MIG-6 suppresses endometrial epithelial cell proliferation by inhibiting phospho-akt

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1 Yoo et l. BMC Cncer (218) 18:65 RESEARCH ARTICLE Open Access suppresses endometril epithelil cell prolifertion y inhiiting phospho-akt Jung-Yoon Yoo 1,2, Hee-Bum Kng 3, Russell R. Broddus 4, John I. Risinger 1, Kyung-Chul Choi 3,5* nd Te Hoon Kim 1* Astrct Bckground: Aerrnt hyperctivtion of epithelil prolifertion, AKT signling, nd ssocition with unopposed estrogen (E2) exposure is the most common endometril cncer dysfunction. In the norml uterus, progesterone () inhiits prolifertion y coordinting stroml-epithelil cross-tlk, which we previously showed is medited y the function of Mitogen-inducile gene 6 (Mig-6). Despite their ttrctive chrcteristics, non-surgicl conservtive therpies sed on progesterone lone hve not een universlly successful. One rrier to this success hs een the lck of understnding of the effect on endometril cells. Method: To further understnd the role of Mig-6 nd in controlling uterine prolifertion, we developed Sprr2f-cre driven mouse model where Mig-6 is specificlly lted only in the epithelil cells of the uterus (Sprr2f cre+ ). We exmined effect nd regultion of AKT signling in the endometrium of mutnt mice. Results: Sprr2f cre+ mice developed endometril hyperplsi. tretment ted the development of endometril hyperplsi nd restored morphologicl nd histologicl chrcteristics of the uterus. tretment reduced cell prolifertion which ws ccompnied y decresed AKT signling nd the restortion of stroml PGR nd ESR1 expression. Furthermore, our in vitro studies reveled n inhiitory effect of on AKT phosphoryltion s well s nd AKT protein interctions. Conclusions: These dt suggest tht endometril epithelil cell prolifertion is regulted y medited Mig-6 inhiition of AKT phosphoryltion, uncovering new mechnisms of ction. This informtion my help guide more effective non-surgicl interventions in the future. Keywords:, Progesterone resistnce, Endometril hyperplsi, AKT Bckground Endometril cncer is the most common gynecologic mlignncy in the United Sttes, nd in the lst severl decdes the incidence of new cses ech yer hs incresed [1]. Endometrioid endometril cncer, the most common type of endometril cncer (8 85%), is ssocited with or preceded y norml multipliction of endometril epithelil cells, known s complex typicl hyperplsi [2 4]. The min tretment for endometril cncer is hysterectomy [5, 6]. Although most * Correspondence: choikc75@mc.seoul.kr; TeHoon.Kim@hc.msu.edu Jung-Yoon Yoo nd Hee-Bum Kng contriuted eqully to this work. 3 Deprtment of Biomedicl Sciences, ASAN Medicl Center, University of Ulsn College of Medicine, Seoul 555, South Kore 1 Deprtment of Ostetrics, Gynecology nd Reproductive Biology, College of Humn Medicine, Michign Stte University, Grnd Rpids, MI 4953, USA Full list of uthor informtion is ville t the end of the rticle endometril cncer dignoses re in post-menopusl women, 5% of cses re dignosed efore ge 4 nd 2~ 25% efore menopuse [7]. Moreover, the incidence of endometril cncer dignoses in younger ptients is likely to increse going forwrd due to increses in oesity, hypertension, dietes mellitus, nd other known endometril cncer risk fctors [8 1]. Therefore, the demnd for non-surgicl pproches to endometril cncer is incresing, especilly for women of reproductive ge with complex typicl hyperplsi nd erly-stge endometrioid endometril cncer who wish to preserve their fertility eyond tretment [8, 1]. Although hysterectomy is key therpy for endometril cncer [5, 6], recent intruterine progestin therpies such s levonorgestrel-relesing intruterine system hve een used for reproductive-ged women with The Author(s). 218 Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Yoo et l. BMC Cncer (218) 18:65 Pge 2 of 1 complex typicl hyperplsi nd erly-stge endometril cncer in cses when there is desire to preserve fertility or when comoridities exclude the possiility of surgery. In ddition, progestin therpy is lso considered for recurrent endometril cncer ecuse it is less toxic thn chemotherpies; however, though the response rte of endometril hyperplsi to progestin tretment is higher thn tht of endometril denocrcinom, the response to progestin in cncer recurrence is worst of ll. Progestin therpies used in the clinic re effective for some ptients ut not ll cses of endometril hyperplsi nd well-differentited endometrioid endometril cncer. Another mjor limittion of progestin therpy is the lck of clinicl stndrd protocol for the type, dose, nd durtion of tretment [11 13]. The moleculr mechnisms underlying progesterone () resistnce in endometril cncer hve not een fully understood. Loss of control over uterine epithelil cell prolifertion nd poptosis y ovrin steroid hormones is the mjor underlying pthogenesis of endometril cncer [14 17]. Progesterone therpy cn prevent this process y locking ctions of unopposed estrogen (E2) [18]. Nonetheless, severl studies indicte tht therpy hs low nd unpredictle response rtes in women with endometril cncer, therefore limiting its potentil use [19 23]. Resistnce to tretment due to loss of either progesterone receptor (PGR) itself or its signling pthwys cuses significnt difficulty in the tretment of dvnced nd recurrent endometril cncer [24]. Identifying moleculr mechnisms involved in resistnce is criticl to effective nd personlized tretment. Unfortuntely, further trnsltionl reserch of endometril cncer is inhiited y the lck of sufficient pre-clinicl niml models. Sequencing nlysis of endometril cncers in the Cncer Genome Atls hs reveled tht upwrds of 9% of cses of endometrioid endometril cncer hve some genetic errtion in the PTEN/PI3K pthwy, which results in incresed AKT ctivity [25]. In ddition, the AKT signling pthwy cn e ctivted y E2 [26] enhncing cell prolifertion [27]. Therefore, n understnding of the moleculr mechnisms etween steroid hormone nd PTEN/PI3K/AKT signling will llow us to e in much etter position to develop new conservtive therpies sed on function. The protein structure of AKT consists of PH domin, linker region, kinse domin, nd regultor domin [28]. These domins undergo vrious protein modifictions including phosphoryltion, cetyltion, uiquityltion, methyltion, hydroxyltion, glycosyltion, nd SUMOyltion which help regulte the proteins ctivity [29]. AKT regultes different pthwys tht id in the promotion of cellulr survivl nd inhiition of poptosis through its serine/threonine kinse ctivity [3, 31]. Inppropritely elevted expression of AKT phosphoryltion is relted to poor prognosis of endometril cncer ptients [32]. Furthermore, inhiition of the AKT pthwy comined with decreses ngiogenesis nd prolifertion in vivo, indicting tht regultion of the AKT pthwy my ply n importnt therpeutic role [33]. Mitogen-inducile gene 6 () functions to suppress endometril cncer in the humn nd mouse uterus [34, 35]. Mig-6 is n importnt meditor of signling in tht it inhiits E2-medited epithelil prolifertion in the uterus [35, 36].lossisuniquelyssocited with infertility nd endometril cncer [35, 37 39], ut the effects of loss hve not een specificlly investigted in regultion of epithelil prolifertion of endometril cncer. In this study, we demonstrte tht Mig-6 is pivotl in the suppression of epithelil prolifertion through its inhiition of AKT ctivtion. Specificlly, we show tht inhiition of endometril tumorigenesis is medited y inhiition of AKT phosphoryltion. Methods Animls nd tretments Mice were mintined for nd used in the designted niml cre fcility ccording to the Michign Stte University institutionl guidelines. All niml procedures were pproved y the Institutionl Animl Cre nd Use Committee of Michign Stte University. Mice were housed in stndrd cges (up to 5 nimls per cge) in rooms with 12 h light/drk cycle, controlled temperture nd humidity under specific pthogen-free conditions. Cmpus Animl Resources t Michign Stte University provides veterinry cre, dily husndry nd helth checks, procurement, nd other dministrtive support for reserch in iomedicl housing fcilities nd ssists with niml helth. Animls re oserved dily y niml cre stff tht hve dditionl trining in lortory niml sciences nd species-specific hndling nd husndry. To generte uterine epithelil specific Mig-6 knockout mice, mice were crossed with Sprr2f cre/+ mice [4]. Control ( ) nd endometril epithelil cell-specific Mig-6 knockout mice (Sprr2f cre/+ ; )[41] were used to investigte the effect of epithelil Mig-6 ltion on the uterus. (eeswx) or (4 mg/pellet) pellets were plced sucutneously into control ( ) nd Sprr2f cre+ mice respectively t 1 weeks of ge for 1 week (n = 6/tretment/genotype). To void ny possiility of pin nd/or distress to the niml, ll surgicl procedures were performed under nesthesi. Mice were nesthetized with isoflurne (3% isoflurne in oxygen y inhltion). All surgeries were conducted in dedicted surgicl suites using septic procedures.

3 Yoo et l. BMC Cncer (218) 18:65 Pge 3 of 1 Recuperting nimls, under close supervision, were kept wrm until full postopertive recovery is chieved. Animls were under nesthetic for mximum of 2 min, nd recovery from surgery normlly occurs within 3 min s evidence y sternl recumency, followed y norml multion, grooming nd feeding. If discomfort is oserved, the nimls were provided Ketoprofen t dose of 5 mg/kg s n nlgesic. At the end of given study, ll mice were humnely euthnized y cervicl disloction under isoflurne nesthesi or y cron dioxide sphyxition nd then the uteri from control nd Sprr2f cre+ mice were collected to investigte the effect of on the development of endometril hyperplsi. Immunohistochemistry nd nlysis Immunohistochemistry nlysis ws performed s previously descried [41]. Briefly, uterine sections were pre-incuted with 1% norml got serum in PBS prior to exposure to nti-pgr (SC-538; Snt Cruz Biotechnology, Dlls, TX), nti-esr1 (SC-543; Snt Cruz Biotechnology, Dlls, TX), nti-akt (CS-4691; Cell Signling, Dnvers, MA), nti- (CS-46; Cell Signling, Dnvers, MA), nd nti-ki67 (BD5569; BD Biosciences, Sn Jose, CA) s pproprite primry ntiodies. Positive signling ws detected with the DAB Peroxidse Sustrte Kit (SK-41; Vector Lortories, Burlingme, CA). The H-score ws clculted s previously reported [42]. The overll H-score rnged from to 3. Cell culture nd trnsient trnsfection Ishikw (99,4,21; Sigm Aldrich, St. Louis, MO) nd HEC1A (HTB-112; ATCC, Mnsss, VA) Cell lines re mintined in Dulecco s modified Egle s medium/ Nutrient Mixture F-12 (DMEM/F12; Gico BRL, Githersurg, MD) with 1% (v/v) fetl ovine serum (FBS; Gico BRL, Githersurg, MD), nd 1% (v/v) penicillin streptomycin (P/S; Gico BRL, Githersurg, MD) t 37 C under 5% CO 2. FLAG-tgging expression vectors were trnsfected using Lipofectmine 2 regent (Invitrogen Crop., Crlsd, CA) ccording to the mnufcturer s instructions. Immunoprecipittion Immunoprecipittion ws performed s descried previously [38]. Briefly, Ishikw nd HEC1A cells were trnsfected with the FLAG- expression vectors. Immunoprecipittion ws performed with Flg ntiody (F184; Sigm Aldrich, St. Louis, MO). Protein interctions were exmined y Western lot nlysis. Western lot nlysis Western lot nlysis ws performed s previously descried [41]. Memrne ws locked with Csein (.5% v/v) prior to exposure to nti-akt (CS-4691; Cell Signling, Dnvers, MA), nti- (CS-46; Cell Signling, Dnvers, MA), nd nti-flg (F184; Sigm-Aldrich, St. Louis, MO) ntiodies. Anti-ctin (SC-1615, Snt Cruz Biotechnology, Dlls, TX) ws used for loding control. Sttisticl nlysis For dt with only two groups, Student s t-test ws used. For dt contining more thn two groups, n nlysis of vrince (ANOVA) test ws used, followed y Tukey or Bonferroni test for pirwise t-tests. All sttisticl nlyses were performed using the Instt pckge from GrphPd (Sn Diego, CA, USA). Results A decrese of stroml PGR nd ESR1 expression in Sprr2f cre+ mice Previously, we reported tht the hyperplstic phenotype of endometril epithelil cell specific Mig-6 knockout (Sprr2f cre+ ; ) mice were oserved t 1 weeks of ge [43]. Endometril cncer displys n imlnce in steroid hormone ction [14 17]. PGR expression hs een shown to e prognostic fctor for endometril cncer [44 46]. Therefore, we first exmined expression of PGR nd ESR1 in mice. Immunohistochemicl nlysis indicted tht levels of PGR nd ESR1 were significntly decresed in the stroml cells of mice compred to control ( ) mice t 1 weeks of ge (n = 6/genotype). However, the expression of PGR nd ESR1 in the epithelium were not chnged in the uteri of mice s compred to control (Fig. 1). These dt suggest tht dysregultion of PGR nd ESR1 expression in the strom my ply n importnt role for the development of endometril hyperplsi. Aerrnt ctivtion of AKT signling in Sprr2f cre+ mice AKT is frequently hyperctivted in humn cncers [47]. To determine if the oserved hyperplstic phenotype ws due to ctivted AKT signling, we exmined the expression of totl AKT, phospho-akt (), nd phospho-s6 (ps6), downstrem mrker of ctive AKT signling in the uteri of control nd mice. First, we exmined cell prolifertion y Ki67 stining (n =6/ genotype). The IHC results reveled significnt increse of uterine epithelil prolifertion in mice (Fig. 2-). Interestingly, we found tht nd ps6 were highly elevted in the epithelil cells of mice t 1 weeks of ge s compred to control mice

4 Yoo et l. BMC Cncer (218) 18:65 Pge 4 of 1 A PGR B 2 PGR strom C ESR1 D 2 ESR1 strom PGR Epithelium 25um ESR1 Epithelium Fig. 1 A decrese of stroml PGR nd ESR1 expression in mice. Immunohistochemicl nlysis for PGR (A) nd ESR1 (C) in control () nd () mice. H-score in strom nd epithelil cells for PGR (B) nd ESR1 (D). The results represent the men ± SEM., p <.1 A Prolifertion c d B % of prolifertive cells Prolifertion * 15 e f ps6 ps6 1 5 Fig. 2 Aerrnt ctivtion of prolifertion nd AKT signling in mice. (A) The expression of Ki67,, nd ps6 in the uteri of (,c,e) nd (, d, nd f) mice. (B) Quntifiction of Ki67 positive cells nd H-score in epithelil cells of nd mice. The results represent the men ± SEM. *, p <.5;, p <.1

5 Yoo et l. BMC Cncer (218) 18:65 Pge 5 of 1 (Fig. 2). However, totl AKT levels were not chnged mong the genotypes (Additionl file 1: Figure S1). These dt suggest tht suppresses endometril epithelil prolifertion vi inhiition of AKT phosphoryltion. The effect of tretment on the development of endometril hyperplsi Exposure to is negtive risk fctor for endometril cncer [48]. Additionlly, it is well known tht endometril cncer is E2-dependent nd tht progestin therpy hs een successful in slowing the growth of endometril tumors in women who re poor surgicl cndidtes nd premenopusl women with complex typicl hyperplsi nd erly-stge endometrioid endometril cncer who hd strong desire to preserve their fertility [22, 23, 49 54]. To ssess the effect of tretment on epithelil ltion of Mig-6, we plced or vehicle pellets into the control nd mice sucutneously t 1 weeks of ge (n = 6/tretment/genotype). After 1 week of the tretment, mice exhiited significntly decresed uterine weight compred to vehicle-treted mice (Fig. 3 nd ). Histologicl nlysis showed tht the development of uterine hyperplsi ws not evident in mice fter tretment (Fig. 3c). tretment lso led to decresed prolifertion in the epithelil cells of mice s compred to vehicle-treted mice (Fig. 3d). These dt suggest tht the hyperplstic phenotype of mice ws responsive to tretment, returning the morphology to norml. The recovery of steroid hormone nd AKT signling y tretment in Sprr2f cre+ mice The expression of PGR nd ESR1 is strongly correlted with the prognosis of endometril cncer [55]. A Uterine/ody weight X 1, week tretment * B c 1cm 1cm d 1cm 1cm C c 1µm d 1µm D % of prolifertive cells % of prolifertive cells Epithelium Veh Strom Fig. 3 Effects of on nd mice. (A) Uterine/ody rtio were significntly decresed in mice compred to vehicletreted Mig-6 KO mice fter tretment. (B) Gross morphology nd (C) Hemtoxylin-eosin stining fter vehicle nd tretment. (D) Immunohistochemicl nlysis nd quntifiction of Ki67 in nd mice. The results represent the men ± SEM. *, p <.5;, p <.1 Veh

6 Yoo et l. BMC Cncer (218) 18:65 Pge 6 of 1 A PGR B PGR strom C ESR1 D ESR1 strom 3 Veh PGR Epithelium 25um 3 Veh ESR1 Epithelium Veh Fig. 4 Recovery of strom PGR nd ESR1 expression in mice fter tretment. Immunohistochemicl nlysis for PGR (A) nd ESR1 (C) in vehicle- () nd -() treted mice. Quntifiction of PGR (B) nd ESR1 (D) y H-score. The results represent the men ± SEM., p < Veh Therefore, we exmined the expression of PGR nd ESR1 using immunohistochemistry (n = 6/tretment). The expression of PGR nd ESR1 were significntly incresed in the strom of mice fter tretment (Fig. 4). These dt indicted tht tretment ctivtes nucler receptors signling t endometril stroml cells of mice. Next, we exmined the expression of totl AKT,, nd ps6 using immunohistochemistry in the uteri of control nd mice fter tretment to investigte whether the suppression of hyperplstic phenotype oserved ws due to recovered AKT signling. Totl AKT levels were not chnged fter tretment (Additionl file 2: FigureS2).However,errntctivtion of AKT signling ws significntly decresed in the uteri of -treted mice s compred to vehicle-treted mice (Fig. 5). These dt suggest tht tretment suppresses errnt ctivtion A B c d 15 Veh ps6 ps6 1 5 Fig. 5 AKT signling is down-regulted fter tretment in mice. (A) Immunohistochemicl nlysis of nd ps6 in vehicle nd -treted mice. (B) Quntifiction of nd ps6 positive cells in epithelil cells of nd mice fter tretment. The results represent the men ± SEM., p <.1 Veh

7 Yoo et l. BMC Cncer (218) 18:65 Pge 7 of 1 of AKT signling in endometril hyperplsi of mice. regultes AKT phosphoryltion dose-dependently nd intercts with AKT In order to exmine effects of on AKT, we performed experiments on endometril cncer cell lines, Ishikw nd HEC1A cells. We trnsfected to Ishikw nd HEC1A cells dose-dependently with FLAG-tgged (FLAG-). Following introduction we exmined levels of AKT nd t 24-h. The levels of AKT phosphoryltion were highly decresed y FLAG- in dose dependent mnner wheres AKT levels were unchnged (Fig. 6). We next exmined whether physiclly intercts with AKT. Ishikw cells were trnsfected with FLAG-, nd the lystes were immunoprecipitted with FLAG ntiody. FLAG immunoprecipittes were then proed with AKT nd specific ntiodies, indicting tht physiclly intercts with AKT (Fig. 6). These results suggest tht inhiits AKT phosphoryltion through protein-protein interction, highlighting its importnt role in the regultion of epithelil prolifertion. Discussion In this study, we evluted whether suppresses endometril epithelil prolifertion vi inhiition of AKT phosphoryltion. plys n inhiitory role on E2 AKT Actin AKT Actin Input Control Ishikw ug Control IgG IP Control AKT Actin HEC1A ug Fig. 6 AKT phosphoryltion were regulted y expression dose-dependently () Western Blot nlysis of,, AKT, nd Actin in FLAG- trnsfected Ishikw nd HEC1A cells. Actin ws used s smple-loding control. () The interction etween nd AKT y immunoprecipittion stimulted prolifertion of uterine epithelil cells [56]. Disruption of steroidl control results in unopposed E2, leding to endometril cncer [17]. Mig-6 is trget of nd PGR, nd its deletion in the uterus leds to enhnced epithelil prolifertion [35]. The mjority of endometril cncers exhiit ctively proliferting epithelil cells nd incresed AKT signling [57 59]. The Cncer Genome Atls nlysis demonstrted n incresed AKT ctivity in endometrioid endometril tumors [25]. Activted AKT signling enhnces cell prolifertion s well s cell survivl through the inhiition of propoptotic proteins [27]. Expression of PGR (PR-A nd PR-B) nd ESRs (ESR1 nd ESR2) hs een reported s prognostic fctors for endometril crcinom [44 46]. We evluted tht stroml PGR nd ESR1 expression ws significntly decresed in the uteri of when compred to control mice (Fig. 1). We showed elevted phosphoryltion of AKT resulting in enhnced epithelil prolifertion (Fig. 2). Stroml PGR nd signling is necessry nd sufficient to medite the ntiprolifertive effects of on E2-induced epithelil cell prolifertion [6, 61]. However, ctivtion of AKT reduces PR-B trnscriptionl ctivity in Ishikw cells nd Pten d/d conditionl mouse model of endometrioid endometril cncer [33]. AKT lso reduces PGR expression levels in rest cncer cells, endometril cncer cells, nd uterine stroml cells ffected y endometriosis [62 64]. However, exctly how signling occurs etween AKT nd resistnce in endometril epithelil nd stroml interction is uncler. Filling this knowledge gp is criticl to understnding resistnce. resistnce is defined y the decresed responsiveness to ioville of trget tissue [65]. Lck of ctivity contriutes significntly to uterine pthophysiology. resistnce is now considered centrl element in women s diseses such s infertility, endometriosis, nd endometril cncer [66 69], ut the mechnism of resistnce in women s diseses remins unknown. We hve demonstrted tht mice exhiiting norml responses nd tretment for 1 week is sufficient to restore endometril hyperplsi to norml (Fig. 3). We treted the mice in the eginning of endometril hyperplsi nd the dt suggest tretment t n erly time point cn e one of the resons to reverse endometril hyperplsi to norml. Therefore, further study on the effects of tretment on endometril turmorigenesis ssocited with its development nd progression re required. Determining the moleculr mechnisms y which steroid hormones control the physiology oftheuterusisofutmost importnce to understnding nd overcoming resistnce. However, resistnce to tretment hs led to limiting the use of therpy in endometril cncer due to its low response rtes [19 23]. The optiml method for

8 Yoo et l. BMC Cncer (218) 18:65 Pge 8 of 1 treting nd surveilling ptients with conservtively treted endometril cncer is not known. Therefore, the identifiction of the moleculr pthwys tht link resistnce to endometril cncer development cn potentilly provide novel trgets for the prevention or tretment of this mlignncy. We showed tht AKT signling is down-regulted fter tretment in mice (Fig. 5). These dt suggest tht tretment with n AKT inhiitor could e vile lterntive for overcoming the -resistnt endometril hyperplsi nd cncer. We found tht decresed AKT phosphoryltion in Ishikw nd HEC1A cell lines in dose-dependent mnner. Immunoprecipittion showed tht there is protein interction etween nd AKT, suggesting tht suppresses E2-induced epithelil cell prolifertion through AKT interctions (Fig. 6). However, the exct moleculr mechnism y which interction regultes the phosphoryltion of AKT is not cler. Further studies will e required to determine exct moleculr mechnism. We hve shown the prevention effect of with mice [43]. We treted mice with efore developing endometril hyperplsi nd found tht prevented the development of endometril hyperplsi y inhiiting epithelil STAT3 phosphoryltion, resulting in decrese of epithelil prolifertion. The moleculr mechnisms in the regultion of epithelil prolifertion y AKT nd STAT3 s well s steroid hormone signling remins to e further studied during endometril tumorigenesis. Our dt support tht the ctivtion of stroml signling y tretment cn contriute to the development of endometril hyperplsi nd the cross-tlk etween AKT/STAT3 nd PGR/ESR1 is criticl to inhiit the endometril hyperplsi. Conclusions Overll, our study suggests tht the negtive regultion of AKT phosphoryltion y ctivted strom signling including Mig-6 hs n importnt role in the regultion of epithelil cell prolifertion during endometril hyperplsi development nd progression. Our results contriute to the understnding of the etiologicl nd moleculr mechnisms of epithelil cell prolifertion nd to the development of new therpeutic pproches for treting endometril hyperplsi nd cncer. Additionl files Additionl file 1: Figure S1 Totl AKT level is not chnged in nd mice. (A) The expression of AKT in the uteri of () nd () mice nd (B) H-score of AKT in the uteri of nd mice. (PPTX 251 k) Additionl file 2: Figure S2 Totl AKT level is not chnged in mice fter tretment. (A) The expression of AKT in the uteri of vehicle () nd () treted mice nd (B) H-score of AKT in the uteri of vehicle nd treted mice. (PPTX 47 k) Arevitions ESR1: Estrogen receptor α; ESR2: Estrogen receptor β; PGR: Progesterone receptor; PI3K: Phosphoinositide 3-kinse; PTEN: Phosphtse nd tensin homolog; S6: Riosoml protein S6 kinse Acknowledgements The Sprr2f-cre mice were cquired from Dr. Diego H. Cstrillon (University of Texs Southwestern Medicl Center, Dlls, TX). We would like to thnk Ryn M. Mrqurdt for mnuscript preprtion. Funding Grnt numers nd sources of support: The design, dt collection, dt nlysis, nd dt interprettion of this study ws supported y Grnt Numer P5CA98258 from the Ntionl Cncer Institute (to R. R. Broddus nd T.H. Kim). The nlysis nd interprettion of in vitro experiments nd writing support of this mnuscript were supported y the Ntionl Reserch Foundtion of Kore (NRF) grnt funded y the Ministry of Eduction, Science nd Technology (No. NRF-216R1D1A1B , to J.Y. Yoo), nd NRF grnt (No. NRF-217R1A2B47971, K.-C. Choi). Avilility of dt nd mterils The dtsets supporting the conclusions of this rticle re included within the rticle. Authors contriutions JYY, nd HBK conceived nd designed the experimentl pproch, performed experiments nd prepred the mnuscript. JIR nlyzed the results. RRB provided pthologicl nlysis. KCC nd THK conceived nd designed the experimentl pproch, performed dt nlysis nd prepred the mnuscript. All uthors hve red nd pproved the finl version of mnuscript. Ethics pprovl All niml procedures were pproved y the Institutionl Animl Cre nd Use Committee of Michign Stte University (Appliction #: 11/ ). Competing interests The uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Deprtment of Ostetrics, Gynecology nd Reproductive Biology, College of Humn Medicine, Michign Stte University, Grnd Rpids, MI 4953, USA. 2 Deprtment of Biochemistry nd Moleculr Biology, Yonsei University College of Medicine, Seoul 3722, South Kore. 3 Deprtment of Biomedicl Sciences, ASAN Medicl Center, University of Ulsn College of Medicine, Seoul 555, South Kore. 4 Deprtment of Pthology, University of Texs M.D. Anderson Cncer Center, Houston, Texs TX 773, USA. 5 Deprtment of Phrmcology, Asn Institute for Life Sciences, Asn Medicl Center, University of Ulsn College of Medicine, Seoul 555, South Kore. Received: 4 Decemer 217 Accepted: 11 My 218 References 1. Siegel RL, Miller KD, Jeml A. Cncer sttistics, 216. CA Cncer J Clin. 216;66(1): Byun JM, Jeong DH, Kim YN, Cho EB, Ch JE, Sung MS, Lee KB, Kim KT. Endometril cncer rising from typicl complex hyperplsi: the significnce in n endometril iopsy nd dignostic chllenge. 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