Research Article Thyroid Hormone Status Interferes with Estrogen Target Gene Expression in Breast Cancer Samples in Menopausal Women

Transcription

1 ISRN Endorinology, Artile ID , 8 pges Reserh Artile Thyroid Hormone Sttus Interferes with Estrogen Trget Gene Expression in Brest Cner Smples in Menopusl Women Sndro José Conde, 1, Rent de Azevedo Melo Luvizotto, Mri Teres de Síio, nd Céli Regin Nogueir 1 Deprtment of Biologil Siene, São Pulo Federl Institute (IFSP), São Roque, SP, Brzil Deprtment of Internl Mediine, Division of Endorinology nd Metolism, UNESP, Botutu, SP, Brzil Correspondene should e ddressed to Sndro José Conde; ondesn@yhoo.om.r Reeived 11 Novemer 13; Aepted 11 Jnury 14; Pulished Ferury 14 Ademi Editors: J. Phuki, V. Pezzi, nd D. F. Skfr Copyright 14 Sndro José Conde et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. We investigted thyroid hormone levels in menopusl BrC ptients nd verified the tion of triiodothyronine on genes regulted y estrogen nd y triiodothyronine itself in BrC tissues. We seleted 15 postmenopusl BrC ptients nd ontrol group of 18 postmenopusl women without BrC. We mesured serum TPO-AB, TSH, FT4, nd estrdiol, efore nd fter surgery, nd used immunohistohemistry to exmine estrogen nd progesterone reeptors. BrC primry tissue ultures reeived the following tretments: ethnol, triiodothyronine, triiodothyronine plus 4-hydroxytmoxifen, 4-hydroxytmoxifen, estrogen, or estrogen plus 4-hydroxytmoxifen. Genes regulted y estrogen (TGFA, TGFB1, nd PGR) nd y triiodothyronine (TNFRSF9, BMP-6, nd THRA) in vitro were evluted. TSH levels in BrC ptients did not differ from those of the ontrol group (1.34 ±.6 versus.41 ± 1.1 μu/ml), ut FT4 levels of BrC ptients were sttistilly higher thn ontrols (1.78 ±. versus.95 ±.16 ng/dl). TGFA ws upregulted nd downregulted fter estrogen nd triiodothyronine tretment, respetively. Triiodothyronine inresed PGR expression; however 4-hydroxytmoxifen did not lok triiodothyronine tion on PGR expression. 4-Hydroxytmoxifen, lone or ssoited with triiodothyronine, modulted gene expression of TNFRSF9, BMP-6, nd THRA, similr to triiodothyronine tretment. Thus, our work highlights the importne of thyroid hormone sttus evlution nd its ility to interfere with estrogen trget gene expression in BrC smples in menopusl women. 1. Introdution For mny yers, ssoitions etween thyroid disorders nd rest ner (BrC) hve rised questions regrding the involvement of thyroid hormone (TH) (either ssoited with estrogen reeptor or not) on the development nd progression of rest ner, nd signifint reserh efforts hvefousedonthisre[1 1]. Reently study first showed tht TH levels in postmenopusl women re positively relted to BrC risk in dose-response mnner [9]. Prognosti nd preditive ftors re indispensle tools in neoplsti disese tretment [11],nd estrogen reeptor (ER) onentrtion is n importnt prmeter in BrC prognosis [1]; ER sttus is n importnt onsidertion for BrC ntiestrogen tretment [13]. Therefore, the presene nd onentrtion of ER provide ruil informtion regrding tumors tht respond to hormonl intervention [14]. Positive ER detetion in BrC tissues is n indition of tumor with hormonl dependene nd indites the enefit of endorine therpy to this type of BrC [15]. Additionlly, identifition of positive or negtive ER tumors n diret therpeuti strtegies nd linil prognosis [16 18]. Given the known effet of TH on BrC, there is little informtion out how this hormone inds with the reeptors of rest tumor ells. Previous reserh demonstrted TH reeptors in the nulei of MCF-7 ells [19], while other works onfirmed the presene of TH reeptors in BrC tissue, ut without orreltion with other hormonl reeptors (ER or progesterone reeptor) or tumor progression []. Sho et l. [1] showed tht triiodothyronine (T3) potentites estrogen tion on ER-positive BrC ell lines.

2 ISRN Endorinology We previously performed in vitro studies in MCF-7 nd MDA-MB-31 ell lines to show tht suprphysiologil onentrtion of T3 indues ell prolifertion nd expression of genes previously stimulted y estrdiol (E) independent of ER, with inhiition of T3 indution y tmoxifen (TAM) [7]. It is reognized tht E nd the hormonl sttus of ptient re importnt in BrC ell prolifertion nd tretment [15], nd with respet to T3, while epidemiologil studies hve produed ontrditory dt regrding its effet on BrC [1, 7], lortory studies hve demonstrted its ility to indue BrC prolifertion in ER-dependent mnner, possily through rosstlk etween the TH nd E pthwys [3, 7, 8]. We investigted thyroid hormones levels in menopusl BrC ptients euse of their lk of estrogen nd verified the tion of T3 on genes regulted y E (TGFA, TGFB1, nd PGR) [7] nd T3 (TNFRSF9, BMP6, nd THRA) [9] in primry BrC tissues exhiiting the erly stges of tumor progression.. Methods.1. Ptients. The study ws pproved y the Cner Hospitl, Antônio Prudente Foundtion, São Pulo, Brzil, nd Ethis Committee, nd ll ptients signed n informed onsent form. Ptients reruited to this study were newly dignosed with rest ner nd underwent surgery t the Cner Hospitl, Antônio Prudente Foundtion, São Pulo, Brzil. All ses were lssified s tumor node metstsis stge I or II. Ages rnged from 48 to 55 yers, nd ll ptients were menopusl (menorrhe for t lest 1 yer). Ptients were exluded for the following resons: rdioor hemotherpy dministrtion efore surgery, hormone replement therpy, ny kind of previously dignosed thyroid disese, hroni kidney filure, or reent elevtion in serum retinine vlues over those normlly expeted for tht prtiulr ge. Other exlusion ftors were norml hepti funtion with sprtte minotrnsferse, lnine minotrnsferse, iliruin, or lkline phosphtse onentrtions higher thn twie the norml upper limit; use of β-loking gents, spirin, heprin, phenytoin, steroids, or dopmine in the month efore or during the study; use of iodine-ontining ontrst gents in the six months efore nd during the study. A ontrol group onsisted of 18 women ged 47 to 57 yers whose reent mmmogrms indited the sene of rest ner. These mmmogrms were performed in the sme week when nmnesis nd lood smples were olleted... Immunohistohemistry. The presene of ER nd progesterone reeptors (PR) in tumors ws determined y immunohistohemil stining using monolonl ntiody to ERα (Upstte Biotehnology In., Lke Plid, NY, USA) nd monolonl nti-pr ntiody 636 (M3569, DkoCytomtion). Biotinylted seondry ntiodies (nti-mouse IgG or nti-rit IgG) were otined from Vetor Lortories (Burlingme, CA, USA). Endogenous peroxidse in tissue setions ws loked y inution with solution of 1% hydrogen peroxide for 3 min, nd ntigen retrievl ws performed y mirowving setions in.1 M itrte uffer (ph 6.) for min t 8 W. Antiodies were diluted individully in PBS ontining 3% BSA. ERα ntiody ws used t dilution of 1 : 5 nd PR ntiody ws used t 1 : 1. Prior to ddition of seondry ntiody, tissue setions were rinsed in PBS ontining.5% Tween. The retions were developed with n vidin-iotin-peroxidse omplex. Tumors known to e positive for the studied mrker were onsidered to e positive ontrols. Tumors were onsidered positive with moderte intensity of stining nd the proportion of this intensity t more thn 1% of ells [3]..3. Serum Dosge. Serum liquots were nlyzed for thyroid peroxidse ntiody (TPO), thyroid-stimulting hormone (TSH), free thyroxine (FT4), nd E using ommerilly ville kits (DPC, Los Angeles, CA, USA). The norml rnges were <35. UI/mL for nti-tpo negtive,.4 4. μu/ml for TSH,.8. ng/dl for FT4, nd. 3. pg/ml for E (fter menopuse)..4. Chemils. E, T3, nd TAM were purhsed from Sigm. Eh ws dissolved in ethnol to give stok solution nd used diluted for use in ulture t the following onentrtions: E t physiologil onentrtion of 1 8 M, T3 t suprphysiologil onentrtion of 1 8 M, nd TAM t phrmologil onentrtion of 1 6 M[7]..5. Primry Culture. Fresh humn rest rinom tissue remining fter pthologil nd prognosti nlysis ws otined from ollorting surgeons nd pthologists; it ws trimmed free of ft nd pled in phosphte-uffered sline.akrumdiektissueslier(almreserhcorportion) ws used to otin.3 mm slies. Slies were divided into six 35 mm dishes nd pled on silionized lens pper floting in ml orgn ulture medium. Eh tretment ws performed in single dish ontining to 3 slies. Primry tissue ws ultured in phenol-red-free RPMI supplemented with 1 U/mL peniillin, 1 μg/ml streptomyin, 5 μg/ml BSA, nd 5 μg/ml insulin (Life Tehnologies Corportion, Crlsd, CA, USA). Dishes reeived the following tretments: dish 1: ethnol; dish : T3 (1 8 M); dish 3: T3 (1 8 M) plus TAM (1 6 M); dish 4: TAM (1 6 M);dish5:E(1 8 M);dish6:E(1 8 M) plus TAM (1 6 M). Cultures were mintined t 37 Cinhumidified tmosphere of 95% ir/5% CO. Medium hnges were performed fter 4 h nd hrvesting ws performed fter 48 h [31]..6. RNA Isoltion nd Reverse Trnsription. Totl RNA ws extrted from slies y the gunidinium thioynte methodndnlyzedyeletrophoresisusing1%grose gels. One mirogrm of totl RNA ws reverse trnsried with SuperSript III First-Strnd Synthesis System for RT- PCR (Invitrogen, no ).

3 ISRN Endorinology 3.7. Mesurement of Gene Expression y Quntittive Rel- Time PCR. The rel-time RT-PCR method with n Assyon-Demnd Gene Expression Produt (Life Tehnologies, P/N ) onsisted of unleled PCR primers nd TqMn MGB proe (FAM dye-leled) optimized to work with the TqMn Universl PCR Mster Mix (P/N ) in n ABI Prism 77 system (PerkinElmer Life Sienes, Boston, MA, USA) nd ws employed to quntittively mesure trnsforming growth ftor lph (TGFA; Hs68187 m1), trnsforming growth ftor et 1 (TGFB1; Hs m1), progesterone reeptor (PGR; Hs15567 m1), tumor nerosis ftor reeptor superfmily memer 9 (TNFRSF9; Hs15551 m1), one morphogeneti protein 6 (BMP6; Hs m1), thyroid hormone reeptors α/β (THRA; Hs6847 m1, nd THRB; Hs3861 m1), nd glyerldehyde-3-phosphte dehydrogense (GAPDH; Hs g1) mrna expression (Applied Biosystems). All ssys were performed in triplite. The mrna ontents were normlized to GAPDH mrna levels, nd differenes in expression were determined y the CT method desried in the ABI user s mnul (Life Tehnologies)..8. Sttistil Anlysis. Serum dosges were ompred y nonprmetri nlysis of vrine for the two-ftor model (P <.5, Mnn-Whitney test). Gene expression omprisons were performed y the nlysis of vrine tehnique for n experiment with ompletely rndomized loks, omplemented y the Tukey multiple omprison test for pirs of mesurements, or the equivlent nonprmetri proedure when dt were not normlly distriuted. The level of signifine ws set t 95% (P <.5)forllpresenteddt. 3. Results 3.1. Hormonl Sttus nd Immunohistohemistry. Fresh tumor smples were used for ER nd PR immunohistohemistry. Two smples were negtive for ER, nd six were negtive for PR. ER-negtive smples were exluded from gene expression nlysis. Serum determintions were performed prior to surgery. After surgery, new serum determintions were performed to onfirm the previously otined dt. Three ptients (numers 1, 5, nd 14) presented with linil hyperthyroidism (low TSH with high FT4), while one ptient (numer 6) wspositivefortpo,ndoneptient(numer11)showed sulinil hypothyroidism (high TSH with norml FT4) (Tle 1). There were no differenes in E levels etween ontrols nd BrC ptients. BrC ptients presented with no signifint differenes in E nd TSH levels ompred with ontrol ptients. Men serum vlues for thyroid hormone were sttistilly higher in BrC thn ontrol ptients. TPO ws not oserved in ontrols. FT4 levels were signifintly higher in BrC ptients thn in ontrols (Tle ). 3.. Primry Culture. All the tissues inluded in this study were positive for estrogen reeptor nd thyroid hormone reeptor expression Figure 1: Totl RNA from primry ulture of rest tumor for different postsurgery tretment periods on 1% grose gel (1 : 8 h; : 16 h; 3 : 4 h; 4 : 3 h; 5 : 4 h; 6 : 48 h). To sertin whether extrted mrna qulity ws ffeted y tretment time, 1% grose gel test ws used to evlute intervls of 8, 16, 4, 3, 4, nd 48 h (Figure 1). Of note, norml rest tissue from ptients undergoing mmmoplsty ws negtive for mrna expression of the studied genes Gene Expression nd Evidene of Thyroid Hormone Influene. Figure shows ox plots tht represent the expression of eh gene following stimultion y E (TGFA, TGFB1, nd PGR) fter dministrtion of the previously desried tretments; Figure 3 detils the respetive dt for T3 (TNFRSF9, BMP-6, nd THRA). Ptients 1, 5, nd 14 (with mended thyroid hormone sttus) nd ptients 3 nd1 (negtive ER) were exluded from these dt. 4. Disussion 4.1. Hormonl Sttus nd Immunohistohemistry. There is muh evidene suggesting reltionship etween thyroid disese nd BrC risk ut it is ontroversil. Numerous studies hveeenonduted,ndsomehvefoundnosignifint reltionship etween thyroid disese [3] or tretment of thyroid disese [33] nd BrC risk, while others orrelted BrCwithhyperthyroidism[1, 1, 34, 35], hypothyroidism [4, 36, 37], nontoxi goiter [8, 38, 39], or thyroid utoimmune diseses [8, 37, 38, 4 4]. With respet to thyroid diseses, previous studies of postmenopusl women hve reported higher hypothyroidism inidene onurrent with BrC ompred with women with other tumor types or norml ontrols [4]. These dt were onfirmed y studies on women with BrC independent of E sttus [35, 36, 4]. However, severl studies were unle to onfirm reltionship etween hypothyroidism nd BrC [39, 43]. Thyroid utoimmune disese is identified y TPO positivity; severl works identified higher TPO in BrC ptients thn ontrols [8, 38, 41, 4]. Autoimmune ses s well s nontoxi goiters re ommon in BrC ses. Some works hve shown signifint inresed thyroid volume in these ses [8, 4].

4 4 ISRN Endorinology Tle 1: Tumor stging, immunohistohemistry, nd serum dosge of rest ner ptients. Cse numer Stging Immunohistohemistry Serum dosge TPOA (1) (UI/mL) TSH () (μi/ml) FT4 (3) (ng/dl) E (4) (pg/ml) 1 T() N(1) M() ER(+); PR(+) <. T() N() M() ER(+); PR( ) < T() N() M() ER( ); PR( ) <. 4 T(1) N() M() ER(+); PR(+) T() N() M() ER(+); PR(+) < <. 6, T(1) N() M() ER(+); PR(+) <. 7 T() N(1) M() ER(+); PR( ) < T() N() M() ER(+); PR( ) T(1) N() M() ER(+); PR(+) T() N(1) M() ER( ); PR(+) T() N() M() ER(+); PR(+) < <. 1 T() N() M() ER(+); PR( ) < <. 13 T() N(1) M() ER(+); PR(+) <. 14 T(1) N() M() ER(+); PR( ) < T() N() M() ER(+); PR(+) (1) Thyroid peroxidse ntiody (TPO): <35. UI/mL = negtive. () Thyroid-stimulting hormone (TSH): normlity etween.4 nd 4. mui/ml. (3) Free thyroxine (FT4): normlity etween.8 nd 1.9 ng/dl. (4) Estrdiol (E): normlity on postmenopuse etween nd 3 pg/ml. Hyperthyroidism. Sulinil hypothyroidism. TPO positive. Tle : Comprison of hormonl dosges etween rest ner nd norml ontrol ptients. Brest ner (N =15) Control(N=18) TSH (μi/ml) 1.34 ±.6.41 ± 1.1 FT4 (ng/dl) 1.78 ±..95 ±.16 E (pg/ml) 3.8 ± ± 3.9 Dt re reported s medin ± totl semirnge. TSH: thyroid-stimulting hormone; FT4: free thyroxine; E: estrdiol. P <.5 ompred with ontrol group (Mnn-Whitney test). In our study, we identified higher prevlene of thyroid disese (33.3%), with hyperthyroidism eing the most frequent ondition (.%), onfirming previous reports [1, 7]. 4.. Primry Culture. One of the mjor onerns for reserhers working with rest ner models is tht these models my exhiit ehvior tht is dissimilr to tht of rest ner tissue in vivo. Burdll nd ollegues [44] evluted the use of rest ner ell ultures nd highlighted the dvntges of these models in tht they exhiit limitless selfreplition, re esily repled with frozen stok, nd demonstrte reltively high degree of homogeneity. However, ell ulture models re prone to genotypi nd phenotypi drift, whih exludes this model when onsidering nd ompring individul hrteristis of ptients. A reent lterntive pproh hs een imed t minimizing the differenes etween in vitro ulture models nd living rest ner tissue. In ontrst, ulture of rest tumor slies does not present the effets of geneti drift, whih my our in ell lines during the ourse of pssging, nd this method mintins some in vivo hrteristis. Furthermore, rest tumor slies hve een primrily useful for evluting responses to hormonl nd phrmologil tretments, showing gene expression results tht re lose to in vivo responses. These dt omplement our understnding of how study models for rest ner my present vritions in results. It ws previously oserved y our group tht TGFA expression in primry orgn ulture ws not reproduile to the results seen in ell lines (5). Cell lines hve dvntges suh s high genomi homogeneity tht led to little vrition in the results when ompred with primry orgn ultures. However, it is now knowledged tht informtion regrding genomi vrition is insignifint in omprison to the vriility introdued during tehnil steps suh s ulture preprtion nd gene expression. In primry orgn ulture, vrition in the dt produed is higher thn the nturl vrition introdued y the tehniques used. These vritions my reflet heterogeneity tht develops in different tumor smples euse of the wide rnge of ftors tht led to genomi instility Gene Expression nd Evidene of Thyroid Hormone Influene. Some gene expression vrition showing mrked tion of E on rest ner hs lredy een estlished in previous works. We previously demonstrte upregulted TGFA nd downregulted TGFB1 fter E nd T3 tretments. We reported this vrition in rest ner ells lines, with

5 ISRN Endorinology 5 Fold hnge in reltive gene expression Fold hnge in reltive gene expression Fold hnge in reltive gene expression TGFA/GAPDH ( ΔΔCt ) TGFB1/GAPDH ( ΔΔCt ) PGR/GAPDH ( ΔΔCt ) d Figure : Gene expression of known estrogen-stimulted genes in primry ultures of rest tumors. Smples were treted s follows: ethnol vehile, triiodothyronine (T3), T3 plus tmoxifen (TAM), TAM, estrdiol (E), nd E plus TAM. After 48 h of tretment, TGFA, TGFB1, nd PGR were quntified y rel-time RT- PCR. Glyerldehyde-3-phosphte dehydrogense (GAPDH) ws used to normlize gene expression. Reltive mrna expression ws lulted using the expression level of the treted ethnol smple s the stndrd set to the dotted line nd represented y letter. Different letters indite P <.5. Fold hnge in reltive gene expression Fold hnge in reltive gene expression Fold hnge in reltive gene expression THRA/GAPDH ( ΔΔCt ) TNFRSF9/GAPDH ( ΔΔCt ) BMP6/GAPDH ( ΔΔCt ) d d Figure 3: Gene expression of known thyroid hormone-stimulted genes in primry ultures of rest tumors. Smples were treted s follows: ethnol vehile, triiodothyronine (T3), T3 plus tmoxifen (TAM), TAM, estrdiol (E), nd E plus TAM. After 48 h of tretment, THRA, TNFRSF9, nd BMP6 were quntified y rel-time RT- PCR. Glyerldehyde-3-phosphte dehydrogense (GAPDH) ws used to normlize gene expression. Reltive mrna expression ws lulted using the expression level in the treted ethnol smple s the stndrd set to the dotted line nd represented y letter. Different letters indite P <.5. estrogen reeptor dependene [7]. When hormonl tretment ws ssoited with TAM, vritions in gene expression did not show sttistil differenes ompred with ontrol ells. However, lthough E nd T3 tretment of primry ultures showed the sme gene expression profile, TAM ssoition did not lok T3 tretment ut rther inresed TGFA nd deresed TGFB1 gene expression during T3 nd TAM ssoition. Here, we hoose PGR s mrker of estrogen tion, s reported elsewhere [45]. As expeted, E tretment inresed PGR expression nd TAM ssoition loked this tion. Although T3 tretment inresed PGR expression, this tion ws less pronouned thn E; however, TAM did not lok T3 tion on PGR expression, diverging from dt previously otined from ell lines [7]. Tretment with TAM n inhiit E tion, funtioning like prtil ntgonist of nulerer,utthereisevideneforthegonistitionof TAM on plsm memrne reeptors for E [46]. Our group hs previously orrelted PGR expression with TH [7], nd

6 6 ISRN Endorinology here we provided evidene for the upregultion of PGR in primry ulture treted with T3 plus TAM. To onfirm the tion of T3 tretment, we verified the expression of previously desried genes exhiiting upregultion (TNFRSF9 nd THRA) or downregultion (4-1BB) under TH tretment [9]. Tretment of primry ultures with T3 nd E reprodued equivlent results otined in ell lines [7]. However, ssoitions of these hormones with TAM did not reprodue these results. Ating s ntgonist on ER, TAM ssoited with E mintined expression of TGFA nd PGR to the ontrol levels, ut when T3 plus TAM ssoition ws pplied, TGFA nd TGFB1 expression ws t the sme levels s those following E tretment. This ssoition my oinide with estrogeni response on geneexpressionndhighlighttheeffetoftamusgein hyperthyroid ptients, espeilly onsidering the prolifertive nd poptoti effets of TGFA nd TGFB1, respetively, on the initil tumor progression stges. This finding ws reinfored y oservtions of upregulted PGR expression under T3 tretment (lthough levels were less thn those produed following E stimultion), whih did not show hnges with TAM ssoition. To ssess whether E ould influene the expression of T3 trget genes, we exmined the effet on TNFRSF9, BMP-6, nd THRA expression nd oserved no differenes in omprison to the ontrol. E nd TAM tretment upregulted THRA expression; however, this effet my e due only to TAM euse it exerted the sme upregultion effets s E plus TAM. Note tht TAM lone or ssoited with T3 modulted gene expression of TNFRSF9, BMP-6, nd THRA relted to the ontrol, similr to T3 tretment, showing tht the TAM n interfere with gene expression modulted y T3. Our results showed tht thyroid dysfuntions orrelte with ER positivity (Tle 1). There re mny possiilities of rosstlk etween T3, E, nd TAM. Some previous reserh onsidered TH nd ER inding, s demonstrted in BrC ell lines [7]. On the other hnd, TH n lter ER-dependent gene trnsription, with ER-thyroid hormone reeptor dimer formtion tht results in flexile regultion of the onsensus ERE [8], or intertion etween thyroid hormone reeptors nd ERE [3]. Most reently, studies hve emphsized the nongenomi tions of T3 nd E, ommening t the reeptors t the plsm memrne or in the ytoplsm nd tivting intrellulr signling pthwys suh s PI3 K or MAPK. The E ntgonist fulvestrnt does not tivte these signlingpthwysythismehnism,utotherseletive estrogen reeptor modultors (SERM), suh s TAM, do it in similr mnner to E, showing tht TAM onfers n ntgonisti tion on nuler ER ut n gonisti tion on memrne ER [47, 48]. AKT nd MAPK phosphoryltion n elevte gonisti TAM nd other SERM tivity [48]. 5. Conlusions Our work identified high inidene of hyperthyroidism in menopusl women with BrC, who exhiited higher degree of TH thn the ontrol group. Primry ulture of tumor smples ws utilized to evlute gene expression modified y T3 or E tretment nd produed similr ut not identil results to those oserved in rest ner ell lines. T3 hd signifint effet on genes lssilly regulted y E, ut the omintion of T3 with TAM did not reverse geneexpressionlevelstothoseoservedintheuntreted ontrol group, in ontrst to E plus TAM, whih resulted in mintined gene expression when E tretment ws pplied. Tking into ount the known ility of TH to mimi the effets of E, prtiulrly in the presene of TAM, our results reinfore previous reports tht the thyroid hormone sttus of BrC ptients n influene E-ontrolled mehnisms, even under TAM intervention nd/or the sene of irulting E in postmenopusl women. Thus, our study highlights the importne of evlution of thyroid hormone sttus when onsidering the prognosis nd tretment options for individul ptients. Arevitions BrC: Brest ner TH: Thyroid hormone ER: Estrogen reeptor T3: Triiodothyronine E: Estrdiol TAM: 4-Hydroxytmoxifen PR: Progesterone reeptor TPO: Thyroid peroxidse ntiody TSH: Thyroid-stimulting hormone FT4: Free thyroxine TGFA: Trnsforming growth ftor lph TGFB1: Trnsforming growth ftor et 1 PGR: Progesterone reeptor TNFRSF9: Tumor nerosis ftor reeptor superfmily 9 BMP-6: Bone morphogeneti protein 6 THRA: Thyroid hormone reeptor lph. Conflit of Interests The uthors delre tht there is no onflit of interests regrding the pulition of this pper. Aknowledgments This study ws supported y FAPESP Grnt 5/ nd 13/ nd supported y CAPES/PNPD Grnt.59158/1. Referenes [1] P. P. Sriv, N. B. Figueiredo, C. R. Pdovni, M. M. Brentni, nd C. R. Nogueir, Profile of thyroid hormones in rest ner ptients, Brzilin Journl of Medil nd Biologil Reserh,vol.38,no.5,pp ,5. [] I. Conde, R. Pnigu, J. Zmor et l., Influene of thyroid hormone reeptors on rest ner ell prolifertion, Annls of Onology,vol.17,no.1,pp.6 64,6.

7 ISRN Endorinology 7 [3] S.Dind,A.Snhez,ndV.Moudgil, Estrogen-likeeffetsof thyroid hormone on the regultion of tumor suppressor proteins, p53 nd retinolstom, in rest ner ells, Onogene, vol.1,no.5,pp ,. [4]S.J.Conde,R.D.A.M.Luvizotto,M.T.deSíio, nd C. R. Nogueir, Humn rest tumor slies s n lterntive pproh to ell lines to individulize reserh for eh ptient, Europen Journl of Cner Prevention, vol.1,no.4,pp , 1. [5] S.J.Conde,R.A.M.Luvizotto,M.T.Siio,M.L.H.Ktym, M. M. Brentni, nd C. R. Nogueir, Tmoxifen inhiits trnsforming growth ftor-α gene expression in humn rest rinom smples treted with triiodothyronine, Journl of Endorinologil Investigtion, vol.31,no.1,pp , 8. [6] S. H. Cestri, N. B. Figueiredo, S. J. Conde et l., Influene of estrdiol nd triiodothyronine on rest ner ell lines prolifertion nd expression of estrogen nd thyroid hormone reeptors, Arquivos Brsileiros de Endorinologi e Metologi, vol.53,no.7,pp ,9. [7] C. R. Nogueir nd M. M. Brentni, Triiodothyronine mimis the effets of estrogen in rest ner ell lines, Journl of Steroid Biohemistry nd Moleulr Biology,vol.59,no.3-4,pp , [8] O. Turken, Y. NrIn, S. DemIrs et l., Brest ner in ssoition with thyroid disorders, Brest Cner Reserh, vol. 5, no. 5, pp. R11 R113, 3. [9] A. Tosovi, A.-G. Bondeson, L. Bondeson, U.-B. Erisson, J. Mlm, nd J. Mnjer, Prospetively mesured triiodothyronine levels re positively ssoited with rest ner risk in postmenopusl women, Brest Cner Reserh,vol.1,no.3, rtile R33, 1. [1] M. Lemire nd L. Bugnet-Mhieu, Thyroid funtion in women with rest ner, Europen Journl of Cner nd Clinil Onology,vol.,no.3,pp.31 37,1986. [11] M. West, C. Blnhette, H. Dressmn et l., Prediting the linil sttus of humn rest ner y using gene expression profiles, Proeedings of the Ntionl Ademy of Sienes of the United Sttes of Ameri, vol. 98, no., pp , 1. [1] M. Lroix, G. Querton, P. Henneert, D. Lrsimont, nd G. Lelerq, Estrogen reeptor nlysis in primry rest tumors y lignd-inding ssy, immunoytohemil ssy, nd northern lot: omprison, Brest Cner Reserh nd Tretment,vol.67,no.3,pp.63 71,1. [13] S. Sommer nd S. A. W. Fuqu, Estrogen reeptor nd rest ner, Seminrs in Cner Biology, vol. 11, no. 5, pp , 1. [14] S. A. W. Fuqu, G. C. Chmness, nd W. L. MGuire, Estrogen reeptor muttions in rest ner, Journl of Cellulr Biohemistry, vol. 51, no., pp , [15] E. V. Jensen, G. Cheng, C. Plmieri et l., Estrogen reeptors nd prolifertion mrkers in primry nd reurrent rest ner, Proeedings of the Ntionl Ademy of Sienes of the United Sttes of Ameri, vol. 98, no. 6, pp , 1. [16] D. P. Edwrds, G. C. Chmness, nd W. L. MGuire, Estrogen nd progesterone reeptor in rest ner, Biohimi et Biophysi At,vol.56,no.4,pp ,1979. [17] L. Bernstein nd R. K. Ross, Endogenous hormones nd rest ner risk, Epidemiologi Reviews, vol. 15, no. 1, pp , [18] L. A. Hel nd J. L. Stnford, Hormone reeptors nd rest ner, Epidemiologi Reviews, vol. 15, no. 1, pp. 9 19, [19] R. E. Burke nd W. L. MGuire, Nuler thyroid hormone reeptors in humn rest ner ell line, Cner Reserh, vol. 38, no. 11 I, pp , [] M.-A. Ceron, M.-F. Pihon, nd E. Milgrom, Thyroid hormone reeptors in humn rest ner, Cner Reserh, vol. 41, no. 1, pp , [1] Z.-M. Sho, M. S. Sheikh, A. K. Rishi et l., Thyroid hormone enhnement of estrdiol stimultion of rest rinom prolifertion, Experimentl Cell Reserh,vol.18,no.1,pp.1 8, [] J. G. Spener, The influene of the thyroid in mlignnt disese, British Journl of Cner,vol.8,no.3,pp ,1954. [3] O. Muhlok nd L. M. Boot, Indution of mmmry ner in miewithoutthemmmrytumorgenty, Cner Reserh, vol.19,no.4,pp.4 41,1959. [4] D. P. Rose nd T. E. Dvis, Plsm thyroid-stimulting hormone nd thyroxine onentrtions in rest ner, Cner, vol. 41, no., pp , [5] B. S. Thoms, R. D. Bulrook, nd M. J. Russell, Thyroid funtion in erly rest ner, Europen Journl of Cner nd Clinil Onology,vol.19,no.9,pp ,1983. [6] H. Vorherr, Thyroid funtion in enign nd mlignnt rest disese, Europen Journl of Cner nd Clinil Onology,vol. 3,no.3,pp.55 57,1987. [7] O. Tktni, T. Okumoto, H. Kosno, M. Nishid, H. Hiride, nd S. Tmkum, Reltionship etween the levels of serum thyroid hormones or estrogen sttus nd the risk of rest ner genesis in Jpnese women, Cner Reserh, vol. 49, no. 11,pp ,1989. [8] N. Vsudevn, N. Koiuhi, W. W. Chin, nd D. W. Pfff, Differentil rosstlk etween estrogen reeptor (ER)α nd ERβ nd the thyroid hormone reeptor isoforms results in flexile regultion of the onsensus ERE, Moleulr Brin Reserh,vol.95,no.1-,pp.9 17,1. [9] T. Ymd-Oke, Y. Stoh, nd H. Ymd-Oke, Thyroid hormone indues the expression of 4-1BB nd tivtion of spses in thyroid hormone reeptor-dependent mnner, Europen Journl of Biohemistry, vol.7,no.14,pp , 3. [3] S. R. Lkhni, M. J. vn de Vijver, J. Jquemier et l., Thepthologyoffmililrestner:preditivevlueof immunohistohemil mrkers estrogen reeptor, progesterone reeptor, HER-, nd p53 in ptients with muttions in BRCA1 nd BRCA, Journl of Clinil Onology, vol., no. 9, pp ,. [31] R. Mir-y-Lopez nd L. Ossowski, Preservtion of steroid hormone reeptors in orgn ultures of humn rest rinoms, Cner Reserh,vol.5,no.1,pp.78 83,199. [3] A. Klhe, M. P. Vessey, nd K. MPherson, Thyroid disese nd rest ner: findings in lrge se-ontrol study, British Journl of Surgery,vol.69,no.7,pp ,198. [33] H. A. Weiss, L. A. Brinton, N. A. Potishmn et l., Brest ner risk in young women nd history of seleted medil onditions, Interntionl Journl of Epidemiology, vol. 8, no. 5,pp ,1999. [34] A. R. Mooss, D. A. Prie Evns, nd A. C. Brewer, Thyroid sttus nd rest ner: repprisl of n old reltionship, AnnlsoftheRoylCollegeofSurgeonsofEnglnd,vol.53,no.3, pp , [35] M. B. Goldmn, Thyroid diseses nd rest ner, Epidemiologi Reviews,vol.1,pp.16 8,199.

8 8 ISRN Endorinology [36] I. Mittr nd J. L. Hywrd, Hypothlmi-pituitry-thyroid xis in rest ner, The Lnet, vol. 1, no. 7863, pp , [37] D. A. Admopoulos, S. Vssilros, nd N. Kpoll, Thyroid disese in ptients with enign nd mlignnt mstopthy, Cner,vol.57,no.1,pp.15 18,1986. [38] C. Gini, P. Fierri, R. Boni et l., Reltionship etween rest ner nd thyroid disese: relevne of utoimmune thyroid disorders in rest mlignny, Journl of Clinil Endorinology nd Metolism,vol.81,no.3,pp ,1996. [39] P. P. A. Smyth, D. F. Smith, E. W. M. MDermott, M. J. Murry, J. G. Gerghty, nd N. J. O Higgins, A diret reltionship etween thyroid enlrgement nd rest ner, Journl of Clinil Endorinology nd Metolism,vol.81,no.3,pp ,1996. [4] K. Itoh nd N. Mruhi, Brest ner in ptients with Hshimoto s thyroiditis, The Lnet, vol., no. 7945, pp , [41] B. Rsmusson, U. Feldt-Rsmussen, nd L. Hegedus, Thyroid funtion in ptients with rest ner, Europen Journl of Cner nd Clinil Onology,vol.3,no.5,pp ,1987. [4] P. P. A. Smyth, S. G. Shering, M. T. Kilne et l., Serum thyroid peroxidse utontiodies, thyroid volume, nd outome in rest rinom, Journl of Clinil Endorinology nd Metolism, vol. 83, no. 8, pp , [43] A. J. Hedley, S. J. Jones, nd D. J. Spiegelhlter, Brest ner in thyroid disese: ft or flly? The Lnet,vol.1,no.81,pp , [44] S. E. Burdll, A. M. Hny, M. R. J. Lnsdown, nd V. Speirs, Brest ner ell lines: friend or foe? Brest Cner Reserh, vol. 5, no., pp , 3. [45] J. Rokiki, P. M. Ds, J. M. Giltnne et l., The ERα otivtor, HER4/4ICD, regultes progesterone reeptor expression in norml nd mlignnt rest epithelium, Moleulr Cner, vol. 9, rtile 15, 1. [46] C. K. Osorne nd R. Shiff, Estrogen-reeptor iology: ontinuing progress nd therpeuti implitions, Journl of Clinil Onology,vol.3,no.8,pp ,5. [47] E. R. Levin, Cellulr funtions of plsm memrne estrogen reeptors, Steroids,vol.67, no.6,pp ,. [48]J.Shou,S.Mssrweh,C.K.Osorneetl., Mehnismsof tmoxifen resistne: inresed estrogen reeptor-her/neu ross-tlk in ER/HER-positive rest ner, Journl of the Ntionl Cner Institute,vol.96,no.1,pp ,4.

9 MEDIATORS of INFLAMMATION The Sientifi World Journl Gstroenterology Reserh nd Prtie Journl of Dietes Reserh Interntionl Journl of Journl of Endorinology Immunology Reserh Disese Mrkers Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Journl of Oesity Journl of Ophthlmology Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Journl of Onology Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine AIDS Behviourl Neurology Reserh nd Tretment Oxidtive Mediine nd Cellulr Longevity