Targeting BIG3 PHB2 interaction to overcome tamoxifen resistance in breast cancer cells

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1 Reeive Fe 13 Aepte 15 Aug 13 Pulishe Sep 13 DOI: 1.13/nomms33 OPEN Trgeting intertion to overome tmoxifen resistne in rest ner ells Tetsuro Yoshimru 1, Msto Komtsu 1, Tisuke Mtsuo 1, Yi-An Chen, Yoihi Murkmi,w, Kenji Mizuguhi, Eiihi Mizoht 3, Tsuyoshi Inoue 3, Miki Akiym, Rui Ymguhi 5, Seiy Imoto, Storu Miyno, Ysuo Miyoshi 7, Mitsunori Ss, Yusuke Nkmur,9 & Toyoms Ktgiri 1 The quisition of enorine resistne is ommon ostle in enorine therpy of ptients with oestrogen reeptor- (ER)-positive rest tumours. We previously emonstrte tht the omplex hs ruil role in the moultion of oestrogen/er signlling in rest ner ells. Here we report ell-permele peptie inhiitor, lle, tht regultes multiple ER-signlling pthwys ssoite with tmoxifen resistne in rest ner ells y inhiiting the intertion etween n. Intrinsi relese from y iretly ins to oth nuler- n memrne-ssoite ER, whih les to the inhiition of multiple ER-signlling pthwys, inluing genomi n nongenomi ER tivtion n ER phosphoryltion, n the growth of ER-positive rest ner ells oth in vitro n in vivo. More importntly, tretment suppresses tmoxifen resistne n enhnes tmoxifen responsiveness in ER-positive rest ner ells. These finings suggest inhiiting the intertion etween n my e new therpeuti strtegy for the tretment of luminl-type rest ner. 1 Division of Genome Meiine, Institute for Genome Reserh, The University of Tokushim, , Kurmoto-ho, Tokushim 77-53, Jpn. Ntionl Institute of Biomeil Innovtion, 7-- Asgi-Sito, Irki-City, Osk 57-5, Jpn. 3 Deprtment of Applie Chemistry, Grute Shool of Engineering, Osk University, -1, Ym-ok, Suit, Osk 55-71, Jpn. Lortory of Moleulr Meiine, University of Tokyo, --1, Shiroknei, Minto-ku, Tokyo 1-39, Jpn. 5 Lortory of Sequene Anlysis, The University of Tokyo, --1, Shiroknei, Minto-ku, Tokyo 1-39, Jpn. Lortory of DNA Informtion Anlysis, Humn Genome Centre, Institute of Meil Siene, The University of Tokyo, --1, Shiroknei, Minto-ku, Tokyo 1-39, Jpn. 7 Division of Brest n Enorine Surgery, Deprtment of Surgery, Hyogo College of Meiine, 1-1, Mukogw-ho, Nishinomiy, Hyogo 3-51, Jpn. Deprtment of Surgery, Tokushim Brest Cre Clini, -7-7, Nkshim, Tokushim 77-5, Jpn. 9 Deprtment of Meiine n Surgery, The University of Chigo, 9 E 57th Street, KCBD1, Chigo, Illinois, USA. w Present ress: Grute Shool of Informtion Sienes, Tohoku University, -3-9, Armki-z-o, Ao-ku, Seni-ity, Miygi 9-579, Jpn. Corresponene n requests for mterils shoul e resse to T.K. (emil: tktgi@genome.tokushim-u..jp). NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

2 NATURE COMMUNICATIONS DOI: 1.13/nomms33 Brest ner is the most ommon ner mong women worlwie 1,. More thn 7% of primry rest tumours re oestrogen reeptor- (ER)-positive, n the intertions etween oestrogen (E) n ER rmtilly enhne the prolifertive n metstti tivity of rest tumour ells 3,. E iologil tions re meite y oth genomi n nongenomi mehnisms; in the former type nuler ER funtions s lign-epenent trnsription ftor tht regultes trget gene expression levels 3,5, wheres in the ltter type E-oun ER in the plsm memrne ssoites with vriety of signlling moleules, inluing IGF reeptor (IGF-1R), phosphoinositie 3-kinse (PI3K) n SH omin ontining (Sh), whih results in Akt n mitogen-tivte protein kinse (MAPK) tivtion or inrese nuler ER phosphoryltion 1. Thus, ER hs pivotl role in the E signlling network n therefore represents n importnt therpeuti trget for rest ner. The seletive ER moultor tmoxifen iretly inhiits E n ER intertions, n is stnr tretment offere to ptients with ER-positive rest ner Nonetheless, tumours often evelop resistne, leving ptients with reurrent tumours tht lk trgete therpeuti options 1,15. The potentil mehnisms for either intrinsi or quire enorine resistne remin poorly unerstoo, ut they lerly inlue ER-oregultory proteins n ross-tlk etween the ER pthwy n other growth ftors n kinse networks 1,11,1. This knowlege hs le to numerous tretment strtegies omining enorine n trgete inhiitor therpies 1719 ; however, omprehensive mesures for this prolem remin unresolve. Therefore, ientifying the ftors n pthwys responsile for resistne n efining wys to overome it represent importnt therpeuti hllenges in rest ner reserh. The novel E/ER signlling regultor refelin A-inhiite gunine nuleotie-exhnge protein 3 (), whih is exlusively overexpresse in mjority of rest ners, ws reently ientifie from genome-wie expression profiles,1. interts n ololizes with prohiitin () in the ytoplsm of rest ner ells,1. is known to funtion s orepressor of ER,3. Our previous stuy emonstrte tht when ws knoke own y smll interfering RNA, E stimultion le to the nuler trnslotion of mjority of the ytoplsmi, enhne the intertion etween n ER, n suppresse ER trnsriptionl tivity 1. Aoringly, we hypothesize tht ptures in the ytoplsm of ner ells n therey inhiits the suppressive ility of in the presene of E, resulting in the onstitutive tivtion of ER signlling pthwys. Here we esrie syntheti, ell-penetrting, ominntnegtive peptie tht inhiits the E/ER signlling network y tivting the tumour suppressive ility of. This peptie lso enhne tmoxifen responsiveness n nti-tumour effets in tmoxifen-resistnt (TAM-R) rest ners. Thus, the regultion of E signlling y trgeting the intertion introues new potentil therpeuti pproh for enorine-resistnt tumours, s well s ER-positive rest ners. Results Ientifition of the interting region. Previous stuies hve shown tht the omplex hs ritil role in rest ner ell growth 1, n strtegies ple of inhiiting this intertion my represent novel therpies for rest ner. Therefore, we first ttempte to etermine the region(s) require for the intertion with through in silio n iohemil nlyses. First, we inepenently otrnsfete five prtil onstruts of FLAG-tgge (Fig. 1) with HA-tgge (HA-) into COS-7 ells. Immunopreipittion with n nti-flag ntioy inite tht HA- o-immunopreipitte with 13, 15 n full-length (Fig. 1), suggesting tht the 115th mino i region of is minimlly require for its intertion with. In prllel with this pproh, we ttempte to preit the protein ining sites on using the PSIVER (Proteinprotein intertion SItes preition server) softwre, n we ientifie luster of nite ining resiues within the 115th mino i region. This luster region ontine three of the highest soring (Z.) resiues (Q15, D19 n Q173; Fig. 1), whih were oriente in the sme iretion (Fig. 1). Inee, the muttions in whih ll of these trget resiues were sustitute with lnine lmost ompletely olishe the intertion with HA- (Fig. 1e), initing the importne of Q15, D19 n Q173 for heteroimeriztion with. Moreover, D19 ws the most ritil site mong these resiues for ining, lthough n lnine muttion on eh resiue resulte in reue ining (Supplementry Fig. S1). Aoringly, we fouse on these resiues s nite -ining resiues. A peptie with ominnt-negtive influene on ER tivity. We next investigte the possiility of ell-penetrting peptie s ominnt-negtive inhiitor trgeting the intertion, n esigne speifi peptie tht inlue these -ining resiues to trget the intertion. This peptie, referre to s ER tivity-regultor syntheti peptie (), ontine the potentil ining resiues (15QMLSDLTLQLRQR177) n memrne-permele polyrginine resiues (11R) t its NH terminus (Fig. ). As negtive ontrols, pepties ontining srmle mino i sequene (sr) n either lnine muttions t key resiues (mt) were onstrute (Fig. ). Inee, o-immunopreipittion experiments revele tht, ut not mt or sr, ompletely inhiite the omplex formtion of enogenous n in the ER-positive rest ner ell lines n KPL-3C, whih strongly express n (Fig. n Supplementry Fig. S). We lso exmine the iret inhiition of the intertion using. As expete, HA- oun to His-tgge reominnt protein n inhiite the intertion in oseepenent mnner, wheres sr i not (Fig. ). In ition, mt exhiite moest ining to the protein t levels sustntilly lower thn (Fig. ). Surfe plsmon resonne (BIAore) intertion nlysis revele tht oun to the His-tgge reominnt with issoition onstnt (K) ¼ 1.9 mm (Fig. ). Thus, our t suggeste tht iretly oun to, resulting in the speifi inhiition of omplex formtion. trnslotes n ttenutes nuler ER tivtion. We investigte the suellulr istriution of enogenous in rest ner ells following tretment y immunoytohemil n iohemil pprohes. In the presene of E, tretment with, ut not with sr, le to signifint inrese in the mount of nuler in timeepenent fshion (Fig. 3). In ition, in the presene of E, tretment le to erese in ytoplsmi, therey sustntilly inresing the intertion etween n ER in the nuleus even fter 1 h (Fig. 3). Furthermore, oimmunopreipitte n ololize with enogenous in the nuleus n the ytoplsm (Supplementry Fig. S3,) ut i not iretly in to ER or. These finings suggeste tht use to e relese from n le to NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.

3 NATURE COMMUNICATIONS DOI: 1.13/nomms33 ARTICLE Se-7 omin DUF omin 1,177 -full , 1, ,177 1,, PSIPRED PSIVER HHHHHHHHHHHHHHHHHHHH A VRA TLSQ MLS D LT LQ L R Q R Q15 D19 Q173 FLAG- IP : nti-flag HA- (kd) (kd) Full 13 35,177 1,,177 IB: FLAG IB: HA IB: FLAG (input) IB: HA (input) FLAG- 5 IP : nti-flag HA- (kd) 5 15 (kd) IB: FLAG IB: HA IB: FLAG (input) IB: HA (input) e HA- FLAG- 13 FLAG- mutnt nti-flag nti-ha IP : FLAG-A IP : HA-A WCL 5 Figure 1 Ientifition of the interting region. () The shemti representtion of humn n the five FLAG- prtil lones lking one of the terminl regions is shown. () Immunolot nlyses were performe to ientify the -ining region in. COS-7 ells were trnsfete with the inite onstruts (full-length, 13, 35,177, 1,177, 11 n 15 ) n HA-. After h, the ells were lyse n FLAG- ws immunopreipitte with n nti-flag ntioy. Immunopreipitte proteins n portion of the originl ell lystes (input) were immunolotte s inite. () The preite intertion sites, s etermine using PSIVER softwre, re shown. The unerline ol letters inite the resiues most likely to e involve in ining. () The puttive -ining sites (Q15, D19 n Q173) on preite three-imensionl struture of protein re shown. (e) Immunolots were performe to ssess the -ining region in protein. The lystes from COS-7 ells trnsfete with 13 or mutnt onstruts were immunopreipitte with nti-flag n nti-ha ntioies to etet n, respetively. Full-length imges of immunolots re shown in Supplementry Fig. S9. E-epenent nuler trnslotion, eventully resulting in the intertion of with nuler ER in ner ells. ER hs een shown to moulte trnsription in two wys: (i) through iret ining to oestrogen-responsive elements (EREs) lote in the promoter n/or enhner regions of trget genes 5 n (ii) y serving s o-tivtor of other trnsription ftors suh s AP-1 (ref. ). Therefore, we explore the impt of tretment on these two moes of ER trnsriptionl tivity. First, we performe hromtin immunopreipittion (ChIP) ssy with E-stimulte ells. The results showe tht tretment inue E- epenent reruitment of the enogenous ER omplex on the ER trget genes, TFF1 n CCND1, respetively, (Fig. 3), suggesting tht i not inhiit the ility of ER to in ERE or AP-1. In luiferse ssys with ERE or AP-1 reporters, signifintly inhiite oth forms of E-inue ER trnsriptionl tivity in ose-epenent mnner in n KPL-3C ells (Supplementry Fig. S3), ut no signifint inhiition ws oserve with sr or mt. These results inite tht suppresse ER trnsriptionl tivity levels through oth nonil ERE- n non-nonil AP-1- ining mehnisms. is known to t s n ER trnsriptionl orepressor y ompeting with the o-tivtor SRC-1 to in ER 3 n y reruiting histone eetylse 1 (HDAC1; ref. 7) n nother orepressor, NoR. Thus, we next explore the effet of on this reruitment in n KPL-3C ells using ChIP ssys. Stimultion with E lone reruite SRC-1 to ER, wheres tretment le to the iret ssoition of with ER in the presene of E, reue SRC-1 ining to ER, n enhne the reruitment of HDAC1 n NoR in (Fig. 3) n KPL-3C ells (Supplementry Fig. S3). Moreover, we performe ChIPquntittive PCR ssy, with E-stimulte ells. The results showe tht tretment signifintly reue the E-epenent reruitment of enogenous SRC-1 on the TFF1 gene ut inrese the E-epenent reruitment of enogenous NoR, HDAC1 n (Supplementry Fig. S3e). In ontrst, tretment h no effet on ChIP ssy using n nti- ntioy (Supplementry Fig. S3e) or on ER expression t the mrna or protein level (Supplementry Fig. S3f). Susequently, we investigte the HDAC tivity of immunopreipittes in ells n foun tht the hromtin-remoelling omplexes reruite y tretment le to signifint inrese in HDAC tivity (Fig. 3e). Moreover, signifintly suppresse E-inue expression of TFF1, CCND1, -My, EF1 n PgR 933 (Fig. 3f). In ition, we vlite the suppressive effet of on ER trnsriptionl tivity. NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

4 NATURE COMMUNICATIONS DOI: 1.13/nomms33 11R-GGG-QMLSDLTLQLRQR sr 11R-GGG-DRQLQLSTLQRML mt 11R-GGG-AMLSALTLALRQR () 1 nm E (kd) () () 5 IP : 5 IP : 5 IP : IgG 5 sr mt (kd) 5 IP : 5 IP : 5 IP : IgG 5 KPL-3C () 1 nm E () () sr mt HA- (μm) Lyste of FLAG- 1 μg His- Pull-own y Ni-resin sr mt Response μm KA(1/M) KD(M) 5.e 1.9e-5 onentrtion Figure inhiits the intertion of with. () The, sr n mt sequenes re shown. () The inhiitory effets of tretment on intertions were evlute in (left) n KPL-3C ells (right). () Diret inhiition of the intertion y ws evlute. The lystes of COS-7 ells, trnsiently trnsfete with FLAG-, were inute with His-tgge reominnt (His-) n HA-, HA-sr or HA-mt for 1 h. Then, His- ws pture with Ni-NTA grose, n the oun frtions were immunolotte s inite. () In vitro iret intertion of n ws evlute y BIAore. epletion use signifint reution in the nonil ERE n non-nonil AP-1 ER trnsriptionl tivities in ERpositive ells ut i not ffet ER-negtive MDA- MB-31 ells (Supplementry Fig. S3g). Tken together, these finings inite tht nuler-trnslote following tretment iretly oun to ER n te s orepressor y reruiting HDAC1 n NoR, therey leing to n lmost omplete suppression of the ER trget gene expression. suppresses E-epenent non-genomi ER signlling. In ition to ER ting s nuler trnsription ftor, E rpily inues IGF-1R tyrosine phosphoryltion followe y the formtion of ternry omplex of IGF-1R, ER n Sh in the ell memrne 9, even though the unne of memrneoun n ytoplsmi ER is low in primry rest ners 3. Inee, we oserve tht portion of relese from y interte with ER in the ytoplsmi/plsm memrne ell frtion, regrless of the presene of E (Fig. 3, n Supplementry Fig. S3,). Therefore, we hypothesize tht oul lso ffet these non-genomi tions of ER. First, we etete E-inue tyrosine phosphoryltion of IGF-1R n o-immunopreipitte IGF-1R, ER n Sh in oth n KLP-3C ells (Fig. ), whih highly expresse IGF- 1R n PI3K (Supplementry Fig. S), s esrie previously 9. In ontrst, tretment remove Sh from this omplex n forme new ternry omplex onsisting of IGF-1R, ER n, n therey suppresse E-inue tyrosine phosphoryltion of IGF-1R (Fig. ). We then exmine the effets of on the phosphoryltion of memrne-ssoite ER (S11), euse its phosphoryltion hs een ssoite with invsive rest ner in linil speimens 35. tretment lerly suppresse the E-inue phosphoryltion (S11) of memrne-ssoite ER in the IGF-1R-preipitte memrne frtion of ells (Supplementry Fig. S). Moreover, lso interfere with the E-inue intertions of ER n PI3K in oth n KLP-3C ells (Fig. ). Next, we exmine the effets of on the phosphoryltion sttus of Akt n p/ MAPK, whih re the ownstrem signlling moleules of IGF-1R n PI3K, respetively. As expete, we oserve tht Akt (S73) n p/ MAPK (T/Y) phosphoryltion levels were lerly inrese in time-epenent mnner fter E stimultion in oth ell lines, wheres tretment with, ut not sr, ompletely suppresse the E-inue phosphoryltion levels of oth proteins (Fig. n Supplementry Fig. S). However, the relese from following tretment i not iretly intert with Akt or p/ MAPK (Supplementry Fig. S). Tken together, these results strongly suggeste tht interfere with E-inue non-genomi ER tivtion pthwys, suh s those meite y IGF-1R. represses E-epenent ER phosphoryltion. Aumulting eviene suggests tht phosphoryltion of ER is n importnt regultor of E-inue ER trnsriptionl tivity, DNA-ining, o-tivtor ining, n protein stility n ell prolifertion in ER-positive rest ner ells 33. Thus, we exmine the effets of on ER phosphoryltion t sites, inluing S1/S1, S11, S17, S35 n Y5. NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.

5 NATURE COMMUNICATIONS DOI: 1.13/nomms33 ARTICLE Reltive signl intensity of nuler-trnslote Merge nm E 1 μm 1 μm sr 1 μm mt IP IgG 1 nm E 1 μm NoR SRC-1 HDAC1 1 () Merge 3 ERE in TFF1 gene 1 nm E 1 μm 1 μm sr 1 Merge (h) 1 nm E 1 μm 1 μm sr 1 μm mt IP IgG Nuler extrt of Merge 1 μm E lone E E sr IP : IP : 1 h h AP-1 in CCND1 gene E : h nm E 1 μm 1 μm sr IP : IP : IP : Tuulin Lmin B IP : IP : IP : Tuulin Lmin B f Reltive gene expression e E : h TFF1 CCND1 Cytoplsm Nuleus (μm) 5 1 P N () 1 nm E EF1 E lone E Figure 3 promotes nuler trnslotion n suppresses E-inue gene expression. () Upper, representtive immunofluoresene imges of the suellulr loliztion of re shown; (re),,-imiino--phenylinole (lue). The rrows inite nuler trnslotion. Lower, sttistil nlyses of the nuler intensity of trnslote. The t re expresse s the fol inrese over untrete ells set t 1. n represent the men±s.e.m. of four inepenent experiments (Po.5, Po.1, Po.1 in two-sie Stuent s t-test). () Immunolot nlysis ws performe to etet the suellulr loliztion of ER, n fter immunopreipittion with the ntioies ginst the inite proteins in the presene of E n or sr. /-Tuulin (tuulin) n lminin B were use s loing ontrols for the ytoplsmi n nuler frtions, respetively. () ChIP ssys were use to etermine the reruitment of ER n to the TFF1 ERE sequene (left) n the CCND1 AP-1 motif (right) fter h of tretment. () Exhnge of hromtin-remoelling omplexes y. After tretment of ells for h with E±, the nuler frtions were immunopreipitte with nti-er or - ntioies n were immunolotte with ntioies ginst the inite proteins. (e) Deetyltion of hromtin-remoelling omplexes ws evlute fter tretment. Nuler extrts from HeL ells n PBS were use s positive (P) n negtive (N) ontrols, respetively. The t represent the men±s.e.m. of three inepenent experiments (Po.1 in two-sie Stuent s t-test). (f) The effets of on ER-trget gene expression levels were evlute using rel-time PCR. The t re expresse the fol inrese over untrete ells t h (set t 1.) n represent the men±s.e.m. of three inepenent experiments (Po.1, Po.1, NS, no signifine in two-sie Stuent s t-test). Fluoresene 5,, 3,, 1, -My PgR NS Phosphoryltion t these five sites within ER ws lerly inrese in response to E stimultion n ontinue for t lest h in n KPL-3C ells. In ontrst, tretment with ompletely rogte these responses in oth ell lines (Fig. n Supplementry Fig. Se). Colletively, these results lerly showe tht relese from following tretment reue E-epenent ER phosphoryltion, leing to ER intivtion. suppresses E-epenent rest ner ell growth. We eluite the inhiitory effet of on the E-epenent growth of or KPL-3C ells. Tretment with, ut not sr or mt, signifintly reue E-stimulte ell growth in ose-epenent mnner (IC 5 ¼. mm n 1.9 mm in n KPL-3C ells, respetively; Fig. 5). Notly, oses greter thn 5 mm ompletely olishe the prolifertive response for up to 3 h fter E stimultion NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

6 NATURE COMMUNICATIONS DOI: 1.13/nomms IP : IGF-1Rβ () E () E P-IGF-1Rβ IGF-1Rβ P-Sh Sh 1 IP : () E () E P-PI3K PI3K 1 1 KPL-3C IP : () E () E P-PI3K PI3K IP : IGF-1Rβ KPL-3C () E () E P-IGF-1Rβ IGF-1Rβ P-Sh Sh 5 IP : PI3K () E () E P-PI3K PI3K 1 1 IP : PI3K () E () E P-PI3K PI3K 1 nm E 1 μm 1 μm sr 3 1 (h) 1 nme 1 μm KPL-3C 3 1 (h) P-Akt (S73) Akt P-MAPK (T/Y) MAPK P-Akt (S73) Akt P-MAPK (T/Y) MAPK Reltive phosphoryltion intensity (S1/1) (S11) P =. P=. 3 1 (h) 3 1 (h) (S17) P= (h) Reltive phosphoryltion intensity (S35) 3 1 (h) (Y5) 3 1 (h) E lone E Figure regultes the E-inue non-genomi pthwy vi IGF-1R. (,) The inhiitory effets of on the intertions of ER with IGF-1R n Sh (), n the intertion of ER n PI3K () in n KPL-3C ells. () Immunolot nlyses were performe to evlute the inhiitory effets of on E-inue Akt (S73) n p/ MAPK (T/Y) tivities in (left) n KPL-3C (right) ells. Representtive results re shown from one of four inepenent experiments. () The inhiitory effets of were evlute on E-inue phosphoryltion of ER (S1/S1, S11, S17, S35 n Y5) in ells. The t re expresse s the fol inrese over untrete ells t h n represent the men±s.e.m. of three inepenent experiments (Po.5, Po.1 in two-sie Stuent s t-test). (Supplementry Fig. S5). The inhiition of oth ell growth (Supplementry Fig. S5, left pnel) n ER trnsriptionl tivity (Supplementry Fig. S5, right pnel) ws mintine for h fter tretment. We onfirme similr growth inhiitory effets of in other rest ner ell lines expressing ER, n (tht is, ZR--1, HCC15, BT-7, YMB-1, T7D, KPL-1 n HBC; Supplementry Fig. S5). In ontrst, h no effet on the growth of norml mmmry epithelil MCF-1 A ells (Fig. 5) tht i not express ER or (Supplementry Fig. S). These finings suggeste tht speifilly inhiite the growth of rest ner ells without ffeting norml mmmry ells. NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.

7 NATURE COMMUNICATIONS DOI: 1.13/nomms33 ARTICLE Cell growth (A 5 ) NS h (μm) sr mt () 1 nm E Cell growth (A 5 )... KPL-3C NS h (μm) sr mt () 1 nm E Cell growth (A 5 ) MCF-1A (μm) h Cell growth (A 5 )... 1 μm 1 nm tmoxifen 1 nm E Reltive ERE tivity 1 μm 1 nm tmoxifen 1 nm E e Counts M1 M M M3 FL-A G1 : 3.3 S : 15.9 G/M :.7 Su-G1 :.5 1, Counts M M1 1 nm E M M3 FL-A G1 :. S : 15.9 G/M : Su-G1 :.17 1, Counts E 1 μm M M1 M M3 FL-A G1 : S : 15. G/M :. Su-G1 :.9 1, Counts E 1 nm tmoxifen 3 M1 G1 :.1 M M S : G/M :.51 M3 Su-G1 :.91 1 FL-A 1, Counts E 1 mm 1 nm tmoxifen M1 G1 : 5. S : 9.3 M M G/M :.7 M3 Su-G1 : 1.7 FL-A 1, 1 Tmoxifen E % of istriution Su-G1 G/M S G1 Figure 5 suppresses growth of ER-epenent rest ner ell lines in vitro. (,) An MTT (3-(,5-imethylthizol--yl)-,5- iphenyltetrzolium romie) ssy ws performe to evlute the inhiitory effet of on E-epenent growth of the -positive n KPL-3C (), n the -negtive mmmry epithelil ell line MCF-1A (). The t represent the men±s.e.m. of three inepenent experiments (Po.1 in two-sie Stuent s t-test). (,) The omine inhiitory tions of n tmoxifen were evlute using MTT ssys () n luiferse ssys (). ells were trete for h with E±, tmoxifen, or omintion of n tmoxifen. The t of ll pnels represent the men±s.e.m. of three inepenent experiments (Po.1, Po.1 in two-sie Stuent s t-test). (e) Flow ytometri nlyses were performe to evlute the effet of tretment on the ell yle. ells were trete for h with E±, tmoxifen, or omintion of n tmoxifen. Furthermore, tretment with omintion of n tmoxifen signifintly suppresse E-inue ell growth (Fig. 5) n ER trnsriptionl tivity (Fig. 5) in ells s ompre with or tmoxifen lone. Next, we exmine the effets of on the ell yle istriution of ells using flow ytometry. The popultion of ells in the G/M phse inrese fter h E stimultion, wheres the popultion in the G1 phse inrese fter or tmoxifen tretment, suggesting tht suppresse ell growth y inuing G1 rrest, similr to tmoxifen (Fig. 5e). Importntly, remrkle inrese in the poptoti (su-g1) ell popultion ws oserve fter tretment with omintion of n tmoxifen (1.7%), lthough no phenotypi ltertions or inreses in the su-g1 popultion were oserve fter tretment with, tmoxifen or E lone (.9%,.91% or.17%, respetively; Fig. 5e). Tken together, our t strongly suggest tht enhne the responsiveness of ER-positive rest ner ells to tmoxifen. Anti-tumour effiy of in xenogrft moels. To etermine whether oul ffet the growth of ER-positive rest ner tumours in vivo, we evelope KPL-3C (Fig. ) n (Supplementry Fig. S) orthotopi rest ner NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

8 NATURE COMMUNICATIONS DOI: 1.13/nomms33 Tumour volume (mm 3 ) Dys E lone E 3.5 mg kg 1 E 7 mg kg 1 E 1 mg kg 1 E mg kg 1 tmoxifen E 1 mg kg 1 mg kg 1 tmoxifen E 1 mg kg 1 sr Reltive gene expression 1 1 TFF1 CCND1 -My E lone E E tmoxifen E tmoxifen Tumour of KPL-3C xenogrft Dy 15 () μg per y E 1 mg kg 1 1 mg kg 1 () () sr P-Akt Akt P-MAPK MAPK P- (S1/1) P- (S11) P- (S17) P- (S35) P- (Y5) Figure inhiits tumour growth in xenogrft moels of humn ER-positive rest ner. () inhiits tumour growth in humn rest ner KPL-3C xenogrft mouse moel. The tumour volume represents the men±s.e.m. of eh group (n ¼ 5; Po.1 in two-sie Stuent s t-test). () Rel-time PCR ws performe to etermine the omine inhiitory effets of n tmoxifen on the expression of ER trget genes. The t re expresse the fol inrese over untrete ells t h (set t 1.) n represent the men±s.e.m. of eh group (n ¼ 5; Po.5, Po.1, Po.1 in two-sie Stuent s t-test). () Immunolot nlyses were performe to evlute the effets of on the phosphoryltion levels of Akt, p/ MAPK n ER proteins in tumours. xenogrfts in nue mie. Dily tretment with E lone inue time-epenent growth of KPL-3C n tumours, wheres tretment with 1 mg kg 1 use signifint inhiition of E-inue tumour growth ompre with mie trete with E lone or sr in oth tumour ell lines (1 mg kg 1 ; n ¼ 5; Po.1 in two-sie Stuent s t-test; Fig. n Supplementry Fig. S). No toxiity or signifint oy weight hnges were oserve in either xenogrft throughout these experiments (Supplementry Fig. S). As expete, signifint reution in mrna levels ws evient for TFF1, CCND1, n -My in tumours trete with ompre with those trete with sr or vehile only (Fig. n Supplementry Fig. S). Furthermore, onsierle suppression of Akt, p/ MAPK n ER phosphoryltion ws oserve in tumours trete with (Fig. n Supplementry Fig. S). More importntly, the omine tretment of n tmoxifen itively inhiite the expression of ER trget genes (Fig. ) n the evelopment of KPL-3C xenogrfts s ompre with tmoxifen or lone (Fig. ). These results emonstrte tht h in vivo nti-tumour tivity n oul enhne the ntitumour effets of tmoxifen. suppresses growth of TAM-R tumours. To onfirm tht h n nti-tumour effet ginst enorine-resistnt rest ner, we first exmine the effets of on the tivtion of the non-genomi signlling pthwy, on the phosphoryltion of ER n on the non-nonil ER trnsriptionl tivtion vi AP-1, whih is responsile for tmoxifen resistne 1,11,1 in TAM-R (ref. 5) n T7D (ref. ) ells. The phosphoryltion levels of Akt, p/ MAPK n ER were enhne in response to tmoxifen lone or omintion of tmoxifen n E, wheres tretment lerly suppresse these responses in oth ell types (Fig. 7, n Supplementry Fig. S7). tretment lso lerly suppresse the phosphoryltion levels of Akt, p/ MAPK n ER in response to omintion of E n IGF- in the presene of tmoxifen in TAM-R T7D ells (Fig. 7). In ition, signifintly inhiite E-inue ER trnsriptionl tivity vi AP-1, s well s ERE (Supplementry Fig. S7), n the E-inue expression of TFF1, CCND1 n -My genes (Supplementry Fig. S7,) in the presene of tmoxifen in TAM-R or TAM-R T7D ells. Next, we teste the ility of to inhiit the E-epenent growth of TAM-R ells n foun tht tretment signifintly reue the growth of TAM-R (Fig. 7) n TAM-R T7D ells (Fig. 7) in the presene of tmoxifen. Furthermore, we exmine the inhiitory effets of on the E-epenent tumour growth of TAM-R n T7D orthotropi rest ner xenogrfts in nue mie. The results emonstrte tht E-inue growth of oth TAM-R tumours ws suppresse y tretment with 1 mg kg 1 (n ¼ ; Po.1, Fig. 7e; n ¼ 5; Po.1 in two-sie Stuent s t-test, Fig. 7f). Furthermore, onsierle suppression of Akt, p/ MAPK n ER phosphoryltion ws oserve in oth TAM-R tumours trete with (Supplementry Fig. S7e,f). Colletively, these t suggeste tht te s n effetive therpeuti gent with respet to enorine-resistnt rest ner. E-epenent iret trnstivtion of y ER. It ws previously reporte tht is upregulte fter E tretment in ells 1. Thus, we hypothesize tht my e potentil trget gene of ER n foun tht its expression ws signifintly upregulte in ells in time-epenent mnner fter E stimultion (Fig. ). Interestingly, we lso note signifint reution in expression t oth the trnsriptionl n protein levels in ose-epenent mnner fter tmoxifen tretment (Fig. ). Aoringly, to otin iret NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.

9 NATURE COMMUNICATIONS DOI: 1.13/nomms33 ARTICLE T7D-WT TAM-R T7D TAM-R 1 μm tmoxifen 1 nm E 1 ng ml 1 IGF- E E 1 μm (μm) nm tmoxifen 5 P-Akt 1 IGF-1Rβ Akt Akt 5 P-Akt Akt P-MAPK Akt 5 MAPK P-MAPK MAPK MAPK MAPK P-S1/ P-S1/1 P-S P-S11 P-S P-S P-S P-S P-Y P-Y Cell growth (A 5 ) (μm) TAM-R () 1 μm tmoxifen nm E Cell growth (A 5 ) T7D-WT NS 1 μm 1 nm tmoxifen 1 nm E 1 ng ml 1 IGF- e f Tumour volume (mm 3 ) 3 1 TAM-R xenogrft Dys T7D-WT xenogrft E 7 mg kg 1 1 mg kg 1 Cell growth (A 5 ) TAM-R T7D μm 1 nm E 1 ng ml 1 IGF- 1 nm tmoxifen TAM-R T7D xenogrft Tumour volume (mm 3 ) Dys E lone E E tmoxifen E tmoxifen Tumour volume (mm 3 ) 1, 1 1 Dys E tmoxifen E tmoxifen Tmoxifen Figure 7 suppresses growth of TAM-R tumours. (,) Immunolot nlyses were performe to evlute the effets of on the phosphoryltion levels of Akt, p/ MAPK n ER proteins in TAM-R (), n prent T7D (T7D-WT) n TAM-R T7D () ells.() MTT(3-(,5- imethylthizol--yl)-,5-iphenyltetrzolium romie) ssys were performe to evlute the inhiitory effet of on the growth of TAM-R ells. TAM-R ells were trete for h with E± in the presene or sene of 1 mm tmoxifen. The t represent the men±s.e.m. of three inepenent experiments (Po.5, Po.1 in two-sie Stuent s t-test). () Inhiitory effet of on the growth of T7D-WT (upper) n TAM-R T7D (lower) ells fter h of tretment with E lone or E n IGF- in the presene n the sene of 1 nm tmoxifen, respetively. The t of ll pnels represent the men±s.e.m. of three inepenent experiments (Po.5, Po.1, Po.1 in two-sie Stuent s t-test). (e) inhiits the tumour growth of TAM-R orthotropi rest ner xenogrfts in nue mie. The tumour volume represents the men±s.e.m. of eh group (n ¼ 5) (Po.5, Po.1 in two-sie Stuent s t-test). (f) inhiits the tumour growth of oth T7D-WT (left) n TAM-R T7D (right) orthotropi rest ner xenogrfts in nue mie. The tumour volume represents the men±s.e.m. of eh group (n ¼ 5; Po.5, Po.1, Po.1 in two-sie Stuent s t-test). NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

10 NATURE COMMUNICATIONS DOI: 1.13/nomms33 Reltive expression Reltive ERE tivity 1 Rel-time PCR 1 nm E 1 3 (h) 5, p 5, p 5, p 5,9 p Up- ERE in Down- E E E Down- ERE Upin ERE onsensus in intron1 of gene E lone f Reltive expression 1 Tmoxifen 1 nm E 1 μm 1 μm sr IP HDAC1 NoR SRC-1 IgG () Rel-time PCR , 1 nm E ERE in gene Positive feek of e Reltive expression 1 Tmoxifen β-atin Non-genomi tions Genomi tions ERE trnsription E ERE (nm) 1 nm E Immunolot nlysis () 1 nm E 1 3 (h) AP-1 trnsription E AP-1 IGF-1Rβ , (nm) E P P PI3K MAPK P Akt E P P P P P P E lone E Figure Positive feek regultion of trnstivtion. () Upregultion of expression ws evlute fter E stimultion. These t re expresse the fol inrese over untrete ells t h (set t 1.) n represent the men±s.e.m. of three inepenent experiments (Po.1 in twosie Stuent s t-test). () The effets of tmoxifen were evlute on expression. For rel-time PCR nlyses (left), the t re expresse s the fol inrese over untrete ells (set t 1.). These t represent the men±s.e.m. of three inepenent experiments (Po.1, Po.1 in two-sie Stuent s t-test). For immunolot nlyses (right), -tin serve s loing ontrol. () Luiferse ssys were performe to evlute the trnstivtion of using luiferse reporter ontining n ERE motif onserve within intron 1 of the gene (5 -TCCAGTTGCATTGACCTGA-3 ; 5,5, p from the trnsriptionl strt site of ) or onstruts ontining the upstrem n ownstrem regions lking this ERE motif. The t represent the men±s.e.m. of three inepenent experiments (Po.1 in two-sie Stuent s t-test). () ChIP ssys show the trnstivtion of through n introni ERE. (e) The effet of on expression using rel-time PCR is shown t the inite time points. The t re expresse the fol inrese over untrete ells t h (set t 1.) n represent the men±s.e.m. of three inepenent experiments (Po.1, Po.1 in two-sie Stuent s t-test). (f) tretment les to multiple levels of inhiition trgete t E-epenent ER tivtion pthwys. ompetitively ins to enogenous, therey preventing its intertions with. Free iretly ins to oth nuler n memrne-ssoite ER, resulting in the repression of E-inue ER tivtion through non-genomi pthwys n its phosphoryltion. Moreover, lso ttenutes trnsription, resulting in the ownregultion of ER trget genes, inluing. eviene for the upregultion of expression y ER, we mesure ER trnsriptionl tivity. E stimultion resulte in roust luiferse tivity only in ells trnsfete with the onstrut ontining n introni ERE from (Fig. ), suggesting potentil trnstivtion of y ER. Next, we exmine the reruitment of ER to n introni ERE motif of the gene y ChIP nlysis (Fig. ). E-epenent reruitment of ER n the o-tivtor SRC-1 ws oserve ssoite with n ERE within intron 1 of the gene in ells. In ontrst, tretment enhne reruitment of HDAC1 n NoR to the ER omplex (Fig. ). In ition, tretment le to signifint suppression of the E-inue expression levels of even fter 3 h (Fig. e). These results emonstrte tht ws iretly trnstivte y ER vi its introni ERE following E tretment, whih suggeste tht ts through positive feek mehnism to enhne ER tivtion in E- epenent rest ner ells. In other wors, loke this positive regultion of, leing to the relese of from ytoplsmi n the inhiition of ER tivity vi multiple mehnisms. Disussion In this stuy, we evelope ominnt-negtive peptie,, se on the resiues Q15, D19 n Q173 in, whih were essentil for its heteroimeriztion with to 1 NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.

11 NATURE COMMUNICATIONS DOI: 1.13/nomms33 ARTICLE suppress the growth of ER-positive rest ner ells, espeilly enorine-resistnt rest ner ells. ompetitively oun enogenous, therey preventing its intertion with n relesing to iretly in to oth nuler- n memrne-ssoite ER. Intrinsi ining to ER le to the repression of numer of E-inue tivtion events, whih resulte in omplete suppression of E-epenent ERpositive rest ner ell growth in vitro n in vivo (Fig. f). These finings suggest tht hs n importnt role in the moultion of multiple spets of the E/ER-signlling network, lthough hs een reporte to e the only trnsriptionl repressor for ER. However, to te, it remins unler how the enogenous tumour suppressor is intivte in ner ells, espite unnt expression n the lk of genomi muttions or methyltions in rest ner linil speimens or ell lines 7. Our t suggest potentil solution to this unnswere prolem; nmely, our results suggest tht my sequester in ner ells, therey using n pprent lossof-funtion of protein. These finings mrk the first step towr unovering new E/ER signlling network in rest ner n she light on novel therpeuti strtegies using protein funtions for E/ER-positive rest ner. The most importnt fining of this stuy ws tht might potentilly t s n effetive inhiitor of TAM-R rest ner ells. Current enorine therpies for rest ner re se minly on trgeting the ER-signlling pthwy, n tmoxifen is the most frequently presrie rug for the tretment of ll stges of rest ner 1,13. However, tmoxifen resistne is mjor linil prolem n leing use of tretment filure n mortlity 1,15. Compelling eviene suggests tht multiply phosphorylte ER n non-nonil ER tivtion vi AP- 1 re linke to tmoxifen or romtse inhiitor resistne in rest ner ells 1,11,1. In ition, the tivtion of the IGF- 1R/AKTERK1/MAPK network is lso linke to tmoxifen resistne in rest ners 1,11,1, even though the unne of memrne-oun n ytoplsmi ER is low in primry rest ners 3. Our stuy emonstrte tht tretment ompletely inhiite ERIGF-1R n/or ERPI3K intertions, ER phosphoryltion t multiple sites n ER trnsriptionl tivtion vi AP-1 in the presene of E in ER-positive rest ner ells. Inee, h signifint nti-tumour effets ginst TAM-R rest ner ells. More importntly, we revele tht the omintion of tmoxifen n inue rpi poptosis n exhiite more potent ntitumour tivity in vivo n in vitro s ompre with either tretment lone, initing enhne tmoxifen responsiveness. This omine effet is thought to e ue to the istint mehnisms of tion of eh rug, suggesting tht relesing intrinsi oul suppress multiftoril mehnisms of enorine resistne. Current enorine therpies, suh s tmoxifen, pinpoint speifi signlling pthwys or moleules in the ER-signlling network. In ontrst, ws shown to moulte multiple spets of the E/ER-signlling network vi the tumour suppressive ility of enogenous, whih ws expresse unntly in ner ells, leing to the omplete suppression of E-epenent rest ner ell growth. Moreover, lthough is trnstivte iretly y ER in positive feek loop, ws shown to ownregulte trnsription, whih reue protein levels in the ytoplsm of ner ells n enle enogenous to suppress the extensive ER signlling network. Moreover, s is speifilly upregulte in rest ner ut is hrly etetle in norml humn tissues, gents suh s, whih re esigne to speifilly isrupt ining, my emonstrte exellent therpeuti inies with miniml off-trget effets. Intrellulr proteinprotein intertions hve een iffiult to trget with smll moleules or syntheti peptie inhiitors, regrless of their ility to regulte mny signlling networks. However, it hs een reporte tht the Nutlins, seletive smll moleule ntgonists of the MDMp53 intertion, possess in vitro n in vivo nti-tumour tivity vi the retivtion of p53 tumour suppressive tivity. In ft, s the inhiitory effet of ws mintine for only h (Supplementry Fig. S5), it will e neessry to improve upon its phrmologi properties using hemil syntheti pprohes, suh s hyroron stpling methos 9,5, or to sreen seletive smll moleule ntgonists trgeting the intertion. In onlusion, trgeting the intertion represents potentil new tretment venue for ER-positive rest ner ptients. This new pproh oul e n importnt supplement therpy n my provie mehnisti insight into the moleulr sis of ER-signlling networks in rest rinogenesis. Thus, omining enorine tretment with these new trgete therpies is promising pproh for improving the urrent tretment strtegies n overoming enorine resistne, n shoul e investigte in future prelinil n linil stuies. Methos ER ntioy. The nti-er (lone AER31) ntioy ws purhse from Thermo Fisher Sientifi (Fremont, CA). This ntioy speifilly reognizes unigeste ER n is equivlent to n nti-er ntioy (SP-1) 3, whih is wiely use for immunohistohemil nlysis (Supplementry Fig. S). Immunolot nlyses. Cells were lyse with lysis uffer (5 mm Tris-HCl, ph.; 15 mm NCl,.1% NP-,.5% CHAPS) ontining.1% protese inhiitor oktil III (Cliohem, Sn Diego, CA). The lystes were eletrophoretilly seprte, lotte onto nitroellulose memrne n loke with % BlokAe solution (Dinippon Phrmeutil, Osk, Jpn) for 1 h. The lots were then inute with ntioies ginst the following proteins: (ref. 1) (1:); (1:5), NoR (1:5) n ER (phospho Y5; 1:5; Am, Cmrige, UK); SRC-1 (1E7; 1:5), Sh (1:5), /-tuulin (1:1,), Akt (1:1,), phospho-akt (S73; 57F11; 1:1,), p/ MAPK (1:5), phospho-p/ MAPK (T/Y; 1:5) n phospho-er (S1/S1; 1:5; Cell Signling Tehnology, Dnvers, MA); HDAC1 (H-11; 1:5), IGF-1R (1:5), PI3-kinse p5 (U13; 1:5), phospho-er (S11; 1:5), phospho-er (S17; 1:5) n lminin B1 (1:1; Snt Cruz Biotehnology, Snt Cruz, CA); phospho-er (S35; 1:5; Millipore, Billeri, MA); phosphotyrosine (1:5; Zyme, Sn Frniso, CA); -tin (AC-15; 1:5,) n FLAG-tg M (1:5,; Sigm, St Louis, MO); n HA-tg (1:3,; Rohe, Mnnheim, Germny). After inution with n horserish peroxise-onjugte seonry ntioy (Snt Cruz Biotehnology, ilution 1:5,) or monolonl nti-rit immunogloulins-peroxise ntioy (RG-1, Sigm, ilition 1:5,) for 1 h, the lots were evelope with n enhne hemiluminesene system (GE Helthre, Bukinghmshire, UK) n were snne using n Imge Reer LAS-3 mini (Fujifilm, Tokyo, Jpn). All experiments were performe more thn three times in triplite. Finlly, the phosphoryltion levels of IGF-1R, Sh, PI3K, Akt, p/ MAPK n ER were ssesse through ensitometri nlysis of immunolot results using n Imge Reer LAS-3 mini 51. Full-length imges of immunolots re shown in Supplementry Fig. S9. Immunopreipittion. The ells were lyse with.1% NP- lysis uffer s esrie ove. The ell lystes were prelere with norml IgG n re-protein G Sephrose B (Zyme) t C for 3 h. Then, the superntnts were inute with ntioies ginst (5 mg), (5 mg) n ER (5 mg) t C for 1 h. Next, the ntigenntioy omplexes were preipitte with re-protein G Sephrose B t C for 1 h. Immunopreipitte protein omplexes were wshe three times with lysis uffer n seprte using SDSPAGE. Immunolot nlyses were performe s esrie ove. Ientifition of the -ining regions in protein. To etermine the -ining region in, we lone five ifferent onstruts orresponing to prtil sequenes ( 13, 35,177, 1,,177, 11 n 15 ) into n mino-terminl FLAG-tgge pcaggs vetor. COS-7 ells were iniviully otrnsfete with vetor n HA- using the FuGENE trnsfetion regent (Rohe). At h fter trnsfetion, the ells were lyse with.1% NP- lysis uffer. The lystes were prelere for 3 h t C n then inute with nti-flag M grose (Sigm) for 1 h t C. Next, the immunopreipitte proteins or intt ell lystes were eletrophorese n lotte NATURE COMMUNICATIONS :3 DOI: 1.13/nomms & 13 Mmilln Pulishers Limite. All rights reserve.

12 NATURE COMMUNICATIONS DOI: 1.13/nomms33 onto nitroellulose. Finlly, the lots were inute with ntioies ginst FLAG M or HA-tg. Intertion site n struture preition. n intertion sites were preite using PSIVER. PSIVER is omputtionl metho to preit resiues tht in to other proteins using only sequene fetures (position-speifi soring mtrix n preite essiility). The metho uses the Nive Byes lssifier with kernel ensity estimtion n ws shown to outperform existing servers ville on the Internet. The efult threshol of.39 ws use in this stuy. Struture preition ws performe using FUGUE 5 n PSIPRED 53. A moel of the puttive -ining helix of (resiues 15717) ws uilt using MODELLER 5 se on the TIP1 protein 55 s templte. intertion inhiition y. The 13 mino i pepties erive from the -ining omin of (oons 15177) were ovlently linke t the NH terminus to memrne trnsuing 11 polyrginine sequene (11R) to onstrut the peptie. Negtive ontrol pepties, sr n mt, were lso synthesize. To exmine the effets of on inhiition of omplex formtion, ells were trete with 1 nm E±1 mm. intertions were ssesse using o-immunopreipittion followe y immunolotting, s esrie ove. Nuler/ytoplsmi frtiontion. ells were trete s esrie ove, n nuler n ytoplsmi/plsm memrne frtions were prepre using the NE-PER nuler n ytoplsmi extrtion regent (Thermo Fisher Sientifi) oring to the mnufturer s instrutions. /-Tuulin n lminin B were use s loing ontrols for the ytoplsmi n nuler frtions, respetively. Cell prolifertion ssy. Cell prolifertion ssys were performe using the Cell Counting Kit- (Dojino, Kummoto, Jpn). The ells were plte in -well pltes t 1 ells per well n mintine t C. At the inite time points, 1:1 ilution of the Cell Counting Kit- solution ws e (into three replite wells) n inute for 1 h. Then, the sorne t 5 nm ws mesure to lulte the numer of vitl ells per well. The t represent the men±s.e.m. of three inepenent experiments. In vivo tumour growth inhiition. Eh suspension (1 1 7 ells per mouse) of KPL-3C ells, ells, T7D ells, TAM-R ells or TAM-R T7D ells ws mixe with n equl volume of Mtrigel (BD, Frnklin Lkes, NJ) n injete ( ml totl) into the mmmry ft ps of -week-ol femle BALB/ nue mie (Chrles River Lortories, Tokyo, Jpn). The mie were house in pthogenfree isoltion fility with 1-h light/rk yle, n were fe roent how n wter liitum. The tumours evelope over perio of 1 week, rehing sizes of B1 mm 3 (lulte s 1/ (with length )). For KPL-3C orthotropi xenogrft experiments, the mie were rnomize into eight tretment groups (five nimls per group): (1) no tretment; () mg per y E; (35) E þ 3.5, 7 or 1 mg kg 1 per y ; () E þ 1 mg kg 1 per y sr; (7) E þ mg kg 1 per y tmoxifen; n () E þ mgkg 1 per y tmoxifen þ 1 mg kg 1 per y. For the orthotopi xenogrft, the mie were rnomize into three tretment groups: (1) mg per y E; () E þ 1 mg kg 1 per y ; n (3) E þ 1 mg kg 1 per y sr. For the T7D orthotopi xenogrft, the mie were rnomize into five tretment groups: (1) no tretment; () mg per y E; (3) E þ 1 mg kg 1 per y ; () E þ 3.7 mgkg 1 per y tmoxifen; n (5) E þ þ tmoxifen. For TAM-R orthotropi xenogrft, the mie were rnomize into four tretment groups: (1) no tretment; () mgkg 1 per y tmoxifen; () mg per y E þ tmoxifen; n (3, ) E þ tmoxifen þ 7or1mgkg 1 per y. For TAM-R T7D orthotopi xenogrfts, the mie were rnomize into four tretment groups: (1) no tretment; () 3.7 mgkg 1 per y tmoxifen; (3) mg per y E þ tmoxifen; n () E þ tmoxifen þ 1 mg kg 1 per y. E ws elivere vi the pplition of solution to the skin t the nek; the other tretments were elivere vi intrperitonel injetion. Tumour volume ws mesure with lipers for weeks, fter whih time the nimls were kille, n the tumours were exise n frozen in liqui nitrogen. All experiments were performe in orne with the guielines of the niml fility t the University of Tokushim. For evlutions of the inhiitory effets of on tumour expression of ER trget genes using rel-time PCR, the t re expresse s the fol inrese in gene expression over the no tretment group (set t 1.) n represent the men±s.e.m. of eh group (five mie). In ition, for evlutions of the effets of on the phosphoryltion of Akt, p/ MAPK n ER proteins in tumours, eh tumour lystes (three to four mie per group) ws immunolotte. Sttistil nlyses. A Stuent s t-test ws use to etermine the signifine of ifferenes mong the experimentl groups. Vlues of Po.5 were onsiere signifint. The other methos re esrie in Supplementry Informtion. Referenes 1. MCrken, M. et l. Cner iniene, mortlity, n ssoite risk ftors mong Asin Amerins of Chinese, Filipino, Vietnmese, Koren, n Jpnese Ethniities. CA Cner J. Clin. 57, 195 (7).. Jeml, A. et l. Cner sttistis,. CA Cner J. Clin. 5, 113 (). 3. Yger, J. D. & Dvison, N. E. Estrogen rinogenesis in rest ner. N. Engl. J. Me. 35, 7 ().. Berry, D. A. et l. Effet of sreening n juvnt therpy on mortlity from rest ner. N. Engl. J. Me. 353, (5). 5. Green, S. & Chmon, P. Nuler reeptors enhne our unerstning of trnsription regultion. Trens Genet., 3931 ().. Khlert, S. et l. Estrogen reeptor rpily tivtes the IGF-1 reeptor pthwy. J. Biol. Chem., (). 7. Simonini, T. et l. Intertion of oestrogen reeptor with the regultory suunit of phosphtiylinositol-3-oh kinse. Nture 7, 5351 ().. Cstori, G. et l. PI3-kinse in onert with Sr promotes the S-phse entry of oestriol-stimulte ells. EMBO J., 559 (1). 9. Song, R. X. et l. The role of Sh n insulin-like growth ftor 1 reeptor in meiting the trnslotion of estrogen reeptor to the plsm memrne. Pro. Ntl A. Si. USA 11, 71 (). 1. Osorne, C. K. & Shiff, R. Estrogen-reeptor iology: ontinuing progress n therpeuti implitions. J. Clin. Onol. 3, 111 (5). 11. Johnston, S. R. New strtegies in estrogen reeptor-positive rest ner. Clin. Cner Res. 1, (1). 1. Fisher, B. et l. Tmoxifen for prevention of rest ner: report of the Ntionl Surgil Ajuvnt Brest n Bowel Projet P-1 Stuy. J. Ntl Cner Inst. 97, 151 (5). 13. Jorn, V. C. Tmoxifen: most unlikely pioneering meiine. Nt. Rev. Drug Disov., 513 (3). 1. Clrke, R., Leoness, F., Welh, J. N. & Skr, T. C. Cellulr n moleulr phrmology of ntiestrogen tion n resistne. Phrmol. Rev. 53, 571 (1). 15. Fisher, B., Dignm, J., Brynt, J. & Wolmrk, N. Five versus more thn five yers of tmoxifen for lymph noe-negtive rest ner: upte finings from the Ntionl Surgil Ajuvnt Brest n Bowel Projet B-1 rnomize tril. J. Ntl Cner Inst. 93, 9 (1). 1. Ring, A. & Dowsett, M. Mehnisms of tmoxifen resistne. Enor. Relt. Cner 11, 35 (). 17. Lery, A. F., Sirohi, B. & Johnston, S. R. Clinil trils upte: enorine n iologil therpy omintions in the tretment of rest ner. Brest Cner Res. 9, 11 (7). 1. Lery, A. F. et l. Lptini restores hormone sensitivity with ifferentil effets on estrogen reeptor signling in ell moels of humn epierml growth ftor reeptor -negtive rest ner with quire enorine resistne. Clin. Cner Res. 1, 1197 (1). 19. Osorne, C. K. et l. Gefitini or pleo in omintion with tmoxifen in ptients with hormone reeptor-positive metstti rest ner: rnomize phse II stuy. Clin. Cner Res. 17, (11).. Nishite, T. et l. Genome-wie gene-expression profiles of rest-ner ells purifie with lser miroem miroissetion: ientifition of genes ssoite with progression n metstsis. Int. J. Onol. 5, (). 1. Kim, J. W. et l. Ativtion of n estrogen/estrogen reeptor signling y through its inhiitory effet on nuler trnsport of /REA in rest ner. Cner Si. 1, 117 (9).. Montno, M. M. et l. An estrogen reeptor-seletive oregultor tht potentites the effetiveness of ntiestrogens n represses the tivity of estrogens. Pro. Ntl A. Si. USA 9, 9795 (1999). 3. Delge-Mourroux, R. et l. Anlysis of estrogen reeptor intertion with repressor of estrogen reeptor tivity (REA) n the regultion of estrogen reeptor trnsriptionl tivity y REA. J. Biol. Chem., ().. Murkmi, Y. & Mizuguhi, K. Applying the nive yes lssifier with kernel ensity estimtion to the preition of protein-protein intertion sites. Bioinformtis, 111 (1). 5. Klinge, C. M. Estrogen reeptor intertion with estrogen response elements. Nulei Ais Res. 9, (1).. Kushner, P. L. et l. Estrogen reeptor pthwys to AP-1. J. Steroi Biohem. Mol. Biol. 7, (). 7. Kurtev, V. et l. Trnsriptionl regultion y the repressor of estrogen reeptor tivity vi reruitment of histone eetylses. J. Biol. Chem. 79, 33 ().. Vrlkhov, N., Snyer, C., Jose, S., Hhm, J. B. & Privlsky, M. L. Estrogen reeptors reruit SMRT n N-CoR orepressors through newly reognize ontts etween the orepressor N terminus n the reeptor DNA ining omin. Mol. Cell Biol. 3, 1315 (1). 9. Brown, A. M., Jeltsh, J. M., Roerts, M. & Chmon, P. Ativtion of ps gene trnsription is primry response to estrogen in the humn rest ner ell line. Pro. Ntl A. Si. USA 1, 33 (19). 1 NATURE COMMUNICATIONS :3 DOI: 1.13/nomms33 & 13 Mmilln Pulishers Limite. All rights reserve.