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

MIG-6 suppresses endometrial epithelial cell proliferation by inhibiting phospho-AKT Background: Aberrant hyperactivation of epithelial proliferation, AKT signaling, and association with unopposed estrogen (E2) exposure is the most common endometrial cancer dysfunction. In the normal uterus, progesterone (P4) inhibits proliferation by coordinating stromal-epithelial cross-talk, which we previously showed is mediated by the function of Mitogen-inducible gene 6 (Mig-6). Despite their attractive characteristics, non-surgical conservative therapies based on progesterone alone have not been universally successful. One barrier to this success has been the lack of understanding of the P4 effect on endometrial cells. Method: To further understand the role of Mig-6 and P4 in controlling uterine proliferation, we developed a Sprr2f-cre cre+ f/f driven mouse model where Mig-6 is specifically ablated only in the epithelial cells of the uterus (Sprr2f Mig-6 ). We examined P4 effect and regulation of AKT signaling in the endometrium of mutant mice. cre+ f/f Results: Sprr2f Mig-6 mice developed endometrial hyperplasia. P4 treatment abated the development of endometrial hyperplasia and restored morphological and histological characteristics of the uterus. P4 treatment reduced cell proliferation which was accompanied by decreased AKT signaling and the restoration of stromal PGR and ESR1 expression. Furthermore, our in vitro studies revealed an inhibitory effect of MIG-6 on AKT phosphorylation as well as MIG-6 and AKT protein interactions. Conclusions: These data suggest that endometrial epithelial cell proliferation is regulated by P4 mediated Mig-6 inhibition of AKT phosphorylation, uncovering new mechanisms of P4 action. This information may help guide more effective non-surgical interventions in the future. Keywords: MIG-6, Progesterone resistance, Endometrial hyperplasia, AKT Background endometrial cancer diagnoses are in post-menopausal Endometrial cancer is the most common gynecologic women, 5% of cases are diagnosed before age 40 and malignancy in the United States, and in the last several 20~ 25% before menopause [7]. Moreover, the incidence decades the incidence of new cases each year has in- of endometrial cancer diagnoses in younger patients is creased [1]. Endometrioid endometrial cancer, the most likely to increase going forward due to increases in obes- common type of endometrial cancer (80–85%), is associ- ity, hypertension, diabetes mellitus, and other known ated with or preceded by abnormal multiplication of endometrial cancer risk factors [8–10]. Therefore, the endometrial epithelial cells, known as complex atypical demand for non-surgical approaches to endometrial can- hyperplasia [2–4]. The main treatment for endometrial cer is increasing, especially for women of reproductive cancer is hysterectomy [5, 6]. Although most age with complex atypical hyperplasia and early-stage endometrioid endometrial cancer who wish to preserve their fertility beyond treatment [8, 10]. * Correspondence: choikc75@amc.seoul.kr; TaeHoon.Kim@hc.msu.edu Although hysterectomy is a key therapy for endomet- Jung-Yoon Yoo and Hee-Bum Kang contributed equally to this work. Department of Biomedical Sciences, ASAN Medical Center, University of rial cancer [5, 6], recent intrauterine progestin therapies Ulsan College of Medicine, Seoul 05505, South Korea such as a levonorgestrel-releasing intrauterine system Department of Obstetrics, Gynecology and Reproductive Biology, College of have been used for reproductive-aged women with Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoo et al. BMC Cancer (2018) 18:605 Page 2 of 10 complex atypical hyperplasia and early-stage endometrial [30, 31]. Inappropriately elevated expression of AKT cancer in cases when there is a desire to preserve fertility phosphorylation is related to poor prognosis of endo- or when comorbidities exclude the possibility of surgery. metrial cancer patients [32]. Furthermore, inhibition of In addition, progestin therapy is also considered for re- the AKT pathway combined with P4 decreases angio- current endometrial cancer because it is less toxic than genesis and proliferation in vivo, indicating that regula- chemotherapies; however, though the response rate of tion of the AKT pathway may play an important endometrial hyperplasia to progestin treatment is higher therapeutic role [33]. than that of endometrial adenocarcinoma, the response Mitogen-inducible gene 6 (MIG-6) functions to sup- to progestin in cancer recurrence is worst of all. Proges- press endometrial cancer in the human and mouse tin therapies used in the clinic are effective for some pa- uterus [34, 35]. Mig-6 is an important mediator of P4 tients but not all cases of endometrial hyperplasia and signaling in that it inhibits E2-mediated epithelial prolif- well-differentiated endometrioid endometrial cancer. eration in the uterus [35, 36]. MIG-6lossisuniquelyasso- Another major limitation of progestin therapy is the lack ciated with infertility and endometrial cancer [35, 37–39], of a clinical standard protocol for the type, dose, and but the effects of MIG-6 loss have not been specifically in- duration of treatment [11–13]. The molecular mecha- vestigated in regulation of epithelial proliferation of endo- nisms underlying progesterone (P4) resistance in endo- metrial cancer. In this study, we demonstrate that Mig-6 is metrial cancer have not been fully understood. pivotal in the suppression of epithelial proliferation through Loss of control over uterine epithelial cell proliferation its inhibition of AKT activation. Specifically, we show that and apoptosis by ovarian steroid hormones is the major P4 inhibition of endometrial tumorigenesis is mediated by underlying pathogenesis of endometrial cancer [14–17]. MIG-6 inhibition of AKT phosphorylation. Progesterone therapy can prevent this process by block- ing actions of unopposed estrogen (E2) [18]. Nonethe- Methods less, several studies indicate that P4 therapy has low and Animals and treatments unpredictable response rates in women with endometrial Mice were maintained for and used in the designated cancer, therefore limiting its potential use [19–23]. Re- animal care facility according to the Michigan State Uni- sistance to P4 treatment due to loss of either progester- versity institutional guidelines. All animal procedures one receptor (PGR) itself or its signaling pathways were approved by the Institutional Animal Care and Use causes significant difficulty in the treatment of advanced Committee of Michigan State University. Mice were and recurrent endometrial cancer [24]. Identifying mo- housed in standard cages (up to 5 animals per cage) in lecular mechanisms involved in P4 resistance is critical rooms with 12 h light/dark cycle, controlled temperature to effective and personalized treatment. Unfortunately, and humidity under specific pathogen-free conditions. further translational research of endometrial cancer is Campus Animal Resources at Michigan State University inhibited by the lack of sufficient pre-clinical animal provides veterinary care, daily husbandry and health models. checks, procurement, and other administrative support Sequencing analysis of endometrial cancers in the for research in biomedical housing facilities and assists Cancer Genome Atlas has revealed that upwards of 90% with animal health. Animals are observed daily by ani- of cases of endometrioid endometrial cancer have some mal care staff that have additional training in laboratory genetic aberration in the PTEN/PI3K pathway, which re- animal sciences and species-specific handling and sults in increased AKT activity [25]. In addition, the husbandry. AKT signaling pathway can be activated by E2 [26]en- To generate uterine epithelial specific Mig-6 knockout f/f cre/+ hancing cell proliferation [27]. Therefore, an under- mice, Mig-6 mice were crossed with Sprr2f mice f/f standing of the molecular mechanisms between steroid [40]. Control (Mig-6 ) and endometrial epithelial cre/+ f/f hormone and PTEN/PI3K/AKT signaling will allow us cell-specific Mig-6 knockout mice (Sprr2f Mig-6 ; d/d to be in a much better position to develop new conser- Mig-6 )[41] were used to investigate the effect of epi- vative therapies based on P4 function. thelial Mig-6 ablation on the uterus. The protein structure of AKT consists of a PH do- Vehicle (beeswax) or P4 (40 mg/pellet) pellets were f/f main, a linker region, a kinase domain, and a regulator placed subcutaneously into control (Mig-6 ) and cre+ f/f domain [28]. These domains undergo various protein Sprr2f Mig-6 mice respectively at 10 weeks of age modifications including phosphorylation, acetylation, for 1 week (n = 6/treatment/genotype). To avoid any ubiquitylation, methylation, hydroxylation, glycosylation, possibility of pain and/or distress to the animal, all surgi- and SUMOylation which help regulate the proteins ac- cal procedures were performed under anesthesia. Mice tivity [29]. AKT regulates different pathways that aid in were anesthetized with isoflurane (3% isoflurane in oxy- the promotion of cellular survival and inhibition of gen by inhalation). All surgeries were conducted in dedi- apoptosis through its serine/threonine kinase activity cated surgical suites using aseptic procedures. Yoo et al. BMC Cancer (2018) 18:605 Page 3 of 10 Recuperating animals, under close supervision, were Western blot analysis kept warm until full postoperative recovery is achieved. Western blot analysis was performed as previously de- Animals were under anesthetic for a maximum of scribed [41]. Membrane was blocked with Casein (0.5% 20 min, and recovery from surgery normally occurs v/v) prior to exposure to anti-AKT (CS-4691; Cell Sig- within 30 min as evidence by sternal recumbency, naling, Danvers, MA), anti-pAKT (CS-4060; Cell Signal- followed by normal ambulation, grooming and feeding. ing, Danvers, MA), and anti-Flag (F1804; Sigma-Aldrich, If discomfort is observed, the animals were provided St. Louis, MO) antibodies. Anti-actin (SC-1615, Santa Ketoprofen at a dose of 5 mg/kg as an analgesic. At the Cruz Biotechnology, Dallas, TX) was used for loading end of a given study, all mice were humanely euthanized control. by cervical dislocation under isoflurane anesthesia or by carbon dioxide asphyxiation and then the uteri from Statistical analysis cre+ f/f control and Sprr2f Mig-6 mice were collected to in- For data with only two groups, Student’s t-test was used. vestigate the effect of P4 on the development of endo- For data containing more than two groups, an analysis metrial hyperplasia. of variance (ANOVA) test was used, followed by Tukey or Bonferroni test for pairwise t-tests. All statistical ana- lyses were performed using the Instat package from Immunohistochemistry and analysis GraphPad (San Diego, CA, USA). Immunohistochemistry analysis was performed as previ- ously described [41]. Briefly, uterine sections were Results pre-incubated with 10% normal goat serum in PBS prior A decrease of stromal PGR and ESR1 expression in cre+ f/f to exposure to anti-PGR (SC-538; Santa Cruz Biotech- Sprr2f Mig-6 mice nology, Dallas, TX), anti-ESR1 (SC-543; Santa Cruz Bio- Previously, we reported that the hyperplastic phenotype technology, Dallas, TX), anti-AKT (CS-4691; Cell of endometrial epithelial cell specific Mig-6 knockout cre+ f/f d/d Signaling, Danvers, MA), anti-pAKT (CS-4060; Cell Sig- (Sprr2f Mig-6 ; Mig-6 ) mice were observed at naling, Danvers, MA), and anti-Ki67 (BD5506090; BD 10 weeks of age [43]. Endometrial cancer displays an im- Biosciences, San Jose, CA) as appropriate primary anti- balance in steroid hormone action [14–17]. PGR expres- bodies. Positive signaling was detected with the DAB sion has been shown to be a prognostic factor for Peroxidase Substrate Kit (SK-4100; Vector Laboratories, endometrial cancer [44–46]. Therefore, we first exam- d/d Burlingame, CA). The H-score was calculated as previ- ined expression of PGR and ESR1 in Mig-6 mice. Im- ously reported [42]. The overall H-score ranged from 0 munohistochemical analysis indicated that levels of PGR to 300. and ESR1 were significantly decreased in the stromal d/d f/f cells of Mig-6 mice compared to control (Mig-6 ) mice at 10 weeks of age (n = 6/genotype). However, the Cell culture and transient transfection expression of PGR and ESR1 in the epithelium were not d/d Ishikawa (99,040,201; Sigma–Aldrich, St. Louis, MO) changed in the uteri of Mig-6 mice as compared to and HEC1A (HTB-112; ATCC, Manassas, VA) Cell lines control (Fig. 1). These data suggest that dysregulation of are maintained in Dulbecco’s modified Eagle’s medium/ PGR and ESR1 expression in the stroma may play an Nutrient Mixture F-12 (DMEM/F12; Gibco BRL, Gai- important role for the development of endometrial thersburg, MD) with 10% (v/v) fetal bovine serum (FBS; hyperplasia. Gibco BRL, Gaithersburg, MD), and 1% (v/v) penicillin cre+ f/f streptomycin (P/S; Gibco BRL, Gaithersburg, MD) at Aberrant activation of AKT signaling in Sprr2f Mig-6 37 °C under 5% CO . FLAG-tagging MIG-6 expression mice vectors were transfected using Lipofectamine 2000 re- AKT is frequently hyperactivated in human cancers [47]. agent (Invitrogen Crop., Carlsbad, CA) according to the To determine if the observed hyperplastic phenotype manufacturer’s instructions. was due to activated AKT signaling, we examined the expression of total AKT, phospho-AKT (pAKT), and phospho-S6 (pS6), a downstream marker of active AKT d/d Immunoprecipitation signaling in the uteri of control and Mig-6 mice. First, Immunoprecipitation was performed as described previ- we examined cell proliferation by Ki67 staining (n =6/ ously [38]. Briefly, Ishikawa and HEC1A cells were trans- genotype). The IHC results revealed a significant in- d/d fected with the FLAG-MIG-6 expression vectors. crease of uterine epithelial proliferation in Mig-6 mice Immunoprecipitation was performed with Flag antibody (Fig. 2a-b). Interestingly, we found that pAKT and pS6 d/d (F1804; Sigma–Aldrich, St. Louis, MO). Protein interac- were highly elevated in the epithelial cells of Mig-6 tions were examined by Western blot analysis. mice at 10 weeks of age as compared to control mice Yoo et al. BMC Cancer (2018) 18:605 Page 4 of 10 PGR stroma ESR1 stroma AC PGRBD ESR1 *** *** 200 200 a a 150 150 100 100 50 50 25µm 25um 0 0 f/f d/d f/f d/d Mig-6 Mig-6 Mig-6 Mig-6 PGR Epithelium ESR1 Epithelium b b 300 300 240 240 180 180 120 120 60 60 25µm 25µm 0 0 f/f d/d f/f d/d Mig-6 Mig-6 Mig-6 Mig-6 d/d Fig. 1 A decrease of stromal PGR and ESR1 expression in Mig-6 mice. Immunohistochemical analysis for PGR (A) and ESR1 (C) in control (a) and d/d Mig-6 (b) mice. H-score in stroma and epithelial cells for PGR (B) and ESR1 (D). The results represent the mean ± SEM. ***, p < 0.001 f/f d/d Proliferation Mig-6 Mig-6 25µm 25µm f/f d/d Mig-6 Mig-6 pAKT *** 25µm 25µm f/f d/d Mig-6 Mig-6 f pS6 *** 25µm 25µm f/f d/d Mig-6 Mig-6 d/d f/f Fig. 2 Aberrant activation of proliferation and AKT signaling in Mig-6 mice. (A) The expression of Ki67, pAKT, and pS6 in the uteri of Mig-6 d/d f/f d/d (a,c,e) and Mig-6 (b, d, and f) mice. (B) Quantification of Ki67 positive cells and H-score in epithelial cells of Mig-6 and Mig-6 mice. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001 d/d f/f Mig-6 Mig-6 pS6 pAKT Proliferation H-Score H-Score d/d f/f Mig-6 Mig-6 H-Score H-Score % of proliferative cells H-Score H-Score Yoo et al. BMC Cancer (2018) 18:605 Page 5 of 10 d/d (Fig. 2). However, total AKT levels were not changed pellets into the control and Mig-6 mice subcutane- among the genotypes (Additional file 1: Figure S1). ously at 10 weeks of age (n = 6/treatment/genotype). d/d These data suggest that MIG-6 suppresses endometrial After 1 week of the P4 treatment, Mig-6 mice exhib- epithelial proliferation via inhibition of AKT ited a significantly decreased uterine weight compared d/d phosphorylation. to vehicle-treated Mig-6 mice (Fig. 3a and b). Histo- logical analysis showed that the development of uterine d/d The effect of P4 treatment on the development of hyperplasia was not evident in Mig-6 mice after P4 endometrial hyperplasia treatment (Fig. 3c). P4 treatment also led to decreased d/d Exposure to P4 is a negative risk factor for endometrial proliferation in the epithelial cells of Mig-6 mice as d/d cancer [48]. Additionally, it is well known that endomet- compared to vehicle-treated Mig-6 mice (Fig. 3d). rial cancer is E2-dependent and that progestin therapy These data suggest that the hyperplastic phenotype of d/d has been successful in slowing the growth of endomet- Mig-6 mice was responsive to P4 treatment, returning rial tumors in women who are poor surgical candidates the morphology to normal. and premenopausal women with complex atypical hyperplasia and early-stage endometrioid endometrial The recovery of steroid hormone and AKT signaling by P4 cre+ f/f cancer who had a strong desire to preserve their fertility treatment in Sprr2f Mig-6 mice [22, 23, 49–54]. To assess the effect of P4 treatment on The expression of PGR and ESR1 is strongly correlated epithelial ablation of Mig-6, we placed P4 or vehicle with the prognosis of endometrial cancer [55]. f/f d/d Mig-6 Mig-6 1 week treatment B 10 a b f/f Mig-6 d/d Mig-6 1cm 1cm Vehicle P4 1cm 1cm C D d/d Vehicle P4 Mig-6 Epithelium a b 100 *** 100µm 100µm 25µm Veh P4 d b Stroma 25µm 25µm 25µm Veh P4 f/f d/d d/d Fig. 3 Effects of P4 on Mig-6 and Mig-6 mice. (A) Uterine/body ratio were significantly decreased in Mig-6 mice compared to vehicle- KO treated Mig-6 mice after P4 treatment. (B) Gross morphology and (C) Hematoxylin-eosin staining after vehicle and P4 treatment. (D) f/f d/d Immunohistochemical analysis and quantification of Ki67 in Mig-6 and Mig-6 mice. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001 Uterine/body weight X 1,000 P4 Vehicle P4 Vehicle % of proliferative cells % of proliferative cells Yoo et al. BMC Cancer (2018) 18:605 Page 6 of 10 PGR stroma ESR1 stroma AC PGRBD ESR1 *** *** 25µm 25um Veh P4 Veh P4 PGR Epithelium ESR1 Epithelium b b 300 300 240 240 180 180 120 120 60 60 25µm 25µm 0 0 Veh P4 Veh P4 d/d Fig. 4 Recovery of stroma PGR and ESR1 expression in Mig-6 mice after P4 treatment. Immunohistochemical analysis for PGR (A) and ESR1 (C) d/d in vehicle- (a) and P4-(b) treated Mig-6 mice. Quantification of PGR (B) and ESR1 (D) by H-score. The results represent the mean ± SEM. ***, p < 0.001 d/d Therefore, we examined the expression of PGR and control and Mig-6 mice after P4 treatment to investi- ESR1 using immunohistochemistry (n = 6/treatment). gate whether the suppression of hyperplastic phenotype The expression of PGR and ESR1 were significantly in- observed was due to recovered AKT signaling. Total d/d creased in the stroma of Mig-6 mice after P4 treat- AKT levels were not changed after P4 treatment ment (Fig. 4). These data indicated that P4 treatment (Additional file 2:FigureS2).However, aberrantacti- activates nuclear receptors signaling at endometrial stro- vation of AKT signaling was significantly decreased in d/d d/d mal cells of Mig-6 mice. the uteri of P4-treated Mig-6 mice as compared to d/d Next, we examined the expression of total AKT, pAKT, vehicle-treated Mig-6 mice (Fig. 5). These data sug- and pS6 using immunohistochemistry in the uteri of gest that P4 treatment suppresses aberrant activation Vehicle P4 AB pAKT a 150 *** 25µm 25µm Veh P4 pS6 *** 25µm 25µm Veh P4 d/d Fig. 5 AKT signaling is down-regulated after P4 treatment in Mig-6 mice. (A) Immunohistochemical analysis of pAKT and pS6 in vehicle and d/d f/f d/d P4-treated Mig-6 mice. (B) Quantification of pAKT and pS6 positive cells in epithelial cells of Mig-6 and Mig-6 mice after P4 treatment. The results represent the mean ± SEM. ***, p < 0.001 P4 Vehicle pS6 pAKT H- H-Score Score H-Score P4 Vehicle H-Score H-Score H-Score H-Score Yoo et al. BMC Cancer (2018) 18:605 Page 7 of 10 of AKT signaling in endometrial hyperplasia of stimulated proliferation of uterine epithelial cells [56]. d/d Mig-6 mice. Disruption of steroidal control results in unopposed E2, leading to endometrial cancer [17]. Mig-6 is a target of MIG-6 regulates AKT phosphorylation dose-dependently P4 and PGR, and its deletion in the uterus leads to en- and interacts with AKT hanced epithelial proliferation [35]. The majority of In order to examine effects of MIG-6 on AKT, we per- endometrial cancers exhibit actively proliferating epithe- formed experiments on endometrial cancer cell lines, lial cells and increased AKT signaling [57–59]. The Can- Ishikawa and HEC1A cells. We transfected to Ishikawa cer Genome Atlas analysis demonstrated an increased and HEC1A cells dose-dependently with FLAG-tagged AKT activity in endometrioid endometrial tumors [25]. MIG-6 (FLAG-MIG-6). Following MIG-6 introduction Activated AKT signaling enhances cell proliferation as we examined levels of AKT and pAKT at 24-h. The well as cell survival through the inhibition of proapopto- levels of AKT phosphorylation were highly decreased by tic proteins [27]. Expression of PGR (PR-A and PR-B) FLAG-MIG-6 in a dose dependent manner whereas and ESRs (ESR1 and ESR2) has been reported as prog- AKT levels were unchanged (Fig. 6a). We next examined nostic factors for endometrial carcinoma [44–46]. We whether MIG-6 physically interacts with AKT. Ishikawa evaluated that stromal PGR and ESR1 expression was d/d cells were transfected with FLAG-MIG-6, and the lysates significantly decreased in the uteri of Mig-6 when were immunoprecipitated with FLAG antibody. FLAG compared to control mice (Fig. 1). We showed elevated immunoprecipitates were then probed with AKT and phosphorylation of AKT resulting in enhanced epithelial MIG-6 specific antibodies, indicating that MIG-6 physic- proliferation (Fig. 2). Stromal PGR and P4 signaling is ally interacts with AKT (Fig. 6b). These results suggest necessary and sufficient to mediate the antiproliferative that MIG-6 inhibits AKT phosphorylation through a effects of P4 on E2-induced epithelial cell proliferation protein-protein interaction, highlighting its important [60, 61]. However, activation of AKT reduces PR-B tran- d/d role in the regulation of epithelial proliferation. scriptional activity in Ishikawa cells and Pten condi- tional mouse model of endometrioid endometrial cancer Discussion [33]. AKT also reduces PGR expression levels in breast In this study, we evaluated whether MIG-6 suppresses cancer cells, endometrial cancer cells, and uterine stro- endometrial epithelial proliferation via inhibition of AKT mal cells affected by endometriosis [62–64]. However, phosphorylation. P4 plays an inhibitory role on E2 exactly how signaling occurs between AKT and P4 re- sistance in endometrial epithelial and stromal interaction is unclear. Filling this knowledge gap is critical to under- standing P4 resistance. Ishikawa HEC1A P4 resistance is defined by the decreased responsive- MIG-6 MIG-6 ness to bioavailable P4 of target tissue [65]. Lack of P4 00.1 0.5 1 2 ug 00.1 0.5 1 2 ug activity contributes significantly to uterine pathophysi- MIG-6 MIG-6 ology. P4 resistance is now considered a central element pAKT pAKT in women’s diseases such as infertility, endometriosis, and endometrial cancer [66–69], but the mechanism of AKT AKT P4 resistance in women’s diseases remains unknown. We d/d Actin Actin have demonstrated that Mig-6 mice exhibiting normal P4 responses and P4 treatment for 1 week is sufficient to restore endometrial hyperplasia to normal (Fig. 3). IP We treated the mice in the beginning of endometrial Input IgG MIG-6 hyperplasia and the data suggest P4 treatment at an early time point can be one of the reasons to reverse endometrial hyperplasia to normal. Therefore, further study on the effects of P4 treatment on endometrial tur- AKT morigenesis associated with its development and pro- MIG-6 gression are required. Actin Determining the molecular mechanisms by which steroid hormones control the physiology of theuterusisofutmost Fig. 6 AKT phosphorylation were regulated by MIG-6 expression dose-dependently (a) Western Blot analysis of MIG-6, pAKT, AKT, and importance to understanding and overcoming P4 resist- Actin in FLAG-MIG-6 transfected Ishikawa and HEC1A cells. Actin was ance. However, resistance to P4 treatment has led to limit- used as sample-loading control. (b) The interaction between MIG-6 ing the use of P4 therapy in endometrial cancer due to its and AKT by immunoprecipitation low response rates [19–23]. The optimal method for Control MIG-6 Control MIG-6 Control MIG-6 Yoo et al. BMC Cancer (2018) 18:605 Page 8 of 10 treating and surveilling patients with conservatively treated Abbreviations ESR1: Estrogen receptor α; ESR2: Estrogen receptor β; PGR: Progesterone endometrial cancer is not known. Therefore, the identifica- receptor; PI3K: Phosphoinositide 3-kinase; PTEN: Phosphatase and tensin tion of the molecular pathways that link P4 resistance to homolog; S6: Ribosomal protein S6 kinase endometrial cancer development can potentially provide Acknowledgements novel targets for the prevention or treatment of this malig- The Sprr2f-cre mice were acquired from Dr. Diego H. Castrillon (University of nancy. We showed that AKT signaling is down-regulated Texas Southwestern Medical Center, Dallas, TX). We would like to thank Ryan d/d after P4 treatment in Mig-6 mice (Fig. 5). These data sug- M. Marquardt for manuscript preparation. gest that treatment with an AKT inhibitor could be a viable Funding alternative for overcoming the P4-resistant endometrial Grant numbers and sources of support: The design, data collection, data hyperplasia and cancer. analysis, and data interpretation of this study was supported by Grant Number P50CA098258 from the National Cancer Institute (to R. R. Broaddus We found that MIG-6 decreased AKT phosphorylation and T.H. Kim). The analysis and interpretation of in vitro experiments and in Ishikawa and HEC1A cell lines in a dose-dependent writing support of this manuscript were supported by the National Research manner. Immunoprecipitation showed that there is protein Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (No. NRF-2016R1D1A1B03934346, to J.Y. Yoo), and interaction between MIG-6 and AKT, suggesting that NRF grant (No. NRF-2017R1A2B4007971, K.-C. Choi). MIG-6 suppresses E2-induced epithelial cell proliferation through AKT interactions (Fig. 6). However, the exact mo- Availability of data and materials lecular mechanism by which interaction regulates the phos- The datasets supporting the conclusions of this article are included within the article. phorylation of AKT is not clear. Further studies will be required to determine exact molecular mechanism. Authors’ contributions We have shown the prevention effect of P4 with JYY, and HBK conceived and designed the experimental approach, d/d d/d performed experiments and prepared the manuscript. JIR analyzed the Mig-6 mice [43]. We treated Mig-6 mice with P4 results. RRB provided pathological analysis. KCC and THK conceived and before developing endometrial hyperplasia and found designed the experimental approach, performed data analysis and prepared that P4 prevented the development of endometrial the manuscript. All authors have read and approved the final version of manuscript. hyperplasia by inhibiting epithelial STAT3 phosphoryl- ation, resulting in a decrease of epithelial proliferation. Ethics approval The molecular mechanisms in the regulation of epithe- All animal procedures were approved by the Institutional Animal Care and lial proliferation by AKT and STAT3 as well as steroid Use Committee of Michigan State University (Application #: 11/16–192-00). hormone signaling remains to be further studied during Competing interests endometrial tumorigenesis. Our data support that the The authors declare that they have no competing interests. activation of stromal signaling by P4 treatment can con- tribute to the development of endometrial hyperplasia Publisher’sNote and the cross-talk between AKT/STAT3 and PGR/ESR1 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. is critical to inhibit the endometrial hyperplasia. Author details Conclusions Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA. Overall, our study suggests that the negative regulation of Department of Biochemistry and Molecular Biology, Yonsei University AKT phosphorylation by activated stroma signaling in- College of Medicine, Seoul 03722, South Korea. Department of Biomedical cluding Mig-6 has an important role in the regulation of Sciences, ASAN Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea. Department of Pathology, University of Texas epithelial cell proliferation during endometrial hyperplasia M.D. Anderson Cancer Center, Houston, Texas TX 77030, USA. Department development and progression. Our results contribute to of Pharmacology, Asan Institute for Life Sciences, Asan Medical Center, the understanding of the etiological and molecular mecha- University of Ulsan College of Medicine, Seoul 05505, South Korea. nisms of epithelial cell proliferation and to the develop- Received: 4 December 2017 Accepted: 11 May 2018 ment of new therapeutic approaches for treating endometrial hyperplasia and cancer. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. Additional files 2016;66(1):7–30. 2. 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MIG-6 suppresses endometrial epithelial cell proliferation by inhibiting phospho-AKT

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Abstract

Background: Aberrant hyperactivation of epithelial proliferation, AKT signaling, and association with unopposed estrogen (E2) exposure is the most common endometrial cancer dysfunction. In the normal uterus, progesterone (P4) inhibits proliferation by coordinating stromal-epithelial cross-talk, which we previously showed is mediated by the function of Mitogen-inducible gene 6 (Mig-6). Despite their attractive characteristics, non-surgical conservative therapies based on progesterone alone have not been universally successful. One barrier to this success has been the lack of understanding of the P4 effect on endometrial cells. Method: To further understand the role of Mig-6 and P4 in controlling uterine proliferation, we developed a Sprr2f-cre cre+ f/f driven mouse model where Mig-6 is specifically ablated only in the epithelial cells of the uterus (Sprr2f Mig-6 ). We examined P4 effect and regulation of AKT signaling in the endometrium of mutant mice. cre+ f/f Results: Sprr2f Mig-6 mice developed endometrial hyperplasia. P4 treatment abated the development of endometrial hyperplasia and restored morphological and histological characteristics of the uterus. P4 treatment reduced cell proliferation which was accompanied by decreased AKT signaling and the restoration of stromal PGR and ESR1 expression. Furthermore, our in vitro studies revealed an inhibitory effect of MIG-6 on AKT phosphorylation as well as MIG-6 and AKT protein interactions. Conclusions: These data suggest that endometrial epithelial cell proliferation is regulated by P4 mediated Mig-6 inhibition of AKT phosphorylation, uncovering new mechanisms of P4 action. This information may help guide more effective non-surgical interventions in the future. Keywords: MIG-6, Progesterone resistance, Endometrial hyperplasia, AKT Background endometrial cancer diagnoses are in post-menopausal Endometrial cancer is the most common gynecologic women, 5% of cases are diagnosed before age 40 and malignancy in the United States, and in the last several 20~ 25% before menopause [7]. Moreover, the incidence decades the incidence of new cases each year has in- of endometrial cancer diagnoses in younger patients is creased [1]. Endometrioid endometrial cancer, the most likely to increase going forward due to increases in obes- common type of endometrial cancer (80–85%), is associ- ity, hypertension, diabetes mellitus, and other known ated with or preceded by abnormal multiplication of endometrial cancer risk factors [8–10]. Therefore, the endometrial epithelial cells, known as complex atypical demand for non-surgical approaches to endometrial can- hyperplasia [2–4]. The main treatment for endometrial cer is increasing, especially for women of reproductive cancer is hysterectomy [5, 6]. Although most age with complex atypical hyperplasia and early-stage endometrioid endometrial cancer who wish to preserve their fertility beyond treatment [8, 10]. * Correspondence: choikc75@amc.seoul.kr; TaeHoon.Kim@hc.msu.edu Although hysterectomy is a key therapy for endomet- Jung-Yoon Yoo and Hee-Bum Kang contributed equally to this work. Department of Biomedical Sciences, ASAN Medical Center, University of rial cancer [5, 6], recent intrauterine progestin therapies Ulsan College of Medicine, Seoul 05505, South Korea such as a levonorgestrel-releasing intrauterine system Department of Obstetrics, Gynecology and Reproductive Biology, College of have been used for reproductive-aged women with Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoo et al. BMC Cancer (2018) 18:605 Page 2 of 10 complex atypical hyperplasia and early-stage endometrial [30, 31]. Inappropriately elevated expression of AKT cancer in cases when there is a desire to preserve fertility phosphorylation is related to poor prognosis of endo- or when comorbidities exclude the possibility of surgery. metrial cancer patients [32]. Furthermore, inhibition of In addition, progestin therapy is also considered for re- the AKT pathway combined with P4 decreases angio- current endometrial cancer because it is less toxic than genesis and proliferation in vivo, indicating that regula- chemotherapies; however, though the response rate of tion of the AKT pathway may play an important endometrial hyperplasia to progestin treatment is higher therapeutic role [33]. than that of endometrial adenocarcinoma, the response Mitogen-inducible gene 6 (MIG-6) functions to sup- to progestin in cancer recurrence is worst of all. Proges- press endometrial cancer in the human and mouse tin therapies used in the clinic are effective for some pa- uterus [34, 35]. Mig-6 is an important mediator of P4 tients but not all cases of endometrial hyperplasia and signaling in that it inhibits E2-mediated epithelial prolif- well-differentiated endometrioid endometrial cancer. eration in the uterus [35, 36]. MIG-6lossisuniquelyasso- Another major limitation of progestin therapy is the lack ciated with infertility and endometrial cancer [35, 37–39], of a clinical standard protocol for the type, dose, and but the effects of MIG-6 loss have not been specifically in- duration of treatment [11–13]. The molecular mecha- vestigated in regulation of epithelial proliferation of endo- nisms underlying progesterone (P4) resistance in endo- metrial cancer. In this study, we demonstrate that Mig-6 is metrial cancer have not been fully understood. pivotal in the suppression of epithelial proliferation through Loss of control over uterine epithelial cell proliferation its inhibition of AKT activation. Specifically, we show that and apoptosis by ovarian steroid hormones is the major P4 inhibition of endometrial tumorigenesis is mediated by underlying pathogenesis of endometrial cancer [14–17]. MIG-6 inhibition of AKT phosphorylation. Progesterone therapy can prevent this process by block- ing actions of unopposed estrogen (E2) [18]. Nonethe- Methods less, several studies indicate that P4 therapy has low and Animals and treatments unpredictable response rates in women with endometrial Mice were maintained for and used in the designated cancer, therefore limiting its potential use [19–23]. Re- animal care facility according to the Michigan State Uni- sistance to P4 treatment due to loss of either progester- versity institutional guidelines. All animal procedures one receptor (PGR) itself or its signaling pathways were approved by the Institutional Animal Care and Use causes significant difficulty in the treatment of advanced Committee of Michigan State University. Mice were and recurrent endometrial cancer [24]. Identifying mo- housed in standard cages (up to 5 animals per cage) in lecular mechanisms involved in P4 resistance is critical rooms with 12 h light/dark cycle, controlled temperature to effective and personalized treatment. Unfortunately, and humidity under specific pathogen-free conditions. further translational research of endometrial cancer is Campus Animal Resources at Michigan State University inhibited by the lack of sufficient pre-clinical animal provides veterinary care, daily husbandry and health models. checks, procurement, and other administrative support Sequencing analysis of endometrial cancers in the for research in biomedical housing facilities and assists Cancer Genome Atlas has revealed that upwards of 90% with animal health. Animals are observed daily by ani- of cases of endometrioid endometrial cancer have some mal care staff that have additional training in laboratory genetic aberration in the PTEN/PI3K pathway, which re- animal sciences and species-specific handling and sults in increased AKT activity [25]. In addition, the husbandry. AKT signaling pathway can be activated by E2 [26]en- To generate uterine epithelial specific Mig-6 knockout f/f cre/+ hancing cell proliferation [27]. Therefore, an under- mice, Mig-6 mice were crossed with Sprr2f mice f/f standing of the molecular mechanisms between steroid [40]. Control (Mig-6 ) and endometrial epithelial cre/+ f/f hormone and PTEN/PI3K/AKT signaling will allow us cell-specific Mig-6 knockout mice (Sprr2f Mig-6 ; d/d to be in a much better position to develop new conser- Mig-6 )[41] were used to investigate the effect of epi- vative therapies based on P4 function. thelial Mig-6 ablation on the uterus. The protein structure of AKT consists of a PH do- Vehicle (beeswax) or P4 (40 mg/pellet) pellets were f/f main, a linker region, a kinase domain, and a regulator placed subcutaneously into control (Mig-6 ) and cre+ f/f domain [28]. These domains undergo various protein Sprr2f Mig-6 mice respectively at 10 weeks of age modifications including phosphorylation, acetylation, for 1 week (n = 6/treatment/genotype). To avoid any ubiquitylation, methylation, hydroxylation, glycosylation, possibility of pain and/or distress to the animal, all surgi- and SUMOylation which help regulate the proteins ac- cal procedures were performed under anesthesia. Mice tivity [29]. AKT regulates different pathways that aid in were anesthetized with isoflurane (3% isoflurane in oxy- the promotion of cellular survival and inhibition of gen by inhalation). All surgeries were conducted in dedi- apoptosis through its serine/threonine kinase activity cated surgical suites using aseptic procedures. Yoo et al. BMC Cancer (2018) 18:605 Page 3 of 10 Recuperating animals, under close supervision, were Western blot analysis kept warm until full postoperative recovery is achieved. Western blot analysis was performed as previously de- Animals were under anesthetic for a maximum of scribed [41]. Membrane was blocked with Casein (0.5% 20 min, and recovery from surgery normally occurs v/v) prior to exposure to anti-AKT (CS-4691; Cell Sig- within 30 min as evidence by sternal recumbency, naling, Danvers, MA), anti-pAKT (CS-4060; Cell Signal- followed by normal ambulation, grooming and feeding. ing, Danvers, MA), and anti-Flag (F1804; Sigma-Aldrich, If discomfort is observed, the animals were provided St. Louis, MO) antibodies. Anti-actin (SC-1615, Santa Ketoprofen at a dose of 5 mg/kg as an analgesic. At the Cruz Biotechnology, Dallas, TX) was used for loading end of a given study, all mice were humanely euthanized control. by cervical dislocation under isoflurane anesthesia or by carbon dioxide asphyxiation and then the uteri from Statistical analysis cre+ f/f control and Sprr2f Mig-6 mice were collected to in- For data with only two groups, Student’s t-test was used. vestigate the effect of P4 on the development of endo- For data containing more than two groups, an analysis metrial hyperplasia. of variance (ANOVA) test was used, followed by Tukey or Bonferroni test for pairwise t-tests. All statistical ana- lyses were performed using the Instat package from Immunohistochemistry and analysis GraphPad (San Diego, CA, USA). Immunohistochemistry analysis was performed as previ- ously described [41]. Briefly, uterine sections were Results pre-incubated with 10% normal goat serum in PBS prior A decrease of stromal PGR and ESR1 expression in cre+ f/f to exposure to anti-PGR (SC-538; Santa Cruz Biotech- Sprr2f Mig-6 mice nology, Dallas, TX), anti-ESR1 (SC-543; Santa Cruz Bio- Previously, we reported that the hyperplastic phenotype technology, Dallas, TX), anti-AKT (CS-4691; Cell of endometrial epithelial cell specific Mig-6 knockout cre+ f/f d/d Signaling, Danvers, MA), anti-pAKT (CS-4060; Cell Sig- (Sprr2f Mig-6 ; Mig-6 ) mice were observed at naling, Danvers, MA), and anti-Ki67 (BD5506090; BD 10 weeks of age [43]. Endometrial cancer displays an im- Biosciences, San Jose, CA) as appropriate primary anti- balance in steroid hormone action [14–17]. PGR expres- bodies. Positive signaling was detected with the DAB sion has been shown to be a prognostic factor for Peroxidase Substrate Kit (SK-4100; Vector Laboratories, endometrial cancer [44–46]. Therefore, we first exam- d/d Burlingame, CA). The H-score was calculated as previ- ined expression of PGR and ESR1 in Mig-6 mice. Im- ously reported [42]. The overall H-score ranged from 0 munohistochemical analysis indicated that levels of PGR to 300. and ESR1 were significantly decreased in the stromal d/d f/f cells of Mig-6 mice compared to control (Mig-6 ) mice at 10 weeks of age (n = 6/genotype). However, the Cell culture and transient transfection expression of PGR and ESR1 in the epithelium were not d/d Ishikawa (99,040,201; Sigma–Aldrich, St. Louis, MO) changed in the uteri of Mig-6 mice as compared to and HEC1A (HTB-112; ATCC, Manassas, VA) Cell lines control (Fig. 1). These data suggest that dysregulation of are maintained in Dulbecco’s modified Eagle’s medium/ PGR and ESR1 expression in the stroma may play an Nutrient Mixture F-12 (DMEM/F12; Gibco BRL, Gai- important role for the development of endometrial thersburg, MD) with 10% (v/v) fetal bovine serum (FBS; hyperplasia. Gibco BRL, Gaithersburg, MD), and 1% (v/v) penicillin cre+ f/f streptomycin (P/S; Gibco BRL, Gaithersburg, MD) at Aberrant activation of AKT signaling in Sprr2f Mig-6 37 °C under 5% CO . FLAG-tagging MIG-6 expression mice vectors were transfected using Lipofectamine 2000 re- AKT is frequently hyperactivated in human cancers [47]. agent (Invitrogen Crop., Carlsbad, CA) according to the To determine if the observed hyperplastic phenotype manufacturer’s instructions. was due to activated AKT signaling, we examined the expression of total AKT, phospho-AKT (pAKT), and phospho-S6 (pS6), a downstream marker of active AKT d/d Immunoprecipitation signaling in the uteri of control and Mig-6 mice. First, Immunoprecipitation was performed as described previ- we examined cell proliferation by Ki67 staining (n =6/ ously [38]. Briefly, Ishikawa and HEC1A cells were trans- genotype). The IHC results revealed a significant in- d/d fected with the FLAG-MIG-6 expression vectors. crease of uterine epithelial proliferation in Mig-6 mice Immunoprecipitation was performed with Flag antibody (Fig. 2a-b). Interestingly, we found that pAKT and pS6 d/d (F1804; Sigma–Aldrich, St. Louis, MO). Protein interac- were highly elevated in the epithelial cells of Mig-6 tions were examined by Western blot analysis. mice at 10 weeks of age as compared to control mice Yoo et al. BMC Cancer (2018) 18:605 Page 4 of 10 PGR stroma ESR1 stroma AC PGRBD ESR1 *** *** 200 200 a a 150 150 100 100 50 50 25µm 25um 0 0 f/f d/d f/f d/d Mig-6 Mig-6 Mig-6 Mig-6 PGR Epithelium ESR1 Epithelium b b 300 300 240 240 180 180 120 120 60 60 25µm 25µm 0 0 f/f d/d f/f d/d Mig-6 Mig-6 Mig-6 Mig-6 d/d Fig. 1 A decrease of stromal PGR and ESR1 expression in Mig-6 mice. Immunohistochemical analysis for PGR (A) and ESR1 (C) in control (a) and d/d Mig-6 (b) mice. H-score in stroma and epithelial cells for PGR (B) and ESR1 (D). The results represent the mean ± SEM. ***, p < 0.001 f/f d/d Proliferation Mig-6 Mig-6 25µm 25µm f/f d/d Mig-6 Mig-6 pAKT *** 25µm 25µm f/f d/d Mig-6 Mig-6 f pS6 *** 25µm 25µm f/f d/d Mig-6 Mig-6 d/d f/f Fig. 2 Aberrant activation of proliferation and AKT signaling in Mig-6 mice. (A) The expression of Ki67, pAKT, and pS6 in the uteri of Mig-6 d/d f/f d/d (a,c,e) and Mig-6 (b, d, and f) mice. (B) Quantification of Ki67 positive cells and H-score in epithelial cells of Mig-6 and Mig-6 mice. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001 d/d f/f Mig-6 Mig-6 pS6 pAKT Proliferation H-Score H-Score d/d f/f Mig-6 Mig-6 H-Score H-Score % of proliferative cells H-Score H-Score Yoo et al. BMC Cancer (2018) 18:605 Page 5 of 10 d/d (Fig. 2). However, total AKT levels were not changed pellets into the control and Mig-6 mice subcutane- among the genotypes (Additional file 1: Figure S1). ously at 10 weeks of age (n = 6/treatment/genotype). d/d These data suggest that MIG-6 suppresses endometrial After 1 week of the P4 treatment, Mig-6 mice exhib- epithelial proliferation via inhibition of AKT ited a significantly decreased uterine weight compared d/d phosphorylation. to vehicle-treated Mig-6 mice (Fig. 3a and b). Histo- logical analysis showed that the development of uterine d/d The effect of P4 treatment on the development of hyperplasia was not evident in Mig-6 mice after P4 endometrial hyperplasia treatment (Fig. 3c). P4 treatment also led to decreased d/d Exposure to P4 is a negative risk factor for endometrial proliferation in the epithelial cells of Mig-6 mice as d/d cancer [48]. Additionally, it is well known that endomet- compared to vehicle-treated Mig-6 mice (Fig. 3d). rial cancer is E2-dependent and that progestin therapy These data suggest that the hyperplastic phenotype of d/d has been successful in slowing the growth of endomet- Mig-6 mice was responsive to P4 treatment, returning rial tumors in women who are poor surgical candidates the morphology to normal. and premenopausal women with complex atypical hyperplasia and early-stage endometrioid endometrial The recovery of steroid hormone and AKT signaling by P4 cre+ f/f cancer who had a strong desire to preserve their fertility treatment in Sprr2f Mig-6 mice [22, 23, 49–54]. To assess the effect of P4 treatment on The expression of PGR and ESR1 is strongly correlated epithelial ablation of Mig-6, we placed P4 or vehicle with the prognosis of endometrial cancer [55]. f/f d/d Mig-6 Mig-6 1 week treatment B 10 a b f/f Mig-6 d/d Mig-6 1cm 1cm Vehicle P4 1cm 1cm C D d/d Vehicle P4 Mig-6 Epithelium a b 100 *** 100µm 100µm 25µm Veh P4 d b Stroma 25µm 25µm 25µm Veh P4 f/f d/d d/d Fig. 3 Effects of P4 on Mig-6 and Mig-6 mice. (A) Uterine/body ratio were significantly decreased in Mig-6 mice compared to vehicle- KO treated Mig-6 mice after P4 treatment. (B) Gross morphology and (C) Hematoxylin-eosin staining after vehicle and P4 treatment. (D) f/f d/d Immunohistochemical analysis and quantification of Ki67 in Mig-6 and Mig-6 mice. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001 Uterine/body weight X 1,000 P4 Vehicle P4 Vehicle % of proliferative cells % of proliferative cells Yoo et al. BMC Cancer (2018) 18:605 Page 6 of 10 PGR stroma ESR1 stroma AC PGRBD ESR1 *** *** 25µm 25um Veh P4 Veh P4 PGR Epithelium ESR1 Epithelium b b 300 300 240 240 180 180 120 120 60 60 25µm 25µm 0 0 Veh P4 Veh P4 d/d Fig. 4 Recovery of stroma PGR and ESR1 expression in Mig-6 mice after P4 treatment. Immunohistochemical analysis for PGR (A) and ESR1 (C) d/d in vehicle- (a) and P4-(b) treated Mig-6 mice. Quantification of PGR (B) and ESR1 (D) by H-score. The results represent the mean ± SEM. ***, p < 0.001 d/d Therefore, we examined the expression of PGR and control and Mig-6 mice after P4 treatment to investi- ESR1 using immunohistochemistry (n = 6/treatment). gate whether the suppression of hyperplastic phenotype The expression of PGR and ESR1 were significantly in- observed was due to recovered AKT signaling. Total d/d creased in the stroma of Mig-6 mice after P4 treat- AKT levels were not changed after P4 treatment ment (Fig. 4). These data indicated that P4 treatment (Additional file 2:FigureS2).However, aberrantacti- activates nuclear receptors signaling at endometrial stro- vation of AKT signaling was significantly decreased in d/d d/d mal cells of Mig-6 mice. the uteri of P4-treated Mig-6 mice as compared to d/d Next, we examined the expression of total AKT, pAKT, vehicle-treated Mig-6 mice (Fig. 5). These data sug- and pS6 using immunohistochemistry in the uteri of gest that P4 treatment suppresses aberrant activation Vehicle P4 AB pAKT a 150 *** 25µm 25µm Veh P4 pS6 *** 25µm 25µm Veh P4 d/d Fig. 5 AKT signaling is down-regulated after P4 treatment in Mig-6 mice. (A) Immunohistochemical analysis of pAKT and pS6 in vehicle and d/d f/f d/d P4-treated Mig-6 mice. (B) Quantification of pAKT and pS6 positive cells in epithelial cells of Mig-6 and Mig-6 mice after P4 treatment. The results represent the mean ± SEM. ***, p < 0.001 P4 Vehicle pS6 pAKT H- H-Score Score H-Score P4 Vehicle H-Score H-Score H-Score H-Score Yoo et al. BMC Cancer (2018) 18:605 Page 7 of 10 of AKT signaling in endometrial hyperplasia of stimulated proliferation of uterine epithelial cells [56]. d/d Mig-6 mice. Disruption of steroidal control results in unopposed E2, leading to endometrial cancer [17]. Mig-6 is a target of MIG-6 regulates AKT phosphorylation dose-dependently P4 and PGR, and its deletion in the uterus leads to en- and interacts with AKT hanced epithelial proliferation [35]. The majority of In order to examine effects of MIG-6 on AKT, we per- endometrial cancers exhibit actively proliferating epithe- formed experiments on endometrial cancer cell lines, lial cells and increased AKT signaling [57–59]. The Can- Ishikawa and HEC1A cells. We transfected to Ishikawa cer Genome Atlas analysis demonstrated an increased and HEC1A cells dose-dependently with FLAG-tagged AKT activity in endometrioid endometrial tumors [25]. MIG-6 (FLAG-MIG-6). Following MIG-6 introduction Activated AKT signaling enhances cell proliferation as we examined levels of AKT and pAKT at 24-h. The well as cell survival through the inhibition of proapopto- levels of AKT phosphorylation were highly decreased by tic proteins [27]. Expression of PGR (PR-A and PR-B) FLAG-MIG-6 in a dose dependent manner whereas and ESRs (ESR1 and ESR2) has been reported as prog- AKT levels were unchanged (Fig. 6a). We next examined nostic factors for endometrial carcinoma [44–46]. We whether MIG-6 physically interacts with AKT. Ishikawa evaluated that stromal PGR and ESR1 expression was d/d cells were transfected with FLAG-MIG-6, and the lysates significantly decreased in the uteri of Mig-6 when were immunoprecipitated with FLAG antibody. FLAG compared to control mice (Fig. 1). We showed elevated immunoprecipitates were then probed with AKT and phosphorylation of AKT resulting in enhanced epithelial MIG-6 specific antibodies, indicating that MIG-6 physic- proliferation (Fig. 2). Stromal PGR and P4 signaling is ally interacts with AKT (Fig. 6b). These results suggest necessary and sufficient to mediate the antiproliferative that MIG-6 inhibits AKT phosphorylation through a effects of P4 on E2-induced epithelial cell proliferation protein-protein interaction, highlighting its important [60, 61]. However, activation of AKT reduces PR-B tran- d/d role in the regulation of epithelial proliferation. scriptional activity in Ishikawa cells and Pten condi- tional mouse model of endometrioid endometrial cancer Discussion [33]. AKT also reduces PGR expression levels in breast In this study, we evaluated whether MIG-6 suppresses cancer cells, endometrial cancer cells, and uterine stro- endometrial epithelial proliferation via inhibition of AKT mal cells affected by endometriosis [62–64]. However, phosphorylation. P4 plays an inhibitory role on E2 exactly how signaling occurs between AKT and P4 re- sistance in endometrial epithelial and stromal interaction is unclear. Filling this knowledge gap is critical to under- standing P4 resistance. Ishikawa HEC1A P4 resistance is defined by the decreased responsive- MIG-6 MIG-6 ness to bioavailable P4 of target tissue [65]. Lack of P4 00.1 0.5 1 2 ug 00.1 0.5 1 2 ug activity contributes significantly to uterine pathophysi- MIG-6 MIG-6 ology. P4 resistance is now considered a central element pAKT pAKT in women’s diseases such as infertility, endometriosis, and endometrial cancer [66–69], but the mechanism of AKT AKT P4 resistance in women’s diseases remains unknown. We d/d Actin Actin have demonstrated that Mig-6 mice exhibiting normal P4 responses and P4 treatment for 1 week is sufficient to restore endometrial hyperplasia to normal (Fig. 3). IP We treated the mice in the beginning of endometrial Input IgG MIG-6 hyperplasia and the data suggest P4 treatment at an early time point can be one of the reasons to reverse endometrial hyperplasia to normal. Therefore, further study on the effects of P4 treatment on endometrial tur- AKT morigenesis associated with its development and pro- MIG-6 gression are required. Actin Determining the molecular mechanisms by which steroid hormones control the physiology of theuterusisofutmost Fig. 6 AKT phosphorylation were regulated by MIG-6 expression dose-dependently (a) Western Blot analysis of MIG-6, pAKT, AKT, and importance to understanding and overcoming P4 resist- Actin in FLAG-MIG-6 transfected Ishikawa and HEC1A cells. Actin was ance. However, resistance to P4 treatment has led to limit- used as sample-loading control. (b) The interaction between MIG-6 ing the use of P4 therapy in endometrial cancer due to its and AKT by immunoprecipitation low response rates [19–23]. The optimal method for Control MIG-6 Control MIG-6 Control MIG-6 Yoo et al. BMC Cancer (2018) 18:605 Page 8 of 10 treating and surveilling patients with conservatively treated Abbreviations ESR1: Estrogen receptor α; ESR2: Estrogen receptor β; PGR: Progesterone endometrial cancer is not known. Therefore, the identifica- receptor; PI3K: Phosphoinositide 3-kinase; PTEN: Phosphatase and tensin tion of the molecular pathways that link P4 resistance to homolog; S6: Ribosomal protein S6 kinase endometrial cancer development can potentially provide Acknowledgements novel targets for the prevention or treatment of this malig- The Sprr2f-cre mice were acquired from Dr. Diego H. Castrillon (University of nancy. We showed that AKT signaling is down-regulated Texas Southwestern Medical Center, Dallas, TX). We would like to thank Ryan d/d after P4 treatment in Mig-6 mice (Fig. 5). These data sug- M. Marquardt for manuscript preparation. gest that treatment with an AKT inhibitor could be a viable Funding alternative for overcoming the P4-resistant endometrial Grant numbers and sources of support: The design, data collection, data hyperplasia and cancer. analysis, and data interpretation of this study was supported by Grant Number P50CA098258 from the National Cancer Institute (to R. R. Broaddus We found that MIG-6 decreased AKT phosphorylation and T.H. Kim). The analysis and interpretation of in vitro experiments and in Ishikawa and HEC1A cell lines in a dose-dependent writing support of this manuscript were supported by the National Research manner. Immunoprecipitation showed that there is protein Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (No. NRF-2016R1D1A1B03934346, to J.Y. Yoo), and interaction between MIG-6 and AKT, suggesting that NRF grant (No. NRF-2017R1A2B4007971, K.-C. Choi). MIG-6 suppresses E2-induced epithelial cell proliferation through AKT interactions (Fig. 6). However, the exact mo- Availability of data and materials lecular mechanism by which interaction regulates the phos- The datasets supporting the conclusions of this article are included within the article. phorylation of AKT is not clear. Further studies will be required to determine exact molecular mechanism. Authors’ contributions We have shown the prevention effect of P4 with JYY, and HBK conceived and designed the experimental approach, d/d d/d performed experiments and prepared the manuscript. JIR analyzed the Mig-6 mice [43]. We treated Mig-6 mice with P4 results. RRB provided pathological analysis. KCC and THK conceived and before developing endometrial hyperplasia and found designed the experimental approach, performed data analysis and prepared that P4 prevented the development of endometrial the manuscript. All authors have read and approved the final version of manuscript. hyperplasia by inhibiting epithelial STAT3 phosphoryl- ation, resulting in a decrease of epithelial proliferation. Ethics approval The molecular mechanisms in the regulation of epithe- All animal procedures were approved by the Institutional Animal Care and lial proliferation by AKT and STAT3 as well as steroid Use Committee of Michigan State University (Application #: 11/16–192-00). hormone signaling remains to be further studied during Competing interests endometrial tumorigenesis. Our data support that the The authors declare that they have no competing interests. activation of stromal signaling by P4 treatment can con- tribute to the development of endometrial hyperplasia Publisher’sNote and the cross-talk between AKT/STAT3 and PGR/ESR1 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. is critical to inhibit the endometrial hyperplasia. Author details Conclusions Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA. Overall, our study suggests that the negative regulation of Department of Biochemistry and Molecular Biology, Yonsei University AKT phosphorylation by activated stroma signaling in- College of Medicine, Seoul 03722, South Korea. Department of Biomedical cluding Mig-6 has an important role in the regulation of Sciences, ASAN Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea. Department of Pathology, University of Texas epithelial cell proliferation during endometrial hyperplasia M.D. Anderson Cancer Center, Houston, Texas TX 77030, USA. Department development and progression. Our results contribute to of Pharmacology, Asan Institute for Life Sciences, Asan Medical Center, the understanding of the etiological and molecular mecha- University of Ulsan College of Medicine, Seoul 05505, South Korea. nisms of epithelial cell proliferation and to the develop- Received: 4 December 2017 Accepted: 11 May 2018 ment of new therapeutic approaches for treating endometrial hyperplasia and cancer. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. Additional files 2016;66(1):7–30. 2. 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BMC CancerSpringer Journals

Published: May 29, 2018

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