ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ ERK signaling pathway

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1 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 RESEARCH Open Aess ERα36 meites ispltin resistne in rest ner ells through /HER2/ signling pthwy Linlin Zhu 1, Jio Zou 1, Yunyin Zho 1, Xiomei Jing 1, Yng Wng 2, Xingwei Wng 3 n Bin Chen 1 Astrt Bkgroun: ERα36, novel ERα66 vrint, hs een emonstrte to promote tmoxifen resistne in rest ner ells. However, the role n mehnisms of ERα36 in ispltin resistne of rest ner ells remin unler. This stuy investigtes the expression n role of ERα36 in ispltin resistne of rest ner ells n eluites its unerlying mehnisms. Methos: The expression of ERα36 n the proteins involve in nongenomi estrogen signling ws evlute y western lot nlysis. Cispltin sensitivity ws explore y CCK8 ssy, monolyer olony formtion ssy n poptosis ssys, respetively. ERα36 sirnas/shrnas n overexpression vetor were trnsfete into ells to ownregulte or upregulte ERα36 expression. Lossn ginof funtion ssys were performe to investigte the role of ERα36 in ispltin sensitivity. The intertion etween ERα36 n /HER2 were etete using CoIP. A mouse xenogrft moel of rest ner ws estlishe to verify the role of ERα36 in vivo. Results: ERα36 is expresse t higher levels in ispltinresistnt rest ner ells ompre to ispltin sensitive ells. Cispltin inue upregultion of ERα36 in oseepenent mnner in rest ner ells. Overexpression of ERα36 lee to ell resistnt to ispltin n knokown of ERα36 in ispltinresistnt rest ner ells restore ispltin sensitivity. The upregultion of ERα36 resulte in inrese tivtion of nongenomi estrogen signling, whih ws responsile for ispltin resistne. Disruption of ERα36meite nongenomi estrogen signling with kinse inhiitors signifintly inhiite ispltininue expression of ERα36 n inrese ispltin sensitivity. The in vivo experiment lso onfirme tht upregultion of ERα36 ttenute ispltin sensitivity in mouse xenogrft moel of rest ner. Conlusions: The results for the first time emonstrte tht ERα36 meites ispltin resistne in rest ner ells through nongenomi estrogen signling, suggesting tht ERα36 my serve s novel trget for ispltin resistne n potentil initor of ispltin sensitivity in rest ner tretment. Keywors: ERα36, Cispltin resistne, Brest ner,, HER2 Bkgroun Cispltin () is the first genertion of pltinum rugs [1, 2]. As ell yle nonspeifi rug, ispltin is wiely use in the tretment of ro rry of soli mlignnies, inluing ler ner, lung ner, ovrin, n rest ner [3 5]. Cispltin exerts ntiner effets minly vi the genertion of DNA lesions followe y Corresponene: innhen@tom.om 1 Deprtment of Biohemistry n Moleulr Biology, Thir Militry Meil University, Chongqing 438, Chin Full list of uthor informtion is ville t the en of the rtile the tivtion of the DNA mge response n the inution of ner ell eth [4, 6, 7]. Although ptients with rest ner usully hve goo initil response to ispltinse hemotherpy, ispltin resistne often ours in linil prtie. Previous stuies hve shown tht tivte /HER2 signling n its ownstrem signling MAPK/ re ssoite with ispltin resistne [4]. Reent stuies hve emonstrte tht RAD5 [8] n Pit1 [9] sensitize humn rest ner ells to ispltin therpy. At present, the well known moleulr mehnisms of ispltin resistne The Author(s). 218 Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( pplies to the t me ville in this rtile, unless otherwise stte.

2 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 2 of 11 inlue inrese DNA repir, ltere rug ellulr umultion, inrese rug ytosoli intivtion, n others [1]. However, the etil mehnisms of ispltin resistne remin to e eluite. Serhing n eveloping new therpeuti strtegies for overoming ispltin resistne is urgently neee to improve the qulity of life n ptient survivl. ERα36 (Estrogen reeptorlph36), memer of ER superfmily with moleulr weight 36 kd, is reently ientifie ERα66 vrint [11]. ERα36 is responsile for estrogenstimulte ell prolifertion n evelopment of ERpositive rest ner [12]. A lrge mount of stuies hve reporte tht highlevel expression of ERα36 is losely relte to tmoxifen resistne in rest ner ells n is one of the unerlying mehnisms of tmoxifen resistne [13 15]. In rest ner ells overexpressing ERα36, tmoxifen isplys estrogenlike effets n tivtes MAPK/ signling meite y ERα36, whih is lso known s ERα36meite nongenomi estrogen signling, n therey stimultes ell survivl n prolifertion [16 18]. Reent stuies hve shown the existene of the ERα36/HER2 positive regultory loops in either ERα negtive or ERα positive rest ner ells [19, 2]. ERα36 n physilly intert with the /HER2. signling n tivte ERα36 trnsription through n AP1 site in the ERα36 promoter, n ERα36 expression stilizes protein or positively regultes HER2 expression [2, 21]. In tmoxifenresistnt MCF7 ells (MCF7/TAM), tmoxifen inues expression of ERα36/HER2 positive regultory loops n the estrution of the loops restores tmoxifen sensitivity [19]. The role of ERα36meite nongenomi estrogen signling in tmoxifen resistne hs ttrte onsierle ttention, however, the signifine of ERα36 in ispltin resistne in rest ner ells hs not een investigte. In our present stuy, we foun tht ispltin inues expression of ERα36 in oseepenent mnner in rest ner ells. The upregultion of ERα36 promotes inrese tivtion of ERα36meite nongenomi estrogen signling, whih finlly resulte in the genertion of ispltin resistne. Bloking ERα36 expression or the tivity of /HER2 or their ownstrem signling MAPK/ signifintly inreses ispltin sensitivity in rest ner ells. Tken together, these results for the first time revel tht ER36 promotes ispltin resistne in rest ner ells through nongenomi estrogen signling, suggesting tht trgeting ERα36 my serve s novel trget for ispltin resistne n potentil initor of ispltin sensitivity in rest ner tretment. Our reserh provies novel insight for improving the therpeuti effet of ispltin, whih my e enefiil to the further linil pplition of ispltin in rest ner tretment. Methos Cell ulture Humn rest ner ell lines MCF7, BT474, n MDAMD231 were purhse from Amerin Type Culture Colletion (ATCC), n hrterize y DNA profiling. These originl ells were ulture in highgluose DMEM (Gio, USA) with 1% FBS (Gio, USA), in humi inutor with 5% CO 2 t 37 C. MCF7/ ells re 5 μg/ml ispltinresistnt strins, whih were estlishe y ulturing MCF7 with high ispltin onentrtions. Cispltin ws e twie week fter reseeing. Every 2 months, ell survivl ws nlyze y MTT ssy. The IC5 vlue of ispltin ginst MCF7 n MCF7/ were 4 μg/ml n 15 μg/ml, respetively. MCF7/ ells were four times s resistnt to the ytotoxi effet of ispltin s ompre with the initil MCF7 ells. MCF7 ells overexpressing ERα36, MCF7/ERα36, n the ontrol ells estlishe s esrie elow were ulture in DMEM meium ontining 1 μg/ml G418 (SigmAlrih, St Louis, MO, USA). Plsmi preprtion n estlishment of ERα36 stle expression or knokown ell lines The oing sequene of ERα36 DNA ws suessfully lone n n ERα36 expression vetor riven y the ytomeglovirus (CMV) promoter ws onstrute, oring to the metho estlishe y Wng et l. [22]. MCF7 ells were trnsfete with either empty vetor or reominnt vetor, using Lipofetmine 3 (Invitrogen, Grn Isln, NY, USA) oring to the mnufturer s instrutions. After trnsfetion for 72 h, 5 μg/ml G418 ws then e to ulture meium for stle lone seletion. MCF7 ells stly overexpressing ERα36 mplifie from single lone were ientifie y western lot, n then use for the experiments. ERα36 shrna lentivirl expression vetor (plvxshrna2puroherα36, LentishERα36) n its ontrol vetor (plvxshrna2puro, LentishNC) were onstrute y GenePhrm (Shnghi, Chin). The ERα36 shrna trget sequenes re 5 ACAUCAUCUCGGUUCCGCA3. MCF7/ n MCF7/ERα36 ells were plte in sixwell pltes (5 1 5 ells/well) n were ulture to 6% onfluene. Approprite volumes of lentiviruses were then e oring to the multipliity of infetion vlues reommene y the mnufture. MCF7/ ells expressing ERα36 shrna n MCF7/ ERα36 ells expressing ERα36 shrna were selete with 5 μg/ml puromyin n ientifie y western lot. Cell prolifertion ssy The ell prolifertion ws ssye y Cell Counting 8 kit (CCK8) (Dojino lortories, Kummoto, Jpn). The ells were seee in triplite into 96well pltes for

3 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 3 of h n given ifferent tretments for the inite time; followe y the ition of 1 μl CCK8 regents to eh well. After inution t 37 C for 1 h, the vlue t OD45 nm ws etermine oring to the mnufturer s instrutions. Monolyer olony formtion ssy Five hunre ells were seee in triplite onto 6well pltes n inute in meium ontining 5 μg/ml ispltin or equivlent (vehile) (Sigm, USA). Every 3 5 ys the meium ws reple with fresh meium. After 2 weeks, the olonies were fixe with 1% methnol, stine with.1% rystl violet n wshe with phosphte uffer solution (PBS). Visile olonies were then ounte for quntifition. Western lot nlysis Isoltion of ell extrts n western lot nlysis were esrie previously [23]. The protein onentrtions were mesure y BCA protein ssy kit (Beyotime Biotehnology, Shnghi, Chin). Immunoetetion ws rrie out using ERα36 (Cell Applitions, Sn Diego, CA, USA), p1/2 (Cell Signling Tehnology, Boston, MA, USA) n totl1/2 (Cell Signling Tehnology, Boston, MA, USA), p (Cell Signling Tehnology, Boston, MA, USA) n totl (Cell Signling Tehnology, Boston, MA, USA), pher2 (Cell Signling Tehnology, Boston, MA, USA) n totlher2 (Cell Signling Tehnology, Boston, MA, USA) ntioies or ntioy. (Beyotime, Shnghi, Chin) ws use s ontrol for equl loing n trnsfer. Hoehst stining The ells were seee in 6well pltes for 12 h n given ifferent tretments for the inite time. Then the ells were stine with Hoehst (Beyotime, Shnghi, Chin) t 1 μg/ml for 1 min in the rk t room temperture n wshe 3 times with PBS n photogrphe uner fluoresene mirosope. Flow ytometry Brest ner ells were hrveste n inute with nnexin VFITC n PI oring to the mnufturer s instrutions (BD, 56112), n then the poptosis ws nlyze y flow ytometer. SiRNA ssy The ERα36 sirna trget sequenes re 5 GCTA GAGATCCTGATGATTGG3. MCF7/ n MCF7/ ERα36 ells were seprtely ulture overnight, n then sirna for ERα36 (sierα36) or ontrol sirna (sinc) ws trnsfete into the ells using lipofetmine 3 oring to the mnufturer s instrutions. Susequently, the ells were trete with or without 5 μg/ml ispltin for 48 h, then hrveste n use for western lot nlysis or ell prolifertion ssys. Coimmunopreipittion (CoIP) nlysis Isoltion of ell extrts were esrie previously [23]. Cell lystes were inute with ntiher2 or nti ntioies or IgG for 1 h t 4 C. Then the Protein A/G plusgrose (Snt Cruz Biotehnology, Dlls, TX, USA) ws e n inute for overnight t 4 C. The preipittes were then extensively wshe with the PBS, resuspene in loing uffer, seprte on SDSpolyrylmie gel eletrophoresis n proe with ntierα36 ntioy s esrie efore [23]. Animl experiments Fiveweekol femle nue mie were purhse from the Lortory Animl Center of Chin (Shnghi, Chin). The mie were rnomize into 2 groups with 1 mie per group n then seprtely inoulte suutneously MCF7/ERα36 n MCF7/V ell suspension. Every mouse ws inoulte ells suspening in.1 ml PBS. When plple tumors forme, every group were rnomly ivie into two groups, n either trete with (ontrol), or ispltin (4 mg/kg) y intrperitonel injetions every other y for 2 weeks, respetively. After tht, the mie were srifie n the xenogrft tumors were hrveste. The volumes were estimte using the following formul: volume = with 2 length 1/2. Tumor tissues were proesse for ERα36 expression nlysis. Sttistil nlysis The t were expresse s the men ± SD unless otherwise stte. Sttistil omprisons etween groups were performe y onewy nlysis of vrine (ANOVA), followe y stuent s ttest. P < 5 ws onsiere signifint. Results Cispltin inues upregultion of ERα36 in rest ner ells MCF7/ ells were foun to e resistnt to 5 μg/ml ispltin, ompre with the wiltype MCF7 ells, s shown in Fig. 1 n. Menwhile, ERα36 expression ws signifintly higher in MCF7/ ells thn in MCF7 ells (Fig. 1 n ), initing tht ispltin my inue ERα36 expression in the proess of ispltin resistne formtion. Corresponing with this oservtion, ispltin tretment inue upregultion of ERα36 in oseepenent mnner in MCF7, BT474, n MDAMB23 ells, respetively (Fig. 1eh), suggesting tht ERα36 my ply n importnt role uring ispltin resistne formtion in rest ner ells.

4 g of ER 36/ OD45(nm) Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 4 of 11 MCF7 MCF7/ Colony numers ER 36 ER 36 (µg/ml) 2.5 (µg/ml) MCF7 MCF7 MCF7/ BT e ER 36 MDAMB231 2 MCF7 5 MCF7 h of ER 36/ MCF7/ 3. f of ER 36/ 3. MCF7 MCF7 BT474 MDAMB231 (µg/ml) Fig. 1 Effet of ispltin on prolifertion n ERα36 expression of rest ner ells. MCF7 n MCF7/ ells were trete with inresing onentrtions of ispltin () for 48 h, n then ell prolifertion ws mesure using CCK8 ssy kit. Cispltin sensitivity of MCF7 n MCF7/ ells ws exmine y monolyer olony formtion ssy. ERα36 protein expression in MCF7 n MCF7/ ells ws nlyze using western lot. The quntittive nlysis of ERα36 expression of (). e MCF7 ells were trete with or without 5 μg/ml ispltin for 48 h, ispltininue expression of ERα36 ws mesure y western lot. f The quntittive nlysis of ispltininue ERα36 expression of (e). g MCF7, BT474 n MDAMB231 ells were trete with ispltin t the inite onentrtions for 48 h n then the protein levels of ERα36 were nlyze y western lot. h The quntittive nlysis of ispltininue ERα36 expression of (g). P < 5, P < 1, P < 1 Overexpression of ERα36 ontriutes to ispltin resistne in rest ner ells To explore the potentil role of ERα36 in ispltin resistne in rest ner ells, MCF7 ells stly overexpressing ERα36 were sreene n ientifie (Fig. 2 n ), n ispltin sensitivity ws etete y ell prolifertion n monolyer olony formtion ssys (Fig. 2 n ). The results revele tht MCF7/ERα36 ells otine resistnt phenotype to 5 μg/ml n 1 μg/ml ispltin, ompre with the ontrol ells. Hoehst stining n flow ytometry nlysis showe tht overexpression of ERα36 in MCF7 ells lso ttenute ispltininue ell poptosis (Fig. 2e n f). Colletively, these results suggeste tht overexpression of ERα36 ontriutes to ispltin resistne in rest ner ells. Knokown of ERα36 in ispltinresistnt rest ner ells restores ispltin sensitivity To further efine the signifine of ERα36 in ispltin resistne, ERα36 in MCF7/ ells or MCF7/ERα36 ells ws silene using sirna or shrna n the hnges of ell prolifertion n monolyer olony formtion were nlyze. Western lot nlysis showe tht trnsfetion of sierα36 or LentishERα36 inue signifint knokown of ERα36 expression ompre with the ontrol trnsfetion (Fig. 3,, e n g). Cell prolifertion ssy revele tht ispltin tretment oviously reue the prolifertion of MCF7/ or MCF7/ERα36 ells trnsfete with sierα36, ompre with the ontrol ells (Fig. 3 n f). Cispltin tretment signifintly erese monolyer olony numer of oth MCF7/ ells expressing ERα36 shrna n MCF7/ERα36 ells expressing ERα36 shrna,

5 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 5 of 11 ER 36 of ER 36/ 3. OD45(nm) MCF7/V MCF7/ER 36 Colony numers e f Q1 1.2% Q2 3.28% MCF7/V MCF7/ER 36 MCF7/V MCF7/ER 36 Q1 2.35% Q2 5.16% Q1 4.4% Q2 3.36% Q1 2.81% Q2 3.51% PI (µg/ml) Q4 93.4% Q3 2.17% Q4 79.% Q3 13.5% Q4 9.6% Q3 1.68% Q4 9% Q3 2.13% Annexin VFITC Fig. 2 Overexpression of ERα36 ontriutes to ispltin resistne in rest ner ells. MCF7 ells stly overexpressing ERα36 were sreene using G418 for 4 weeks n ientifie y western lot. The quntittive nlysis of ERα36 expression of (). MCF7/V n MCF7/ERα36 ells were trete with inresing onentrtions of ispltin () for 48 h, n then ell prolifertion ws mesure with CCK8 ssy kit. Cispltin sensitivity of MCF7/V n MCF7/ERα36 ells ws exmine y monolyer olony formtion ssy. e, f MCF7/V n MCF7/ERα36 ells were trete with or without 5 μg/ml ispltin for 48 h. The ell nuleus were stine with Hoehst n then oserve uner fluoresene mirosope. The representtive imges were shown n the typil poptoti oies were mrke with white rrows (e). The ells were stine with nnexin VFITC/PI. Then the perentge of poptoti ells ws lulte using flow ytometry (f). P <5, P < 1, P <1 ompre with the ontrol ells (Fig. 3 n h). These t suggeste tht ERα36 is n importnt eterminnt for ispltin resistne in rest ner ells n ownregultion of ERα36 expression n enhne ispltin sensitivity. Upregultion of ERα36 les to inrese tivtion of nongenomi estrogen signling To lrify the mehnism of ERα36 involve in ispltin resistne, we investigte the hnges of ERα36 expression n ERα36meite nongenomi estrogen signling fter ispltin tretment. In MCF7 ells, ispltin tretment inue upregulte expression of ERα36 (Fig. 1eh, 4, n ) n tivte ERα36meite nongenomi estrogen signling or the ERα36//HER2/ pthwy (Fig. 4e). Cispltininue upregultion of ERα36 n tivtion of ERα36meite nongenomi estrogen signling oul lso e oserve in other rest ner ell lines inluing BT474, n

6 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 6 of 11 e ER 36 ER 36 MCF7/ MCF7/ER 36 OD45(nm) sinc sier 36 f OD45(nm) sinc sier 36 MCF7/ MCF7/ER 36 g ER 36 ER 36 MCF7/ MCF7/ER 36 Colony numers LentishNC LentishER 36 h Colony numers LentishNC LentishER 36 MCF7/ MCF7/ER 36 Fig. 3 Knokown of ERα36 restores ispltin sensitivity in ispltinresistnt rest ner ells., MCF7/ ells were trnsfete with sierα36 n negtive ontrol sirna (sinc) for 48 h. The ells were ollete n nlyze for ERα36 protein expression using western lot (). The trnsfete MCF7/ ells were trete with 5 μg/ml ispltin () for 48 h, n ell prolifertion ws mesure with CCK8 ssy kit (). ERα36 expression in MCF7/ ells expressing ERα36 shrna n the ontrol ells nlyze using western lot. Cispltin sensitivity of MCF 7/ ells expressing ERα36 shrna n the ontrol ells ws exmine y monolyer olony formtion ssy. e, f MCF7/ERα36 were trete s in (, ), then the peotein level of ERα36 ws etete y western lot (e). The prolifertion of the trnsfete MCF7/ERα36 ells ws evlute using CCK8 ssy kit (f). g ERα36 expression in MCF7/ERα36 ells expressing ERα36 shrna n the ontrol ells nlyze using western lot. h Cispltin sensitivity of MCF7/ERα36 ells expressing ERα36 shrna n the ontrol ells ws exmine y monolyer olony formtion ssy. P < 5, P < 1, P < 1 MDAMB231(Fig. 4 n e). These t suggeste tht upregultion of ERα36 les to inrese tivtion of nongenomi estrogen signling. In tion, ispltin tretment inue the inrese intertion etween ERα36 n /HER2 (Fig. 4f), whih my further inrese the tivtion of nongenomi estrogen signling. Inrese tivtion of ERα36meite nongenomi estrogen signling is responsile for ispltin resistne in rest ner ells The previous t hve shown tht overexpression of ERα36 in MCF7/ n MCF7/ERα36 ells ontriutes to ispltin resistne (Figs. 1, 2, 3). Next, we investigte the signifine of ERα36meite nongenomi estrogen signling in ispltin resistne. Mthing with ispltinresistnt phenotypes of MCF7/ or MCF7/ERα36 ells (Figs. 1, 2, 3), the ERα36/ /HER2/ signling in these two types of ells ws pprently tivte ompre with the ontrol ells (Fig. 5 n ). However, knokown of ERα36 in oth MCF7/ ells n MCF7/ERα36 ells ttenute ERα36meite nongenomi estrogen signling (Fig. 5 n ), n restore ispltin sensitivity (Fig. 3). These t suggeste tht inrese tivtion of ERα36meite nongenomi estrogen signling is responsile for ispltin resistne in rest ner ells. Disruption of ERα36meite nongenomi estrogen signling inreses ispltin sensitivity in rest ner ells Sine inrese tivtion of ERα36meite nongenomi estrogen signling is involve in ispltin resistne in rest ner ells, we oserve whether loking ERα36meite nongenomi estrogen signling oul inrese ispltin sensitivity in rest ner ells. Consistent with the previous oservtion (Fig. 1eh n Fig. 4e), the upregultion expression of ERα36 n the tivtion of ERα36meite nongenomi estrogen signling in MCF7 ells were inue gin y ispltin tretment (Fig. 6). However, ispltin omine with the tyrosine kinse inhiitor AG1478 (SigmAlrih, St Louis, MO, USA) or the ul n HER2 tyrosine kinse inhiitor Lptini (MCE, Monmouth Juntion, NJ, USA) or the MAPK/ kinse inhiitor U126 (SigmAlrih, St Louis, MO, USA), signifintly inhiite ispltininue expression of ERα36 n tivtion of ERα36meite nongenomi estrogen signling in MCF7 ells, respetively (Fig. 6). Menwhile, the omintion of ispltin n

7 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 7 of 11 f HER2 ER 36 P Tuuin HER2 ER 36 P Tuuin (µg/ml) ER 36 MCF7 MCF7 Input IgG IP(Her2) IP() of trget protein/ BT HER2 ER 36 MCF7 MDAMB231 2 MCF7 Fig. 4 Upregultion of ERα36 les to inrese tivtion of nongenomi estrogen signling. MCF7 ells were trete with or without 5 μg/ml ispltin () for 48 h. Then the protein levels of, HER2, ERα36, totl () n phosphorylte (P) were etete using western lot., The quntittive nlysis of ispltininue expression of ERα36,, HER2 n P/ of (). MCF7, BT474 n MDAMB231 ells were trete with ispltin t the inite onentrtions for 48 h n then the protein levels of, HER2, ERα36, n P were nlyze y western lot. e The quntittive nlysis of ispltininue expression of P/ of (). f MCF7 ells were trete s in (). The ell lystes were immunopreipitte with ntiher2 or nti ntioies. Then the immunopreipittes were seprte y SDSPAGE n proe with ntierα36 ntioies. Immunopreipittion of IgG ws use s negtive ontrol 5 e of P/ 2.5 (µg/ml) of P/ 3. MCF7 BT474 MCF7 MDAMB either of the three inhiitors inrese ispltin sensitivity ompre with ispltin tretment lone (Fig. 6). In ispltinresistnt MCF7/ERα36 ells, the omintion of ispltin n either of the three inhiitors resulte in mrkely inhiite phosphoryltion of n enhne ispltin sensitivity ompre with ispltin tretment lone (Fig. 6 n ). These results suggeste tht loking ERα36 expression or the tivity of /HER2, or their ownstrem signling MAPK/ oul estroy ERα36meite nongenomi ER 36 HER2 P ER 36 HER2 P ER 36 HER2 P MCF7/ ER 36 HER2 P MCF7/ER 36 Fig. 5 Inrese tivtion of ERα36meite nongenomi estrogen signling is responsile for ispltin resistne., MCF7 n MCF7/ ells () or MCF7/V n MCF7/ERα36 ells () were hrveste n the protein levels of ERα36,, HER2, totl () n phosphorylte (P) were etete using western lot., MCF7/ () n MCF7/ERα36 () ells were trnsfete with sierα36 n negtive ontrol sirna (sinc) for 48 h. Then the ells were ollete n the levels of ERα36,, HER2, P, were nlyze y western lot

8 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 8 of 11 AG1478(1µM) Lptini(2µM) P PHER2 HER2 ER 36 P U126(1µM) OD45(nm) AG1478(1µM) Lptini(2µM) U126(1µM) Inhiitor P Inhiitor AG1478(1µM) Lptini(2µM) U126(1µM) MCF7 MCF7/ER 36 Inhiitor OD45(nm) Inhiitor AG1478(1µM) Lptini(2µM) U126(1µM) MCF7/ER 36 Fig. 6 Disruption of ERα36meite nongenomi estrogen signling inreses ispltin sensitivity in rest ner ells. MCF7 ells were trete with or without 5 μg/ml ispltin () for 48 h fter preinute with or without AG1478, Lptini, n U126 t the inite onentrtions for 6 h, respetively. Then the levels of ERα36, totl () n phosphorylte (P), totl HER2 (HER2) n phosphorylte HER2 (PHER2), totl () n phosphorylte (P) were evlute using western lot. MCF7 ells were trete s in (), n then the ell prolifertion ws mesure with CCK8 ssy kit. MCF7/ERα36 ells were trete with 5 μg/ml ispltin for 48 h fter preinute with or without AG1478, Lptini, n U126 t the inite onentrtions for 6 h, respetively. Then the totl () n phosphorylte (P) ws etete y western lot. MCF7/ERα36 ells were trete s in (), n then the ell prolifertion ws exmine using CCK8 ssy kit. P < 5, P < 1, P < 1 MCF7 estrogen signling n therey inrese ispltin sensitivity in rest ner ells. Upregultion of ERα36 ttenutes ispltin sensitivity in nue mouse xenogrft moel MCF7/ERα36 humn rest ner xenogrfts in Nue Mie were use to evlute the effet of ERα36 expression on ispltin sensitivity in vivo. Cispltin tretment signifintly inhiite the ontrol ut not MCF7/ ERα36 xenogrft growth (Fig. 7 n ). ERα36 expression in MCF7/ERα36 xenogrft tissues were mrkely higher thn in ontrol tissues (Fig. 7), n ispltin tretment oviously inue upregultion of ERα36 in ontrol xenogrft tissues (Fig. 7). These results suggeste tht upregultion of ERα36 n reue ispltin sensitivity in vivo. Disussion To the est of our knowlege, this stuy presents the first eviene tht ERα36 promotes ispltin resistne in rest ner ells, whih is meite y inrese tivtion of nongenomi estrogen signling. Our results suggest tht ERα36 my serve s novel trget to overome ispltin resistne s well s potentil iomrker of ispltin sensitivity in the tretment of rest ner. Aumulting eviene hs emonstrte tht ERα36 regultes the multiple physiologil funtions of vrious tissues. ERα36 is neessry for ovry evelopment n ooyte meioti mturtion [24] n mintining the one ensity of postmenopusl women [25]. Dysregultion of ERα36 uses vrious ysfuntions n iseses, suh s osteoporosis, irwy hyperresponsiveness, n even ners [12, 26]. In rest ner, knokown of ERα36 inhiits prolifertion, migrtion, n invsion n inreses sensitivity to plitxel in MDAMB231 ells [27]. ERα36 lso ontriutes to the prolifertion n mintenne of stemlike ells [21, 28]. Speifilly, extensive reserh hs shown tht ERα36meite nongenomi estrogen signling is involve in tmoxifen

9 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 9 of 11 (4µg/mg) (4µg/mg) ER 36 MCF7/V MCF7/ER 36 ER 36 MCF7/V Tumor volume(mm 3 ) MCF7/V MCF7/ER 36 Fig. 7 Upregultion of ERα36 ttenutes ispltin sensitivity in nue mouse xenogrft moel., The nue mie ering MCF7/V ellerive n MCF7/ERα36 ellerive suutneous tumors were trete intrperitonelly with or without ispltin () for 2 weeks, the xenogrft tumors were hrveste () n the tumor volume ws lulte s esrie in Methos (). ERα36 protein levels in eh group were evlute y western lot. MCF7/V tumors trete with or without ispltin were nlyze for ERα36 protein levels using western lotting. P < 5, P < 1, P < 1 resistne in rest ner ells [13 15]. In spite of ll these investigtions, more reserh is neee to lrify ERα36 iologil funtion n mehnism. Our urrent results emonstrte tht ERα36 promotes ispltin resistne in rest ner ells, whih revels new iologil funtion of ERα36 in the tretment of rest ner. The possile mehnism of ERα36 involve in ispltin resistne in rest ner ells ws explore in this stuy. Our urrent t suggeste tht ERα36 promotes ispltin resistne through nongenomi estrogen signling. The tivtion of /HER2/ signling is well known ispltin resistnt mehnisms [4, 29, 3]. For exmple, overexpression of HER2 les to the ylinepenent kinse inhiitor 1A nuler exlusion whih ontriutes to ispltin resistne [4] n it hs een relte to ispltin resistne in NSCLC ptients [31]. The MAPK/ signling hs een ssoite with oth inrese n erese sensitivity to ispltin in ifferent experiment moels [32, 33]. Although the reltionship etween /HER2/ signling n ispltin resistne in rest ner ells remins to e efine, the inhiition of the MAPK pthwys sensitizes sllike MDAMB468 ells to ispltin tretment [34]. The high expression of mphiregulin, speifi lign of the, shows highly signifint orreltion with ispltin resistne in vriety of humn rest ner ell lines [35]. More importntly, the use of rhu MA HER2 in omintion with ispltin in ptients with HER2/neuoverexpressing metstti rest ner results in ojetive linil response rtes higher thn those reporte previously for ispltin lone, or rhuma HER2 lone [36]. These stuies inite tht tivtion of /HER2/ signling my e involve in ispltin resistne in rest ner ells. ERα36meite nongenomi estrogen signling is hrterize y tivte /HER2/ signling. In our stuy, we foun tht ispltin tretment inue expression of ERα36 n the intertion etween ERα36 n / HER2. Cispltininue upregultion of ERα36 enhne ERα36meite nongenomi estrogen signling n therey resulte in ispltin resistne in rest ner ells. However, loking ERα36 expression or the tivity of /HER2, or their ownstrem signling MAPK/ oul estroy ERα36meite nongenomi estrogen signling n therey inrese ispltin sensitivity. These t suggeste tht inrese tivtion of ERα36meite nongenomi estrogen signling is the mehnism of ERα36 promoting ispltin resistne in rest ner ells. Aitionl, sine isruption of ERα36meite nongenomi estrogen signling oul inrese ispltin sensitivity in rest ner ells, these finings lso revel new therpeuti trgets for overoming ispltin resistne in rest ner ells. Interestingly, oth ispltininue expression of ERα36 n /HER2 were oserve in our stuy. It is very importnt to lrify the potentil mehnisms involve in the regultion of ERα36 n /HER2 expression y ispltin in future. Aitionl, our urrent t only emonstrte tht ispltininue expression of ERα36 enhnes nongenomi estrogen signling n onfer ispltin resistne. Consiering tht there is regultive reltionship etween ERα36 n /HER2 [2, 21], it remins unler whether ispltininue expression of ERα36 oul promote the formtion of the

10 Zhu et l. Journl of Experimentl & Clinil Cner Reserh (218) 37:123 Pge 1 of 11 ERα36/HER2 positive regultory loops n whether these loops my e invovle in ispltin resistne. The etile mutul regultory mehnisms of ERα36 n /HER2 in ispltin resistne in rest ner ells nee further stuy. Conlusion In onlusion, our urrent stuy emonstrtes for the first time tht overexpression of ERα36 promotes ispltin resistne through nongenomi estrogen signling. These finings revel new importnt mehnism for the reserh on ispltin resistne in rest ner ells n my provie new strtegies to overome ispltin resistne y trgeting ERα36. Future reserh will further explore the signifine of ERα36 expression in rest ner ptients trete with ispltin. Arevitions : Cispltin; : Epierml growth ftor reeptor; ERα36: Estrogen reeptorlph36; HER2: Humn epierml growth ftor reeptor2; MAPK: Mitogentivte protein kinse; : Extrellulr signlregulte kinse; TAM: Tmoxifen; G418: Genetiin; CCK8: Cell Counting 8 kit; CMV: Cytomeglovirus; CoIP: Coimmunopreipittion Aknowlegments We re grteful to Dr. ZhoYi Wng in Deprtment of Meil Miroiology n Immunology, Creighton University Meil Shool, 25 Cliforni Plz, Omh, NE, US for proviing prt of ERα36 ntioies. Funing This work ws supporte y grnts from the Ntionl Nturl Siene Fountion of Chin (No , No n No ). Avilility of t n mterils The tsets supporting the finings of this stuy re inlue within the rtile. Authors ontriutions BC is orresponing uthor n orgnize the stuy. LZ performe experiments. JZ n YZ nlyze results. XJ, YW, n XW eite the mnusript. All uthors hve re n pprove the finl mnusript. Ethis pprovl This stuy hs een onute in orne with ethil stnrs n the ntionl n interntionl guielines. All niml experiments were rrie out oring to the protool pprove y the Thir Militry Meil University Guielines for Use n Cre of Animls. No humn smples were involve in this stuy. Consent for pulition Not pplile. Competing interests The uthors elre tht they hve no ompeting interests. Pulisher s Note Springer Nture remins neutrl with regr to jurisitionl lims in pulishe mps n institutionl ffilitions. Author etils 1 Deprtment of Biohemistry n Moleulr Biology, Thir Militry Meil University, Chongqing 438, Chin. 2 Deprtment of Clinil Lortory, Institute of Surgery Reserh, Dping Hospitl, Thir Militry Meil University, Chongqing 438, Chin. 3 Deprtment of Urology, Shenzhen University Generl Hospitl, Shenzhen 5186, Gungong, Chin. Reeive: 27 April 218 Aepte: 15 June 218 Referenes 1. Ugur S, Ulu R, Dogukn A, Gurel A, Yigit IP, Gozel N, Aygen B, Ilhn N. The renoprotetive effet of urumin in ispltininue nephrotoxiity. 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