivity (Figure 2d).

6 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 6 of 16 Reltive Expression MK167 KRT19 e Reltive Expression STAT5A STAT5B PRLR,d e e +MPA Lo Ti Hi Ti +MPA Lo Ti Hi Ti Reltive Expression EGF AREG TGFA e e e d Reltive Expression TGFB2 BMP2 DCN +MPA Lo Ti Hi Ti Figure 2 Hormone therpy effets on gene mrkers relted to mmmry glnd prolifertion. () mrkers inluded ell prolifertion (MKI67) nd epithelil density (KRT19) nd genes involved in the following signling pthwys; () STAT5; () EGFR; nd (d) TGF-et. mrna levels were determined y quntittive PCR. Vertil lines indite 9% onfidene intervl. Letters indite signifint differenes ompred with ontrol (P <.5, P <.1, P <.1 ) nd onjugted equine estrogens plus medroxyprogesterone ette ( + MPA), (P <.1 d, P <.1 e ) groups. +MPA Lo Ti Hi Ti Adding progestin to ET inhiits ER tivity We next exmined whether ptterns of ER tivity were ssoited with tretment differenes in prolifertion nd growth ftor expression. Tretment with mrkedly indued gene mrkers of ER tivity, wheres the ddition of MPA ompletely or prtilly ntgonized this effet (depending on the mrker). For exmple, inresed trefoil ftor 1 (TFF1) expression y 82-fold, wheres + MPA ws not different from pleo (Figure 3). For other ERindued mrkers suh s progesterone reeptor (PGR) nd growth regultion y estrogen in rest ner 1 (GREB1), the ddition of MPA loked 75% nd 71% of -indued expression, respetively. This pttern ws lso present on mirorry nlysis, where the primry luster of genes differentilly ltered etween nd + MPA groups were relted to ER signling. The gene luster inluded TFF1, PGR, GREB1, nd other ER-sensitive genes suh s insulin-like growth ftor inding protein 1 (IGFBP1), rest rinom mplified sequene, nd fiulin. Tiolone hd mixed pttern of effets on ER tivity, induing PGR nd GREB1 (P <.1forothompredwithontrol)ut not TFF1 t the higher dose (Figure 3). Tretment effets on ER tivity were not diretly ssoited with hnges in expression of ER-lph (ESR1), ER-et (ESR2) (Figure 3), or key genes relted to estrdiol metolism (Figure 3, 3d). However, ER immunoleling ws lower for ESR1 following + MPA ut not s desried previously [25]. In ontrst to growth ftors, mrkers of ER tivity showed modest (PGR nd GREB1) or no(tff1) signifint orreltion with prolifertion [see Additionl file 2: Figure S1].

7 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 7 of TFF1 GREB1 PGR,e 2.5 ESR1 ESR2 Reltive Expression ,e,e Reltive Expression MPA Lo Ti Hi Ti. +MPA Lo Ti Hi Ti 3. CYP19 HSD17B1 STS d 2. HSD17B2 SULT1E Reltive Expression Reltive Expression d. +MPA Lo Ti Hi Ti. +MPA Lo Ti Hi Ti Figure 3 Hormone therpy effets on gene mrkers relted to ER expression nd tivtion. () Hormone therpy effets on gene mrkers relted to oestrogen reeptor (ER) tivtion; () ER expression; () estrdiol synthesis nd iotivtion; nd (d) estrdiol detivtion. mrna expression levels were determined y quntittive PCR. Vertil lines indite 9% onfidene intervl. Letters indite signifint differenes ompred with ontrol (P <.5, P <.1, P <.1 ) nd onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) (P <.1 d, P <.1 e ) groups. Tiolone tretment does not indue growth ftor signls The min trnsriptionl pttern mong genes ltered y the Hi Ti dose relted to ER signling; no other ler ptterns were noted. Of the 24 identified genes with more thn three FC nd P <.5 ompred with ontrol, 2 genes were upregulted. Among these, there were seven known ER-sensitive genes (PGR, GREB1, stnniolin 2 [STC2], seretogloin fmily 1D memer 2 [SCGB1D2], kllikreins 11 [KLK11] nd 12 [KLK12], nd IGFBP1); two genes involved in steroid metolism (ytohrome p45 fmily 2 sufmily A polypeptide 7 [CYP2A7] nd 3-ethydroxysteroid dehydrogense Δ-5-Δ-4 isomerse type II [HSD3B2]); two seretogloins (SCGB3A1 nd SCGB1D2); nd three peptidses (disintegrin nd metlloproteinse with thromospondin motifs 8 [ADAMTS8], KLK11, nd KLK12). Notly, isoforms of HSD3B re the primry enzymes driving prodution of the progestogeni delt-4 tiolone metolite [13]. Despite 6.5 FC inrese in HSD3B2 expression in the Hi Ti group, no mrkers of progestogeni tivity were noted. EPT inreses RANK/RANKL pthwy expression The RANK pthwy exhiits rosstlk with oth STAT5 [26] nd EGFR [27] signling nd ontriutes to progestogen-indued ell prolifertion in the mouse mmmry glnd [28,29]. Mouse mmmry expression of RANKL is inresed sustntilly fter progestin tretment [3], nd inresed RANK expression is oserved t dutl side

8 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 8 of 16 rnhes nd lveoli during pregnny-indued mmmry morphogenesis [31]. To further explore hormonl regultion of this pthwy in the primte mmmry glnd, we evluted ET nd EPT effets on expression of mrnas enoding RANK, RANKL, nd the endogenous inhiitor of RANKL, osteoprotegerin (OPG). No signifint group differenes in RANK, RANKL, or OPG expression were noted in nlysis y mirorry. By qpcr, tretment with lone did not result in signifint hnges in RANK, RANKL, or OPG expression (Figure 4), wheres tretment with + MPA resulted in signifintly higher expression of RANK nd lower expression of OPG reltive to RANK RANKL 5 OPG : P <.5 reltive to Reltive Expression : P <.1reltiveto : P <.1 reltive to +MPA RANK/OPG RANKL/OPG 8 : P <.5 reltive to : P <.1 reltive to : P <.1reltiveto Rtio of mrna Expression d : P <.5 reltive to +MPA d +MPA Figure 4 Hormone therpy effets on gene mrkers of RANK/RANKL signling. () mrna expression of RANK, RANKL, nd the endogenous inhiitor of RANKL signling (OPG) nd () rtios etween these mrkers. mrna levels were determined y quntittive PCR nd expressed s () reltive mens or s () rtios of RANK/OPG or RANKL/OPG. Vertil lines indite 9% onfidene intervl. Letters indite signifint differenes etween groups s indited.

9 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 9 of 16 ontrol (P <.5 nd P <.1, respetively; Figure 4). Tretment with + MPA lso inresed RANKL mrna more thn three-fold versus ontrol nd the differenes etween RANKL mrna levels fter + MPA versus tretment were sttistilly signifint (P <.1).Anlysis of RANKL mrna hnges ws onfounded y one niml in the -treted ohort whih hd more thn 5-fold higher levels of RANKL mrna thn ll other nimls. Tretment with + MPA resulted in greter rtios of RANK nd RANKL to OPG ompred with ontrol (P <.1 nd P <.1, respetively; Figure 4) nd greter rtio of RANKL to OPG ompred with the group (P <.5; Figure 4). Although RANKL mrna levels did not orrelte with MKI67 [see Additionl file 3: Figure S2], the rtios of RANKL:OPG nd RANK:OPG expression showed signifint positive ssoition with MKI67 (Figure 5), with the strongest orreltions oserved in the + MPA group (R =.59, P =.7 nd R =.64, P =.2, respetively). The RANK/OPG rtio expression lso showed signifint positive ssoition with STAT5A (R =.69, P <.1) nd KRT19 (R =.55, P <.1), wheres RANKL/OPG rtio showed signifint positive, yet modest, orreltions with STAT5A nd KRT19 (R =.35, P =.8 nd R =.29, P =.6, respetively) [see Additionl file 4: Figure S3] MPA R P-vlue Not orrelted Not orrelted +MPA.59.7 Log 1 MKI67 RQ Log 1 RANKL/OPG Log 1 MKI67 RQ MPA R P-vlue Not orrelted MPA Log 1 RANK/OPG Figure 5 Regression nlysis for expression of the prolifertion mrker MKI67 versus RANKL/OPG or RANK/OPG mrna rtios. Correltion of MKI67 nd the RANKL/OPG mrna rtio ws only oserved within the onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) group nd not in the ontrol or groups. The strongest orreltion of MKI67 nd RANK/OPG mrna rtio ws oserved in the + MPA group.

10 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 1 of 16 RANKL nd RANK protein show distint ptterns of expression in the mmmry glnd We next utilized IHC with monolonl ntiodies ginst hurankl or hurank to verify tht the oserved hnges in mrna expression were trnslted into protein nd to determine preise ellulr loliztion for eh protein. RANKL protein ws not oserved within the mmmry epithelium of postmenopusl monkeys in the ontrol group ut ws seletively inresed in the + MPA group (Figures 6; 7, ). In ontrst, RANK protein in the mmmry glnd ws prevlent ross ll groups (>5% of monkeys in eh group; Figures 6; 7) with modestly lower omposite sore for the nd + MPA groups ompred with ontrol (P <.5 nd P <.1,respetively;Figure7).RANKLprotein expression ws oserved exlusively within luminl epithelil ells of duts nd loulolveolr strutures, suh tht RANKL-positive ells were djent to RANKLnegtive ells (Figure 6). Dul immunostining of smples from + MPA-treted monkeys indited tht RANKL protein ws lolized in PGR-expressing luminl epithelil ells of duts nd loulolveolr strutures [see Additionl file 5: Figure S4], similr to wht hs een desried in mie [28] nd humns [32]. Expression of RANK protein ws not uniform within the mmmry glnd, showing segmentl foi of positive stining predomintely within duts nd loulolveoli (Figure 6). Furthermore, RANK protein expression ws oserved in sl ells nd other epithelil ells tht extended from the sl omprtment to the lumen. Immunostining of oth RANK nd RANKL ws predomintely limited to mmmry epithelium, with rre expression in infiltrting ells (presumed lymphoytes). Stining ws ytoplsmi nd memrnous for oth RANK nd RANKL, often with grnulr ytoplsmi pperne for RANKL. This ellulr distriution of RANK nd RANKL protein within the monkey mmmry glnd ws similr to tht oserved in mie [28,31] nd tissue from norml humn rest (Brnstetter nd Dougll, mnusript in preprtion). RANKL nd RANK protein expression is ssoited with mmmry epithelil ell prolifertion The intensity of RANKL protein expression determined y IHC showed signifint positive orreltion with RANKL mrna within the + MPA group (R =.62, P =.4) ut not the ontrol nd groups [see Additionl file 6: Figure S5]. Previous nlysis using Ki-67 IHC defined mmmry epithelil prolifertive response speifilly in the + MPA group, with the mjority of leling in the loulolveolr omprtment nd miniml Ki-67 inreses oserved in lrge duts [21]. Here, RANKL proteinexpressionwithin the + MPA group ws signifintly orrelted with the degree of prolifertion s determined y Ki-67 IHC in oth lveoli (R =.48, P =.9; Figure 8) nd duts (R =.6, P =.2; Figure 8); there were no signifint positive orreltions of RANKL protein nd Ki-67 IHC within ontrol or treted groups (Figure 8, 8). RANK protein nd mrna were not signifintly orrelted in ny group [see Additionl file 6: Figure S5]. Although the intensity of RANK protein ws not orrelted with the degree of prolifertion in ny group [see Additionl file 7: Figure S6], dul leling of RANK nd Ki-67 ws oserved in suset of proliferting rest epithelil ells from + MPAtreted monkeys. Segmentl foi of rest epithelium tht stined positively for RANK were lso frequently positive for Ki-67 wheres RANK-negtive regions of the sme rest tissue often hd few or no Ki-67 leled ells [see Additionl file 8: Figure S7]. In ddition, ler exmples of individul ells positive for oth RANK nd Ki-67 were oserved in duts nd loulolveolr strutures [see Additionl file 8: Figure S7]. Disussion The ddition of progestin to ET is ssoited with inresed rest tissue prolifertion [33], mmmogrphi density [4], nd rest ner risk in postmenopusl women [4,7,8]. Moleulr mehnisms driving these effets re not lerly defined. In this study we show tht dding the progestin MPA to drmtilly ltered the mrna profile in the norml primte mmmry glnd. This hnge ws ssoited with greter mmmry glnd prolifertion, deresed mrkers of ER tivity, nd inresed mrkers of growth ftor signling. Mny of the progestin-dependent hnges oserved here re lso seen in the mouse mmmry glnd nd hve een ssoited with mmmry rinogenesis. These findings identify key differenes mong ommon types of menopusl HTs nd highlight speifi pthwys relevnt to hormonl promotion of mmmry epithelil ell growth. Our results show ler differenes etween ET nd EPT effets on rest tissue nd support the hypothesis tht EPT inreses ell prolifertion eyond tht of ET lone due in prt to speifi growth ftor signls. Three primry progestin-regulted pthwys were identified in this study: proltin reeptor (PRLR)/STAT5, EGFR, nd RANK/RANKL. The STAT5 pthwy hs een shown to medite PRLR tivity nd regulte mmmry glnd development, differentition, nd prolifertion [26,34], wheres EGFR is entrl growth ftor pthwy in mmmry glnd development nd suset of rest ners [35]. Both PRLR/STAT5 nd EGFR pthwys re lso known trgets of progestogen tion in the mouse mmmry glnd [36,37]. RANK/RANKL is the third pthwy seletively modified y the omintion of + MPA. This pthwy hs importnt roles in lymph node development during

11 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 11 of 16 RANKL 2 m 2 m Dut Alveoli +MPA 2 m 2 m Dut Alveoli RANK 2 m 2 m Dut Alveoli +MPA Dut 2 m 2 m Alveoli Figure 6 Expression nd loliztion of RANKL nd RANK protein within the mmmry glnd. (,) Representtive imges of RANKL nd RANK immunohistohemil stining in ontrol nd onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) groups. There is no detetle stining for RANKL in the ontrol group ut RANKL protein is lerly evident exlusively within the luminl epithelil ells of the loulolveolr nd dutl epithelium in the + MPA group. RANKL ws not deteted within myoepithelil, stroml, or infiltrting immune ells. RANK expression ws oserved in oth dutl nd loulolveolr strutures in ll groups. RANK protein ws predomintely oserved in sl ells in lose proximity to the sement memrne nd lso in some epithelil ells, whih extended from the sement mtrix/sl lyer to the lumen. The oserved stining of intrluminl seretory mteril using the RANK ntiodies ws due to non-speifi inding to proteineous mteril in serum (dt not shown).

12 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 12 of 16 Inidene of RANK nd RANKL Expression N RANK RANKL % % % 11% +MPA % 71.4% Composite IHC Sore MPA : P <.5 reltive to : P <.1 reltive to : P <.1reltiveto d : P <.1reltiveto RANK RANKL Figure 7 Hormone therpy effets on inidene of expression nd intensity of IHC stining for RANK nd RANKL. () Inidene sore for RANK or RANKL immunohistohemil (IHC) positivity in eh group. () Men omposite IHC sore +/ stndrd error of the men in eh tretment group. Letters indite signifint differenes versus ontrol (P <.5, P <.1, P<.1 ) or onjugted equine estrogens () (P <.1 d ) groups. emryogenesis nd is essentil for the formtion, funtion, nd survivl of one-resoring osteolsts [38]. Modultion of the ltter mehnism is the sis for the development of the fully humn monolonl ntiody to RANKL, denosum, reently pproved for the prevention of skeletl-relted events in ptients with one metstses from solid tumors [39]. Anlysis of RANK- nd RANKLknokout mie reveled defetive mmmry lveologenesis [4], whih resemled the mmmry morphogeni defet oserved in PGR-knokout mie [41]. Trnsription of RANKL is rpidly indued upon progesterone exposure in mie [3] nd o-lolized with PGR within trnsmitter ER/PGR-positive luminl mmmry epithelil ells [42]. Susequent studies hve shown tht RANKL is n essentil prrine meditor of progesterone funtion in the mouse mmmry glnd, leding to oth mmmry epithelil prolifertion [43] nd the trnsient expnsion nd inresed regenertive potentil of mmmry stem ells [44,45] during pregnny nd the estrous yle. Importntly, these,d Log 1 Ki67 Positive Cells (%) Log 1 Ki67 Positive Cells (%) Composite RANKL IHC Sore MPA +MPA Loulolveolr Dutl R=.6 P = Composite RANKL IHC Sore R=.48 P =.9 Figure 8 Correltion of RANKL protein expression with Ki-67 protein expression. Regression nlysis for expression of the prolifertion mrker Ki-67 (immunohistohemil (IHC)) versus RANKL protein (IHC) in () loulolveolr or () dutl ells. Correltion etween RANKL IHC sore nd perent Ki-67 positive ells is oserved in onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) treted nimls (R =.48, P =.9 for loulolveolr epitheli, R =.6, P =.2 for dutl epitheli). The mjority of nimls within the ontrol nd groups (1% nd 88%, respetively) did not express RANKL y IHC, preluding n urte orreltion nlysis etween RANKL nd Ki-67 in these groups. funtionlities re not limited to norml mmmry morphogenesis nd oservtions in rodent models hve now shown tht RANKL, vi tivtion of RANK within mmmry epithelium, medites progesterone-dependent mmmry tumor formtion [28,29]. Currently, it is unler whether the RANK/RANKL pthwy funtions similrly in humn rest tissue. In the urrent study, we show tht key omponents of the

13 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 13 of 16 RANKL pthwy re expressed in the norml primte mmmry glnd nd modulted y long-term EPT exposure t linilly relevnt doses. Protein expression ptterns of RANKL nd RANK protein were highly similr to those seen in the mouse [28] nd humn mmmry glnd (dt not shown) [32]. In eh speies, RANKL protein is folly expressed in disrete luminl epithelil ells of the duts nd loules often seprted y djent RANKL-negtive ells. Dul immunoleling reveled tht RANKL protein expression ws highly ololized within PGR-positive luminl epithelil ells in this study, similr to wht hs een reently desried in mie nd humns [28,32]. In ontrst, RANK protein is segmentlly expressed, spordilly in lveoli nd often in ells loted long the sl spet of duts ut lso in epithelil ells tht extend from the sl omprtment to the lumen. Similr to oservtions in mie, RANKL protein levels within the mmmry epithelium of mques were lerly elevted upon exposure to estrogen with progestin ut not estrogen lone. In the + MPA group, we lso oserved deresed mrna expression levels of OPG, the negtive regultor of RANKL, therey inresing the rtio of either RANKL/OPG or RANK/OPG. Inresed RANKL protein expression ws positively ssoited with inresed dutl nd lveolr prolifertion driven y EPT. RANK protein ws ololized with Ki-67 in suset of ells, suggesting tht RANKexpressing rest ells diretly respond to the RANKL signl nd omprise t lest prt of the prolifertive omponent fter progestin exposure. Altogether, multiple mehnisms of hormone-dependent ontrol ontriuting to the net inrese in RANKL signl were identified nd shown to e positively ssoited with inresed epithelil prolifertion (MKI67) nd density(krt19), suggesting tht this pthwy my e utilized ross mmmlin speies for progestogen-dependent rest prolifertion. In premenopusl women, ovrin-produed progesterone my lso ontriute to the estlished reltion etween rest ner risk nd numer of menstrul yles or reprodutive history [46], potentilly vi inresed rest prolifertion [47] nd the non-prolifertive expnsion of norml or trnsformed mmmry stem ells [44,45,48]. Using ndidte gene pproh, reent study identified RANKL, -Kit, nd gene signtures representing MSC or luminl progenitors s eh eing ssoited with younger ge t rest ner dignosis [49]. Although the reltive ontriution of RANKL mrna from norml rest versus tumor tissue ws not speified in this nlysis of ptients with rest ner, the uthors onluded tht the strong orreltion of these gene sets (inluding RANKL expression)wsindependentofrestnersutypendinsted represented unique iologil pthwys ommon to rest ner in young women nd perhps relted to the poor prognosis in these ptients. Given the present evidene, the inresed mmmry mitogenesis oserved during the progesterone-dominnt lutel phse of the humn menstrul yle [47] ould involve n opertive role of RANKL. Reent gene expression nlysis of fine-needle spirtes of humn rest tissue demonstrting signifint upregultion of RANKL mrna during the lutel phse [5] is onsistent with this notion. In ft, reent pulition [32] hs demonstrted tht RANKL levels in the humn rest re orrelted with serum progesterone levels. Furthermore, RANKL ws not only suffiient to indue humn rest ell prolifertion ut ws lso required for progesterone-indued rest ell prolifertion. These dt, with oservtions presented in this primte study, suggest tht the inresed RANKL signl in humn rest tissue is onsequene of progestogen exposure in postmenopusl women or lutel phse ovrin progesterone in premenopusl women. Moreover, this inresed RANKL my e orrelted with the prolifertive sttus nd overll density of the mmmry epithelium nd ontriute to hormone-dependent rest tumor formtion. The three signling pthwys identified here s eing seletively inresed y EPT ll exhiit signling ross-tlk tht my e funtionlly importnt in rest ner. Prior studies hve shown tht the indution of RANKL y MPA requires expression of PRLR nd tht proltin signling is neessry for nuler trnslotion of STAT5A fter EPT [29,51]. These findings lso indite tht the interferongmm responsive elements identified within the RANKL promoter re essentil for tivtion of the JAK2/STAT5A response [51] nd potentilly importnt for progestogendependent inreses. Other studies hve shown tht nuler phosphorylted STAT5A is o-lolized with PGR nd RANKL in ells fter EPT [26], further suggesting tht progestogen-dependent inreses in RANKL trnsription my e governed t the RANKL promoter, t lest in prt, y omplex of PGR nd STAT5A, similr to tht oserved with the β-sein promoter [52]. Finlly, EGFR lignds hve een shown to strongly derese OPG expression in n EGFR-dependent mnner [27] nd tivte STAT5A in mmmry tissue [53]. Colletively, these dt support model in whih progestogen tivity in rest tissue my inrese RANKL protein expression either diretly, or indiretly, vi PRLR/STAT5 signling, wheres OPG protein expression my e deresed vi EGFR signling. Future studies re wrrnted to determine if multiftoril onvergenes of the PRLR/STAT5, EGFR, nd RANK/RANKL pthwys my ontriute to rest ner risk. Unlike EPT, tiolone did not show ler indution of mrna mrkers in rest tissue relted to growth ftor signling. This finding is onsistent with the lk of inresed mmmry epithelil Ki-67 leling reported previously [21] nd the lk of inresed rest ner risk mong older postmenopusl women reeiving tiolone in the LIFT linil tril [12]. In ontrst, tiolone tretment

14 Wood et l. Brest Cner Reserh 213, 15:R62 Pge 14 of 16 in the LIBERATE linil tril ws ssoited with 4% inresed risk of reurrene mong rest ner ptients with ER-positive tumors nd vsomotor symptoms [15]. Su-group nlysis mong women reeiving djuvnt endorine therpy indited tht tiolone interferene ws greter for romtse inhiitors (whih derese systemi nd lol estrogen) thn for tmoxifen (whih loks ERs). The uthors of the study suggest tht these findings point to potentil ER gonist effets of tiolone on oult, estrogen-sensitive metstses [15]. Results from the urrent study provide limited support for this ide, showing modest estrogeni effets of tiolone on some gene mrkers of ER tivity ut no ler progestogeni effets or tivity relted to speifi growth ftor pthwys. lusions Minimizing rest ner risk y mitigting potentil dverse effets of hormonl gents is entrl hllenge in women s helth. Results of this study expnd the urrent understnding of trnsriptionl ptterns nd signling pthwys underlying HT effets in mmmlin rest tissue. Findings presented here identify PRLR/STAT5, EGFR, nd RANK/RANKL s moleulr pthwys tht my e relevnt to inresed rest tissue prolifertion, mmmogrphi density, nd rest ner risk in postmenopusl women tking EPT. These pthwys re potentil trgets for ssessing nd preventing progestogen-ssoited risk, nd this informtion should help inform linil strtegies to etter prevent hormone-ssoited rest ner nd reurrene. Additionl files Additionl file 1: Tle S1. Primer/proe sets for trget genes evluted y quntittive PCR. Additionl file 2: Figure S1. Regression nlysis for expression of the prolifertion mrker MKI67 nd mrkers of STAT5, epiderml growth ftor reeptor (EGFR), nd estrogen reeptor (ER) signling pthwys. () MKI67 mrna versus STAT5 mrkers; () MKI67 mrna versus EGFR mrkers; nd () MKI67 mrna versus ER mrkers. Aross ll groups, the strongest positive orreltions were oserved etween MKI67 versus STAT5A, PRLR, mphiregulin (AREG), nd trnsforming growth ftor-lph (TGFA) (P <.1 for ll). Additionl file 3: Figure S2. Regression nlysis for MKI67 versus RANKL nd RANK. () RANKL mrna versus MKI67. () RANK mrna versus MKI67. Signifint positive orreltions were oserved etween RANK versus MKI67 (R =.48, P <.1). Additionl file 4: Figure S3. Regression nlysis for STAT5A or mrker for rest density (KRT19)versusRANKL/OPG or RANK/OPG mrna rtios. () RANKL/OPG mrna rtio versus STAT5A.()RANK/OPG mrna rtio versus STAT5A.()RANKL/OPG mrna rtio versus KRT19.(d)RANK/OPG mrna rtio versus KRT19. Signifint positive orreltions were oserved etween RANK/ OPG versus STAT5A (R =.69, P <.1) nd RANK/OPG versus KRT19 (R =.55, P <.1). RANKL/OPG versus STAT5A or KRT19 show modest positive orreltions (R =.35, P =.8 nd R =.29, P =.6, respetively). Additionl file 5: Figure S4. RANKL is lolized in PGR-positive mmmry luminl epithelil ells. Representtive exmples of immunohistohemil oleling of RANKL nd PGR in rest tissue from onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) treted monkeys. RANKL is red nd PGR is rown. () RANKL nd PGR leling in rest dut (4X); () RANKL nd PGR leling in loulolveolr struture (6X). RANKL nd PGR protein is lerly evident exlusively within the luminl epithelil ells of the loulolveolr nd dutl epithelium. Cytoplsmi nd memrne RANKL expression is lolized in PGR expressing ells. Additionl file 6: Figure S5. Correltion of either RANKL or RANK protein expression with orresponding mrna expression. () RANKL protein immunohistohemil (IHC) sore versus RANKL mrna. A signifint positive orreltion ws oserved in the onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) group (R =.62, P =.4) only. The mjority of nimls within the ontrol nd groups (1% nd 88%, respetively) did not express RANKL y IHC, preluding orreltion nlysis etween protein nd mrna expression in these groups. () RANK protein IHC sore versus RANK mrna. There were no signifint orreltions in ny group. Additionl file 7: Figure S6. Correltion of RANK protein expression with Ki-67 protein expression. RANK omposite immunohistohemil (IHC) sore versus log 1 Ki-67 positive ells (%) in () loulolveolr or () dutl ells. No orreltion etween RANK IHC sore nd Ki-67 positive ells (%) ws oserved. Additionl file 8: Figure S7. Dul leling of RANK protein expression nd Ki-67 in rest epithelium from onjugted equine estrogens plus medroxyprogesterone ette ( + MPA) treted monkeys. ( to d) Representtive exmples of immunohistohemil (IHC) o-leling of RANK nd Ki-67 in rest tissue from + MPA-treted monkeys. RANK is red nd Ki-67 is rown. Nuler stining of Ki-67 ws oserved in suset of ells with ytoplsmi nd memrne expression of RANK. Low mgnifition views (, 1X;, 4X) of rest tissue demonstrte segmentl foi of rest epithelium stined positively for RANK tht were lso frequently positive for Ki-67 (s irumsried y dshed lines). versely, RANK-negtive regions of the sme rest tissue often hd few or no Ki-67 leled ells. ( nd d) Higher mgnifition views (6X) show ler exmples of individul ells positive for oth RANK nd Ki-67 in () duts nd (d) loulolveolr strutures. Individul dutl ells positive for oth Ki-67 nd RANK re indited y rrows. In the exmple of stining in loulolveolr tissue, the mjority of this prtiulr segment stins positively for RANK (similr to tht shown in Figure 6 nd Additionl file 8: Figure S7, ) with Ki-67 stining in suset of these RANK-positive ells. Arevitions ACTB: Bet-tin; ANOVA: Anlysis of vrine; AREG: Amphiregulin; : jugted equine estrogens; EGFR: Epiderml growth ftor reeptor; EPT: Estrogen + progestin therpy; ER: Estrogen reeptor; ESR1: Estrogen reeptor-lph; ESR2: ER-et; ET: Estrogen-lone therpy; FC: Fold hnge; GAPDH: Glyerldehyde-3-phosphte dehydrogense; GREB1: Growth regultion y estrogen in rest ner 1; HSD17B1: 17-et hydroxysteroid dehydrogense type 1; HSD17B2: 17-et hydroxysteroid dehydrogense type 2; HSD3B: 3-et-hydroxysteroid dehydrogense/delt-5-delt-4 isomerse; HT: Hormone therpy; IGFBP1: Insulin-like growth ftor inding protein 1; IHC: Immunohistohemil; KRT19: Kertin 19; MKI67: Antigen identified y monolonl ntiody Ki-67; MPA: Medroxyprogesterone ette; OPG: Osteoprotegerin; PCA: Prinipl omponents nlysis; PGR: Progesterone reeptor; PRLR: Proltin reeptor; qpcr: Quntittive rel-time polymerse hin retion; RANK: Reeptor tivtor of nuler ftor kpp B; RANKL: Reeptor tivtor of nuler ftor kpp B lignd; SA-HRP: Streptvidin-horserdish peroxidse; STAT5: Signl trnsduer nd tivtor of trnsription 5; TFF1: Trefoil ftor 1; TGF: Trnsforming growth ftor; Ti: Tiolone; WHI: Women s Helth Inititive. Competing interests CW hs reeived reserh funding from Amgen In nd Pfizer; DB, AJ, KR, LH, nd WD re employees nd hve reeived stok from Amgen In; JC hs onsulted nd reeived reserh funding from Amgen In; TR nd HB hve no dislosures. Authors ontriutions eption nd design: CW, WD, nd DB. Development of methodology: CW, WD, DB, HB, LH, KR, TR. Aquisition of dt: CW, WD, LH, DB, nd JC. Anlysis nd interprettion of dt: CW, AJ, WD, DB, nd JC. Writing, review, nd/or revision of the mnusript: CW, AJ, WD, DB, TR, nd JC. All uthors red nd pproved the mnusript.