REPRODUCTION-DEVELOPMENT
Activated AKT Pathway Promotes Establishment of Endometriosis Tae Hoon Kim, Yanni Yu, Lily Luo, John P. Lydon, Jae-Wook Jeong, and J. Julie Kim Department of Obstetrics, Gynecology, and Reproductive Biology (T.H.K., J.-W.J.), Michigan State University, College of Human Medicine, Grand Rapids, Michigan 49503; Division of Reproductive Biology Research (Y.Y., L.L., J.J.K.), Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611; and Department of Molecular and Cellular Biology (J.P.L.), Baylor College of Medicine, Houston, Texas 77030
The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from diseasefree patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)glycogen synthase kinase 3 did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PRcre/⫹Ptenf/⫹ heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ⫾ 0.33 lesions per Ptenf/⫹ mouse and 7.5 ⫾ 0.43 lesions in the PRcre/⫹Ptenf/⫹ mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206 – treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues. (Endocrinology 155: 1921–1930, 2014)
E
ndometriosis is a disease that affects approximately 10% of women of reproductive age in the United States (1). It is identified by the presence of endometrial tissue outside of the uterine cavity, which is responsible for the severe pain that often accompanies this disease as well as problems in fertility (2). The pathogenesis of endome-
triosis is poorly understood due to the limited accessibility to tissue specimens and limited availability of animal models. As a result, current options for the treatment of endometriosis are suboptimal (3–7). AKT (v-akt murine thymoma viral oncogene homolog) is hyperactivated in a number of solid tumors due to mu-
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received October 15, 2013. Accepted January 16, 2014. First Published Online February 26, 2014
Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; BrdU, 5-bromo-2⬘deoxyuridine; DMSO, dimethylsulfoxide; E2, estradiol; ER, estrogen receptor; FOXO1, forkhead box O1; GSK3, glycogen synthase kinase 3; HRP, horseradish peroxidase; p, phosphorylated; PR, progesterone receptor; SRC-1, steroid receptor coactivator-1; TBST, Trisbuffered saline with Tween 20.
doi: 10.1210/en.2013-1951
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tations in PTEN, PI3K, and other receptors that activate the AKT pathway. In endometriosis, we and others have demonstrated that the AKT pathway is overactive compared with endometrial cells from endometriosis-free patients (8 –11). Increased levels of the phosphorylated activated forms of AKT have been demonstrated in eutopic endometrial stromal cells from women with endometriosis compared with those from disease-free women (9, 12). Levels of phosphorylated (p)(Ser473)-AKT are consistently higher in stromal cells from ovarian endometriomas compared with eutopic endometrial stromal cells from disease-free women (8). Even in the presence of the decidualizing stimuli (medroxyprogesterone acetate and a cAMP analog, dibutyryl cAMP), which decreases pAKT in endometrial stromal cells, levels of pAKT were much higher in endometriotic stromal cells (8). Immunohistochemical staining of endometriotic tissues from the adnexa of women with endometriosis showed that p(Ser473)-AKT levels are higher in the stroma compared with that of normal endometrial tissues (8). Recently, we demonstrated that inhibiting AKT with MK-2206 decreased cell viability and increased apoptosis in endometriotic stromal cells (13). In addition, inhibition of AKT resulted in an increase in total and nuclear progesterone receptor (PR) A and B protein levels. Although we demonstrated that AKT in human endometriotic cells and tissues affect their ability to proliferate and survive, it is unknown what the role of AKT is in the establishment of disease. In this study, we used the bigenic PRcre/⫹Ptenf/⫹ mouse, which has a deletion of Pten in cells that express PR (14). In these mice, PR-expressing cells occur in the myometrium and endometrium as well as other reproductive tissues including ovary and mammary gland. Deletion of Pten increases activation of AKT, because phosphatase activity that negatively regulates AKT is absent. Specifically, the PRcre/⫹Ptenf/⫹ heterozygote mice were used because only 1 allele has a Pten deletion and the homozygote animals develop endometrial carcinoma as early as 1 month of age (14). Using these mice, autologous implantation of uterine tissues was done to establish endometriosis. Here, we demonstrate that increased AKT activity promotes formation of ectopic lesions, and inhibition of this pathway using the AKT inhibitor, MK-2206, prevents establishment of ectopic lesions.
Materials and Methods Tissue acquisition This study was approved by the Northwestern University Institutional Review Board. Written informed consent was obtained from all patients. Endometrioma cyst walls were obtained
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from patients undergoing ovarian cystectomy or salpingo-oophorectomy for endometriosis at Prentice Women’s Hospital. Endometrial tissues were obtained from patients without endometriosis undergoing hysterectomy for benign indications. All patients reported regular menses. Patients who had undergone hormonal therapy or had been pregnant within the 3 months before surgery were excluded. All samples were histologically confirmed by a pathologist.
Stromal cell isolation and cell culture Stromal cells were isolated as previously described (15). Endometriotic stromal cells were given the designation OSIS, and normal endometrial stromal cells were designated ESC. Cells were cultured in DMEM/F12 (1:1) (Invitrogen) with 10% fetal bovine serum and penicillin (100 U/mL) with streptomycin (100 U/mL) at 37°C in a humidified atmosphere with 5% CO2. Culture medium was changed every 3 days. Cells were used for experiments until passage 7, at which point they were discarded. Cells were treated with designated concentrations of MK-2206 (generously provided by Merck Sharp and Dohme, Corp, and the National Cancer Institute, National Institutes of Health) or dimethylsulfoxide (DMSO) (vehicle).
Immunoblotting OSIS and ESC were washed with ice-cold PBS, and whole-cell lysates were obtained on ice using the M-PER mammalian lysis solution (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Sigma). Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Scientific). Equal amounts of protein were resolved on a 10% polyacrylamide gel and transferred to a polyvinyl difluoride membrane. The membranes were blocked with 5% BSA in Trisbuffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature and then incubated overnight at 4°C in 1% BSA with antibodies against pAKT (Ser473), total AKT, phosphoglycogen synthase kinase 3 (pGSK3) (Ser9), total GSK3, phospho-forkhead box O1 (FOXO1) (Ser256) (Cell Signaling), total FOXO1 (Bethyl Laboratories), and -actin (Sigma). All primary antibodies used for Western blots were at a 1:2000 dilution except FOXO1 and -actin, which were diluted 1:5000. The blots were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit or antimouse secondary antibodies (1:10 000) for 1 hour and then detected with a chemiluminescent detection kit (Thermo Scientific). Digital images of the Western blots were captured using the Fujifilm LAS-3000 Imager.
Immunohistochemistry Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Embedded tissues were cut into 4-m sections and then mounted on glass slides. Tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed by heating sections in citrate buffer at pH 6.0. Endogenous peroxidase activity was blocked by incubating sections with 0.03% H2O2 for 10 minutes. Sections were blocked with protein block for 30 minutes (Dako) followed by rinsing with TBST and then incubated with primary antibodies to FOXO1 (1:100; Bethyl Laboratories) overnight at 4°C in a humidified chamber. Slides were rinsed with TBST and HRP-conjugated secondary antibodies were applied for 30 minutes. HRP activity was detected using diamino-
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benzidine tetrahydrochloride as substrate and then counterstained with hematoxylin. For p(Ser473)-AKT (1:500), Ki67 (Dako; 1:31 250), and PR (Dako; 1:1000), tissue sections were processed for immunohistochemistry at the Mouse Histology and Phenotyping Core at Northwestern University.
Cell viability and proliferation OSIS or ESC were plated in 96-well plates at 5 ⫻ 104 cells per well and allowed to attach overnight. Cells were then washed with PBS and incubated in phenol red-free DMEM/F12 (1:1)
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with 10% fetal bovine serum and penicillin (100 U/mL) with streptomycin (100 U/mL) and various concentrations of MK2206 or DMSO for 24 hours. The WST-1 cell proliferation assay kit (Clontech) was used to measure cell viability according to the manufacturer’s protocol. Fold change relative to vehicle control was calculated. The 5-bromo-2⬘-deoxyuridine (BrdU) incorporation immunoassay (Roche Applied Sciences) was used to measure cell proliferation according to the manufacturer’s protocol. Fold change relative to vehicle control was calculated.
Figure 1. Inhibition of AKT with MK-2206 in ESC and OSIS cells. ESC and OSIS cells were treated with increasing concentrations of MK-2206 and levels of p(Ser473)-AKT and total AKT (A and B), p(Ser256)-FOXO1 and total FOXO1 (C), and p(Ser9)-GSK3 and total GSK3 (D) were measured by Western blot. Western blots from 4 experimental replicates (4 patients) were quantitated using ImageJ and presented graphically as the mean ⫾ SEM (n ⫽ 4). For pAKT, data were transformed and linear regression analysis was done to determine IC50. For pFOXO1, FOXO1, pGSK3, and GSK3, densitometric values were statistically analyzed for differences between groups. *, P ⬍ .05.
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Induction of endometriosis in mice Animals were maintained in a designated animal care facility according to the Michigan State University’s Institutional Guidelines for the care and use of laboratory animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. PRcre mice were crossed with Ptenf/f mice to generate PRCre/⫹Ptenf/⫹ bigenics in which 1 allele of Pten is abrogated specifically in PR-expressing cells. Ptenf/⫹ and PRCre/⫹Ptenf/⫹ mice were ovariectomized at 6 weeks of age. Two weeks later, ovariectomized Ptenf/⫹ and PRCre/⫹Ptenf/⫹ mice were injected with estradiol (E2) (0.1 g/ mouse) every 24 hours for 3 days, and surgical induction of endometriosis was performed at 72 hours (see Figure 4). Endometriotic lesions were established by inoculating endometrial tissue into the peritoneal cavity. Animals were deeply anesthetized with avertin. To access the peritoneal cavity, mice underwent laparotomy by midventral incision (1 cm) to expose the uterus and intestine. The left uterine horn was removed and placed in a petri dish containing Eagle’s basal medium (Invitrogen). The average weight of the left uterine horn was 56.70 ⫾ 5.47 mg from the Ptenf/⫹ mice and 64.44 ⫾ 6.11 mg from PRCre/⫹Ptenf/⫹mice. There was no significant difference in uterine weights between the genotypes (n ⫽ 10 per genotype). The uterine horn was opened longitudinally and then cut into small fragments. The fragments suspended in 0.5 mL Eagle’s basal medium were returned to the peritoneal cavity of the same mouse from which the uterus was taken for an autologous implantation, and a gentle massage was given to disperse the tissue. Estrogen pellets (0.36 mg/d for 60 days; SE-121; Innovative Research of America) were placed sc into mice to support the formation of endometriosis-like lesions. Vehicle (30% captisol; Cydex) (16) or MK-2206 (360 mg/kg) (16) was administered once a week for 1 month from 2 days after E2 injection by oral gavage. Captisol was used as the vehicle, as recommended by Merck, because DMSO is toxic to the mice. After 4 weeks, mice were euthanized, the peritoneal cavity was opened, and endometriosis-like lesions were counted and removed. Endometriosis-like lesions and uterine tissues were fixed with 4% (vol/vol) paraformaldehyde for histological analysis.
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allosteric AKT inhibitor MK-2206 decreased levels of p(Ser473)-AKT in both OSIS and ESC in a dose-dependent manner (Figure 1, A and B). Western blots from 4 experimental replicates were quantitated, and the IC50 for MK2206 in ESC and OSIS were calculated to be 136.1nM and 141.7nM, respectively. Slopes of the curves were not statistically different. Total AKT levels were not affected. Two proteins that are directly phosphorylated by AKT are FOXO1 and GSK3. Levels of p(Ser256)-FOXO1 were high in OSIS cells, which decreased with MK-2206 treatment (Figure 1C). In contrast, levels of p(Ser256)-FOXO1 were low in ESC. Total levels of FOXO1 also varied between ESC and OSIS; however, variability in responses in cells from 4 patients did not render significant differences (Figure 1C). Levels of p(Ser9)-GSK3 were also higher in OSIS cells compared with ESC; however, MK-2206 did not significantly decrease levels of p(Ser9)-GSK3 in either ESC or OSIS cells, suggesting that other kinases are able to phosphorylate Ser9 despite the presence of MK2206 (Figure 1, C and D). Next, the effect of MK-2206 on cell viability and proliferation were assessed in OSIS and ESC grown in culture (Figure 2). Cell viability decreased in response to increasing concentrations of MK-2206 (0M–10M) for both OSIS and ESC in a similar manner (Figure 2A). Cell pro-
Statistical analysis Statistical analyses were performed with Graphpad Prism. Unpaired two-tailed t tests between ESC and OSIS groups, or paired, two-tailed t tests between control and MK-2206 groups were used to analyze the differences between 2 groups. Western blots were quantitated using Image J. Data were transformed, and a linear regression was done. IC50 was calculated, and the slopes of the curves for ESC and OSIS were compared. P values ⬍ .05 were considered statistically significant.
Results MK-2206 inhibits the AKT pathway in human OSIS cells Stromal cells from human ovarian endometriomas (OSIS) as well as the endometrium from disease-free women (ESC) exhibit increased levels of p(Ser473)-AKT in the presence of growth medium in vitro. Addition of the
Figure 2. Effect of MK-2206 on viability and proliferation of ESC and OSIS. ESC or OSIS cells were treated with increasing concentrations of MK-2206 and viability using the WST assay (A) or proliferation using the BrdU assay (B) were measured. Data are expressed as fold change relative to vehicle and are the mean ⫾ SEM of experimental replicates from 4 patients. *, P ⬍ .05 for comparison between ESC and OSIS at each concentration of MK-2206.
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liferation as measured by the BrdU assay was significantly reduced by MK-2206 in OSIS, whereas effects on ESC were more modest (Figure 2B). Decreases were also dosedependent (0M–1M) for OSIS cells. These data suggest that the AKT pathway plays an important role in OSIS cell proliferation. Induction of endometriosis in PRcre/ⴙPtenf/ⴙ mice Although the above studies demonstrate that the AKT pathway plays an important role in OSIS cell proliferation in culture, it remains unknown whether increased activation of the AKT pathway observed in endometriosis contributes to the pathogenesis of the disease. To address this question, we used the bigenic PRcre/⫹Ptenf/⫹ mice in which Pten is deleted in 1 allele in only cells expressing PR. PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway, and its absence due to deletions or mutations
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result in hyperactivation of the AKT pathway (17). Specifically, a line of mice in which cre recombinase is under the control of the PR promoter (PRcre) was crossed with a mouse line containing the floxed Pten allele. This allowed tissue-specific Pten heterozygotes in all PR-expressing cells, which includes cells of the uterus (14). These heterozygous mice are distinct from the PRCre/⫹Ptenf/f homozygotes, which have been shown to develop endometrial carcinoma by 1 month of age (14). In contrast, the PRcre/⫹Ptenf/⫹ heterozygotes have a normal endometrium even at 2 months of age (Figure 3A). Immunohistochemical analysis for p(Ser473)-AKT in the endometrium of intact mice at 2 months of age showed very little staining (Figure 3B). Staining of a few stromal cells was evident at the higher magnification. Interestingly, the endometrium from mice that were ovariectomized
Figure 3. Histological analysis of the uterus from PRcre/⫹Ptenf/⫹ mice. A, Gross morphology of a uterus from a 2-month-old PRcre/⫹Ptenf/⫹ mouse is shown. A cross-section of uterine tissue is visualized with hematoxylin and eosin staining. B, Immunohistochemical staining was performed for p(Ser473)-AKT of uterine tissue from 2-month-old PRcre/⫹Ptenf/⫹ mice that are intact or ovariectomized (OVX) and treated with E2.
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and treated with exogenous E2 stained for p(Ser473)AKT in a number of glandular cells, implicating estradiol in increasing activation of AKT in these heterozygous Pten mice. Autologous implantation of uterine tissues has been used to demonstrate establishment of ectopic lesions in the peritoneal cavity in the mouse strains ICR, C57BL/6, and BALB/c (18 –21). Following similar methods, 1 horn from PRcre/⫹Ptenf/⫹ or the control, Ptenf/⫹ was minced and placed back into the peritoneal cavity of the same mouse. Mice were given E2 2 days before tissue implantation (Figure 4A). After 4 weeks, the peritoneum was examined and
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found to contain an average of 4.33 ⫾ 0.33 lesions per Ptenf/⫹ mouse compared with 7.50 ⫾ 0.43 lesions in the PRcre/⫹Ptenf/⫹ mice (Figure 4B). Hematoxylin and eosin staining of ectopic lesions showed endometrial tissues resembling that of the eutopic endometrium (Figure 4C). These data imply that increased activation of the AKT pathway is promoting the establishment of ectopic lesions. To determine that increased AKT was the primary mechanism by which ectopic lesions were established in the PRcre/⫹Ptenf/⫹ mice, the allosteric AKT inhibitor MK2206 was given to both Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice on the same day as tissue injection into the peritoneum. After 4 weeks of MK-2206 treatment, the number of lesions counted was on average 2.50 ⫾ 0.22 and 2.60 ⫾ 0.51 for both Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice, respectively, which is significantly lower than the 4.50 ⫾ 0.34 or 7.25 ⫾ 0.48 lesions, respectively, in the untreated mice (Figure 5A and B) . Histological analysis of ectopic tissues did not show any significant differences between vehicle- and MK2206 –treated Ptenf/⫹ or PRcre/⫹Ptenf/⫹ mice (Figure 5B). The eutopic endometrium did not show any uncharacteristic histological changes either. Despite no obvious differences in tissue histology, immunohistochemical staining of the ectopic tissues revealed differences between vehicle- and MK-2206 –treated mice. Levels of p(Ser473)-AKT were lower in ectopic tissues of MK-2206 –treated mice compared with vehicle-treated mice (Figure 5C). Levels of FOXO1, a downstream target of AKT, were higher with MK-2206 mainly in the stroma. Ki-67 staining was modest in the vehicle-treated tissues, yet levels were even lower in ectopic tissues in MK-2206 – treated mice. Finally, we previously reported that inhibition of AKT using MK-2206 of human endometriotic stromal cells increased protein levels of PR (13). In the PRcre/⫹ Ptenf/⫹ mice treated with MK-2206, levels of PR were higher in the stroma compared with ectopic tissues in vehicle-treated mice. Thus, although uterine tissues from PRcre/⫹Ptenf/⫹ mice that did attach to peritoneal tissues were indistinguishable morphologically between vehicle and MK-2206 treatment, biochemically, they expressed different levels of proteins, signifying that the AKT inhibitor was affecting the ectopic uterine tissues.
Discussion Figure 4. Established endometriosis lesions in PRcre/⫹Ptenf/⫹ mice. A, Experimental schematic of induction of endometriosis in control Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice. B, After 4 weeks of tissue injection, the peritoneal cavity was examined for lesions and counted. Data are mean ⫾ SEM of 6 mice per genotype. ***, P ⬍ .001. C, Hematoxylin and eosin staining of ectopic lesions and eutopic endometrium was done for Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice.
In this study, we have demonstrated that the AKT inhibitor MK-2206 decreased AKT activity and preferentially inhibited proliferation of human endometriotic stromal cells in culture compared with disease-free endometrial stromal cells. Furthermore, mice with Pten deleted in 1 allele in uterine cells (PRcre/⫹Ptenf/⫹) formed more ectopic lesions after
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Figure 5. Effect of AKT inhibition on establishment of endometriotic lesions. A, Mice were treated as depicted in Figure 4A with the administration of vehicle or 360 mg/kg MK-2206 at the time of tissue injection and then once a week for 4 weeks. Lesions in the peritoneal cavity were counted. Data are mean ⫾ SEM of 4 to 6 mice per group (vehicle Ptenf/⫹, n ⫽ 6; vehicle PRcre/⫹Ptenf/⫹, n ⫽ 4; MK-2206 Ptenf/⫹, n ⫽ 6; MK2206 PRcre/⫹Ptenf/⫹, n ⫽ 5). B, Hematoxylin and eosin staining of ectopic lesions and eutopic endometrium was done for Ptenf/⫹ and PRcre/⫹ Ptenf/⫹ mice treated with vehicle or MK-2206. C, Immunohistochemical staining of ectopic lesions was done for p(Ser-473)-AKT, FOXO1, Ki-67, and PR for vehicle- or MK-2206 –treated PRcre/⫹Ptenf/⫹ mice.
autologous implantation of uterine tissues compared with Pten intact mice. Treatment of the PRcre/⫹Ptenf/⫹ mice with MK-2206 significantly decreased the number of ectopic lesions formed in these mice, demonstrating the major role that AKT plays in establishment of endometriosis. Although in vitro studies of primary cells from endometriosis and xenografted human tissues in mice are essential to study cellular and molecular mechanisms associated with its pathobiology, mimicking establishment of disease outside of a woman’s body, let alone deciphering the role of a pathway such as AKT in the establishment of disease, has remained a challenge. Animal models of endometriosis range from stitching uterine tissues to the peritoneal wall (22–25) to injecting endometrial fragments or menstrual tissue into the peritoneal cavity (21, 26) to study the early events of disease establishment and progression. Syngeneic and autologous models in mouse strains, ICR, C57BL/6, and BALB/c, where endometrial fragments are autotransplanted into the peritoneal cavity, have been used to test various compounds (18 –22). The major advantage of using such models is the presence of an intact immune system, which is lost in xenotransplant models. These models do not, however, allow for the study of how a particular gene or pathway influences endometriosis,
unless the endometrial tissues are manipulated before implantation. For example, overexpression or knockdown of genes or even labeling of the cells in the tissues can be done to determine how a gene influences establishment of disease. Efficient delivery of gene constructs to all cells remains a limitation to this type of manipulation. Transgenic mice have been used to study pathogenic progression of endometriosis (22). Endometriosis was induced in steroid receptor coactivator-1 (SRC-1)–null mice as well as Tnf⫺/⫺ and Mmp9⫺/⫺ mice by stitching uterine tissues to the peritoneum (22). This group demonstrated that a 70kDa SRC-1 proteolytic isoform forms from activation of TNF␣-induced matrix metallopeptidase 9 (MMP9) activity in endometriotic mouse tissue. The endometriotic 70kDa SRC-1 C-terminal fragment prevented TNF␣-mediated apoptosis in human endometrial epithelial cells and caused epithelial-mesenchymal transition and the invasion of human endometrial cells. In another study, estrogen receptor-␣ (ER␣)-knockout and ER-knockout mice were used to study the role of ER and E2 in endometriosis (27). They induced endometriosis in these immunocompetent mice using the donor/recipient transplantation strategy and demonstrated the importance of ER␣ for establishment of endometriosis lesions. To our knowledge,
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Figure 5. Continued.
ours is the first study using a bigenic mouse to determine the role of a signaling pathway in the establishment of endometriosis. Specifically, only cells expressing PR have Pten deleted in 1 allele, which means the uterus and not the peritoneal tissues have Pten deletion. This bypasses the need for gene manipulation in cells before induction of endometriosis, does not require surgical stitching of tissues, and does not require transplantation of donor transgenic tissues into wild-type recipients. With our autologous implantation of uterine tissues from PRcre/⫹Ptenf/⫹ mice, we demonstrate that the AKT pathway promotes establishment of lesions, specifically, that aberrant signaling within the eutopic endometrium causes ectopic lesion formation. Improvements to this model can be made in terms of detecting and quantitating ectopic lesions. As only those visible by dissecting scopes were identified, there could have been smaller lesions that formed that were not visible. Labeling of uterine tissues before transplantation into the peritoneum, whether it be through flu-
orescent labels such as calcein AM fluorescent dye or by crossing mice with mice harboring green fluorescent protein, would improve detection of lesions. The decreased number of lesions found in both Ptenf/⫹ or PRcre/⫹Ptenf/⫹ mice in response to MK-2206 treatment could be due to either the inability to attach or rapid regression after attachment. In other words, increased AKT activity could promote attachment of tissues through aberrant expression of adhesion molecules, or inactivation of this pathway compromises its survival and causes attached lesions to regress. Additional studies will be required to understand how inhibiting AKT results in fewer ectopic lesions. We have demonstrated recently that levels of PR increase in human endometriotic stromal cells in response to MK-2206 (13). Interestingly, we see an increase in PR protein in the stroma of ectopic tissues in the PRcre/⫹ Ptenf/⫹ mice treated with MK-2206. The consequence of this is unknown at this time, because the mice in this study
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were ovariectomized and only E2 was given during the duration of the experiment. Unliganded PR activity could contribute to the observations in this study. This requires further experimentation potentially with PR antagonists to better understand the role of unliganded PR in AKTmediated attachment and survival of lesions. If this occurs in the eutopic endometrium, modulation of progesterone function could affect fertility. Given our longstanding interest in AKT/PR crosstalk in the endometrium (13, 28 – 32), this mouse model is ideal to study progesterone sensitivity in the eutopic endometrium in the PRcre/⫹Ptenf/⫹ mice and its effects on fertility. Whether the presence of ectopic lesions can affect fertility in this mouse model, whether inhibition of AKT sensitizes these mice to progesterone, and identification of the endometrial markers involved remain to be investigated. The use of mouse models such as the one reported here provides invaluable information regarding the functional role of a specific gene or pathway in a disease process. Here we have demonstrated that deletion of Pten in 1 allele in PR-expressing cells promotes the establishment of endometriosis-like lesions in an autologous implantation model of endometriosis. The ability of a specific AKT inhibitor to decrease the number of lesions established indicates that the AKT pathway is critical for the establishment of this disease in this mouse model. As with any mouse model for human disease, data should be properly interpreted within the boundaries of its context. As such, questions remain as to whether women with hyperactivated AKT signaling in their eutopic endometrium are more prone to developing endometriosis, because retrograde menstruation occurs in most premenopausal women. Transgenic models such as the one presented in this study provide evidence that if there is an inherent defect in the eutopic endometrium that is placed in the peritoneum, this increases the likelihood of establishment of lesions. This is in contrast to the animal models where normal endometrial tissue or menstrual effluent are placed in the peritoneal cavity. Although there is no perfect model for human endometriosis aside from the human model, studies such as these provide the proof-of-principle evidence to explore and consider potential targets of therapy for endometriosis.
Acknowledgments We are grateful to the Mouse Histology and Phenotyping Core facilities at the Robert Lurie Cancer Center at Northwestern University. The MK-2206 was generously provided by Merck Sharp and Dohme Corp and the National Cancer Institute, National Institutes of Health (NIH).
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Address all correspondence and requests for reprints to: J. Julie Kim, PhD, Department of Obstetrics and Gynecology, Robert H. Lurie Comprehensive Cancer Center, 303 East Superior, Room 4 –117, Northwestern University, Chicago, IL 60611. Email: [email protected]. This work was supported by NIH Grants R01HD044715 (to J.J.K.), U54 HD07495 (to J.P.L.), and R01HD057873 (to J.W.J.); the Friends of Prentice Grants Initiative (to J.J.K.), and the Medical Student Summer Research Program (to L.L.). Disclosure Summary: The authors have nothing to disclose.
References 1. Giudice LC. Clinical practice. Endometriosis. N Engl J Med. 2010; 362:2389 –2398. 2. Bulun SE. Endometriosis. N Engl J Med. 2009;360:268 –279. 3. Kauppila A, Rönnberg L. Naproxen sodium in dysmenorrhea secondary to endometriosis. Obstet Gynecol. 1985;65:379 –383. 4. Crosignani P, Olive D, Bergqvist A, Luciano A. Advances in the management of endometriosis: an update for clinicians. Hum Reprod Update. 2006;12:179 –189. 5. Vercellini P, Trespidi L, Colombo A, Vendola N, Marchini M, Crosignani PG. A gonadotropin-releasing hormone agonist versus a low-dose oral contraceptive for pelvic pain associated with endometriosis. Fertil Steril. 1993;60:75–79. 6. Bianchi S, Busacca M, Agnoli B, Candiani M, Calia C, Vignali M. Effects of 3 month therapy with danazol after laparoscopic surgery for stage III/IV endometriosis: a randomized study. Hum Reprod. 1999;14:1335–1337. 7. Child TJ, Tan SL. Endometriosis: aetiology, pathogenesis and treatment. Drugs. 2001;61:1735–1750. 8. Yin X, Pavone ME, Lu Z, Wei JJ, Kim JJ. Increased Activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis. J Clin Endocrinol Metab. 2012;97:E35–E43. 9. Cinar O, Seval Y, Uz YH, et al. Differential regulation of Akt phosphorylation in endometriosis. Reprod Biomed Online. 2009;19: 864 – 871. 10. Leconte M, Nicco C, Ngô C, et al. The mTOR/AKT inhibitor temsirolimus prevents deep infiltrating endometriosis in mice. Am J Pathol. 2011;179:880 – 889. 11. Zhang H, Zhao X, Liu S, Li J, Wen Z, Li M. 17E2 promotes cell proliferation in endometriosis by decreasing PTEN via NFB-dependent pathway. Mol Cell Endocrinol. 2010;317:31– 43. 12. Velarde MC, Aghajanova L, Nezhat CR, Giudice LC. Increased mitogen-activated protein kinase kinase/extracellularly regulated kinase activity in human endometrial stromal fibroblasts of women with endometriosis reduces 3⬘,5⬘-cyclic adenosine 5⬘-monophosphate inhibition of cyclin D1. Endocrinology. 2009;150:4701– 4712. 13. Eaton JL, Unno K, Caraveo M, Lu Z, Kim JJ. Increased AKT or MEK1/2 activity influences Progesterone Receptor levels and localization in endometriosis. J Clin Endocrinol Metab. 2013;98:E1871– E1879. 14. Daikoku T, Hirota Y, Tranguch S, et al. Conditional loss of uterine Pten unfailingly and rapidly induces endometrial cancer in mice. Cancer Res. 2008;68:5619 –5627. 15. Ryan IP, Schriock ED, Taylor RN. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab. 1994;78:642– 649. 16. Hirai H, Sootome H, Nakatsuru Y, et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol Cancer Ther. 2010;9:1956 –1967.
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17. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A. 1999;96:4240 – 4245. 18. Körbel C, Menger MD, Laschke MW. Size and spatial orientation of uterine tissue transplants on the peritoneum crucially determine the growth and cyst formation of endometriosis-like lesions in mice. Hum Reprod. 2010;25:2551–2558. 19. Pelch KE, Schroder AL, Kimball PA, Sharpe-Timms KL, Davis JW, Nagel SC. Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women. Fertil Steril. 2010; 93:1615–1627.e18. 20. Umezawa M, Tanaka N, Tainaka H, Takeda K, Ihara T, Sugamata M. Microarray analysis provides insight into the early steps of pathophysiology of mouse endometriosis model induced by autotransplantation of endometrium. Life Sci. 2009;84:832– 837. 21. Somigliana E, Viganò P, Rossi G, Carinelli S, Vignali M, PaninaBordignon P. Endometrial ability to implant in ectopic sites can be prevented by interleukin-12 in a murine model of endometriosis. Hum Reprod. 1999;14:2944 –2950. 22. Han SJ, Hawkins SM, Begum K, Jung SY, Kovanci E, Qin J, Lydon JP, DeMayo FJ, O’Malley BW. A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis. Nat Med. 2012;18:1102–1111. 23. Becker CM, Sampson DA, Short SM, Javaherian K, Folkman J, D’Amato RJ. Short synthetic endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse model. Fertil Steril. 2006;85:71–77.
24. Becker CM, Wright RD, Satchi-Fainaro R, et al. A novel noninvasive model of endometriosis for monitoring the efficacy of antiangiogenic therapy. Am J Pathol. 2006;168:2074 –2084. 25. Olivares C, Ricci A, Bilotas M, Barañao RI, Meresman G. The inhibitory effect of celecoxib and rosiglitazone on experimental endometriosis. Fertil Steril. 2011;96:428 – 433. 26. Fazleabas AT. A baboon model for inducing endometriosis. Methods Mol Med. 2006;121:95–99. 27. Burns KA, Rodriguez KF, Hewitt SC, Janardhan KS, Young SL, Korach KS. Role of estrogen receptor signaling required for endometriosis-like lesion establishment in a mouse model. Endocrinology. 2012;153:3960– 3971. 28. Kim JJ, Buzzio OL, Li S, Lu Z. Role of FOXO1A in the regulation of insulin-like growth factor-binding protein-1 in human endometrial cells: interaction with progesterone receptor. Biol Reprod. 2005;73:833– 839. 29. Neubauer NL, Ward EC, Patel P, et al. Progesterone receptor-B induction of BIRC3 protects endometrial cancer cells from AP1– 59-mediated apoptosis. Horm Cancer. 2011;2:170 –181. 30. Pant A, Lee II, Lu Z, Rueda BR, Schink J, Kim JJ. Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin. PloS One. 2012;7:e41593. 31. Ward EC, Hoekstra AV, Blok LJ, et al. The regulation and function of the forkhead transcription factor, Forkhead box O1, is dependent on the progesterone receptor in endometrial carcinoma. Endocrinology. 2008;149: 1942–1950. 32. Hoekstra AV, Sefton EC, Berry E, et al. Progestins activate the AKT pathway in leiomyoma cells and promote survival. J Clin Endocrinol Metab. 2009;94:1768 –1774.
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Activated AKT Pathway Promotes Establishment of Endometriosis Tae Hoon Kim, Yanni Yu, Lily Luo, John P. Lydon, Jae-Wook Jeong, and J. Julie Kim Department of Obstetrics, Gynecology, and Reproductive Biology (T.H.K., J.-W.J.), Michigan State University, College of Human Medicine, Grand Rapids, Michigan 49503; Division of Reproductive Biology Research (Y.Y., L.L., J.J.K.), Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611; and Department of Molecular and Cellular Biology (J.P.L.), Baylor College of Medicine, Houston, Texas 77030
The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from diseasefree patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)glycogen synthase kinase 3 did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PRcre/⫹Ptenf/⫹ heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ⫾ 0.33 lesions per Ptenf/⫹ mouse and 7.5 ⫾ 0.43 lesions in the PRcre/⫹Ptenf/⫹ mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206 – treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues. (Endocrinology 155: 1921–1930, 2014)
E
ndometriosis is a disease that affects approximately 10% of women of reproductive age in the United States (1). It is identified by the presence of endometrial tissue outside of the uterine cavity, which is responsible for the severe pain that often accompanies this disease as well as problems in fertility (2). The pathogenesis of endome-
triosis is poorly understood due to the limited accessibility to tissue specimens and limited availability of animal models. As a result, current options for the treatment of endometriosis are suboptimal (3–7). AKT (v-akt murine thymoma viral oncogene homolog) is hyperactivated in a number of solid tumors due to mu-
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received October 15, 2013. Accepted January 16, 2014. First Published Online February 26, 2014
Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; BrdU, 5-bromo-2⬘deoxyuridine; DMSO, dimethylsulfoxide; E2, estradiol; ER, estrogen receptor; FOXO1, forkhead box O1; GSK3, glycogen synthase kinase 3; HRP, horseradish peroxidase; p, phosphorylated; PR, progesterone receptor; SRC-1, steroid receptor coactivator-1; TBST, Trisbuffered saline with Tween 20.
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tations in PTEN, PI3K, and other receptors that activate the AKT pathway. In endometriosis, we and others have demonstrated that the AKT pathway is overactive compared with endometrial cells from endometriosis-free patients (8 –11). Increased levels of the phosphorylated activated forms of AKT have been demonstrated in eutopic endometrial stromal cells from women with endometriosis compared with those from disease-free women (9, 12). Levels of phosphorylated (p)(Ser473)-AKT are consistently higher in stromal cells from ovarian endometriomas compared with eutopic endometrial stromal cells from disease-free women (8). Even in the presence of the decidualizing stimuli (medroxyprogesterone acetate and a cAMP analog, dibutyryl cAMP), which decreases pAKT in endometrial stromal cells, levels of pAKT were much higher in endometriotic stromal cells (8). Immunohistochemical staining of endometriotic tissues from the adnexa of women with endometriosis showed that p(Ser473)-AKT levels are higher in the stroma compared with that of normal endometrial tissues (8). Recently, we demonstrated that inhibiting AKT with MK-2206 decreased cell viability and increased apoptosis in endometriotic stromal cells (13). In addition, inhibition of AKT resulted in an increase in total and nuclear progesterone receptor (PR) A and B protein levels. Although we demonstrated that AKT in human endometriotic cells and tissues affect their ability to proliferate and survive, it is unknown what the role of AKT is in the establishment of disease. In this study, we used the bigenic PRcre/⫹Ptenf/⫹ mouse, which has a deletion of Pten in cells that express PR (14). In these mice, PR-expressing cells occur in the myometrium and endometrium as well as other reproductive tissues including ovary and mammary gland. Deletion of Pten increases activation of AKT, because phosphatase activity that negatively regulates AKT is absent. Specifically, the PRcre/⫹Ptenf/⫹ heterozygote mice were used because only 1 allele has a Pten deletion and the homozygote animals develop endometrial carcinoma as early as 1 month of age (14). Using these mice, autologous implantation of uterine tissues was done to establish endometriosis. Here, we demonstrate that increased AKT activity promotes formation of ectopic lesions, and inhibition of this pathway using the AKT inhibitor, MK-2206, prevents establishment of ectopic lesions.
Materials and Methods Tissue acquisition This study was approved by the Northwestern University Institutional Review Board. Written informed consent was obtained from all patients. Endometrioma cyst walls were obtained
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from patients undergoing ovarian cystectomy or salpingo-oophorectomy for endometriosis at Prentice Women’s Hospital. Endometrial tissues were obtained from patients without endometriosis undergoing hysterectomy for benign indications. All patients reported regular menses. Patients who had undergone hormonal therapy or had been pregnant within the 3 months before surgery were excluded. All samples were histologically confirmed by a pathologist.
Stromal cell isolation and cell culture Stromal cells were isolated as previously described (15). Endometriotic stromal cells were given the designation OSIS, and normal endometrial stromal cells were designated ESC. Cells were cultured in DMEM/F12 (1:1) (Invitrogen) with 10% fetal bovine serum and penicillin (100 U/mL) with streptomycin (100 U/mL) at 37°C in a humidified atmosphere with 5% CO2. Culture medium was changed every 3 days. Cells were used for experiments until passage 7, at which point they were discarded. Cells were treated with designated concentrations of MK-2206 (generously provided by Merck Sharp and Dohme, Corp, and the National Cancer Institute, National Institutes of Health) or dimethylsulfoxide (DMSO) (vehicle).
Immunoblotting OSIS and ESC were washed with ice-cold PBS, and whole-cell lysates were obtained on ice using the M-PER mammalian lysis solution (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Sigma). Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Scientific). Equal amounts of protein were resolved on a 10% polyacrylamide gel and transferred to a polyvinyl difluoride membrane. The membranes were blocked with 5% BSA in Trisbuffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature and then incubated overnight at 4°C in 1% BSA with antibodies against pAKT (Ser473), total AKT, phosphoglycogen synthase kinase 3 (pGSK3) (Ser9), total GSK3, phospho-forkhead box O1 (FOXO1) (Ser256) (Cell Signaling), total FOXO1 (Bethyl Laboratories), and -actin (Sigma). All primary antibodies used for Western blots were at a 1:2000 dilution except FOXO1 and -actin, which were diluted 1:5000. The blots were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit or antimouse secondary antibodies (1:10 000) for 1 hour and then detected with a chemiluminescent detection kit (Thermo Scientific). Digital images of the Western blots were captured using the Fujifilm LAS-3000 Imager.
Immunohistochemistry Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Embedded tissues were cut into 4-m sections and then mounted on glass slides. Tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed by heating sections in citrate buffer at pH 6.0. Endogenous peroxidase activity was blocked by incubating sections with 0.03% H2O2 for 10 minutes. Sections were blocked with protein block for 30 minutes (Dako) followed by rinsing with TBST and then incubated with primary antibodies to FOXO1 (1:100; Bethyl Laboratories) overnight at 4°C in a humidified chamber. Slides were rinsed with TBST and HRP-conjugated secondary antibodies were applied for 30 minutes. HRP activity was detected using diamino-
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benzidine tetrahydrochloride as substrate and then counterstained with hematoxylin. For p(Ser473)-AKT (1:500), Ki67 (Dako; 1:31 250), and PR (Dako; 1:1000), tissue sections were processed for immunohistochemistry at the Mouse Histology and Phenotyping Core at Northwestern University.
Cell viability and proliferation OSIS or ESC were plated in 96-well plates at 5 ⫻ 104 cells per well and allowed to attach overnight. Cells were then washed with PBS and incubated in phenol red-free DMEM/F12 (1:1)
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with 10% fetal bovine serum and penicillin (100 U/mL) with streptomycin (100 U/mL) and various concentrations of MK2206 or DMSO for 24 hours. The WST-1 cell proliferation assay kit (Clontech) was used to measure cell viability according to the manufacturer’s protocol. Fold change relative to vehicle control was calculated. The 5-bromo-2⬘-deoxyuridine (BrdU) incorporation immunoassay (Roche Applied Sciences) was used to measure cell proliferation according to the manufacturer’s protocol. Fold change relative to vehicle control was calculated.
Figure 1. Inhibition of AKT with MK-2206 in ESC and OSIS cells. ESC and OSIS cells were treated with increasing concentrations of MK-2206 and levels of p(Ser473)-AKT and total AKT (A and B), p(Ser256)-FOXO1 and total FOXO1 (C), and p(Ser9)-GSK3 and total GSK3 (D) were measured by Western blot. Western blots from 4 experimental replicates (4 patients) were quantitated using ImageJ and presented graphically as the mean ⫾ SEM (n ⫽ 4). For pAKT, data were transformed and linear regression analysis was done to determine IC50. For pFOXO1, FOXO1, pGSK3, and GSK3, densitometric values were statistically analyzed for differences between groups. *, P ⬍ .05.
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Induction of endometriosis in mice Animals were maintained in a designated animal care facility according to the Michigan State University’s Institutional Guidelines for the care and use of laboratory animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. PRcre mice were crossed with Ptenf/f mice to generate PRCre/⫹Ptenf/⫹ bigenics in which 1 allele of Pten is abrogated specifically in PR-expressing cells. Ptenf/⫹ and PRCre/⫹Ptenf/⫹ mice were ovariectomized at 6 weeks of age. Two weeks later, ovariectomized Ptenf/⫹ and PRCre/⫹Ptenf/⫹ mice were injected with estradiol (E2) (0.1 g/ mouse) every 24 hours for 3 days, and surgical induction of endometriosis was performed at 72 hours (see Figure 4). Endometriotic lesions were established by inoculating endometrial tissue into the peritoneal cavity. Animals were deeply anesthetized with avertin. To access the peritoneal cavity, mice underwent laparotomy by midventral incision (1 cm) to expose the uterus and intestine. The left uterine horn was removed and placed in a petri dish containing Eagle’s basal medium (Invitrogen). The average weight of the left uterine horn was 56.70 ⫾ 5.47 mg from the Ptenf/⫹ mice and 64.44 ⫾ 6.11 mg from PRCre/⫹Ptenf/⫹mice. There was no significant difference in uterine weights between the genotypes (n ⫽ 10 per genotype). The uterine horn was opened longitudinally and then cut into small fragments. The fragments suspended in 0.5 mL Eagle’s basal medium were returned to the peritoneal cavity of the same mouse from which the uterus was taken for an autologous implantation, and a gentle massage was given to disperse the tissue. Estrogen pellets (0.36 mg/d for 60 days; SE-121; Innovative Research of America) were placed sc into mice to support the formation of endometriosis-like lesions. Vehicle (30% captisol; Cydex) (16) or MK-2206 (360 mg/kg) (16) was administered once a week for 1 month from 2 days after E2 injection by oral gavage. Captisol was used as the vehicle, as recommended by Merck, because DMSO is toxic to the mice. After 4 weeks, mice were euthanized, the peritoneal cavity was opened, and endometriosis-like lesions were counted and removed. Endometriosis-like lesions and uterine tissues were fixed with 4% (vol/vol) paraformaldehyde for histological analysis.
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allosteric AKT inhibitor MK-2206 decreased levels of p(Ser473)-AKT in both OSIS and ESC in a dose-dependent manner (Figure 1, A and B). Western blots from 4 experimental replicates were quantitated, and the IC50 for MK2206 in ESC and OSIS were calculated to be 136.1nM and 141.7nM, respectively. Slopes of the curves were not statistically different. Total AKT levels were not affected. Two proteins that are directly phosphorylated by AKT are FOXO1 and GSK3. Levels of p(Ser256)-FOXO1 were high in OSIS cells, which decreased with MK-2206 treatment (Figure 1C). In contrast, levels of p(Ser256)-FOXO1 were low in ESC. Total levels of FOXO1 also varied between ESC and OSIS; however, variability in responses in cells from 4 patients did not render significant differences (Figure 1C). Levels of p(Ser9)-GSK3 were also higher in OSIS cells compared with ESC; however, MK-2206 did not significantly decrease levels of p(Ser9)-GSK3 in either ESC or OSIS cells, suggesting that other kinases are able to phosphorylate Ser9 despite the presence of MK2206 (Figure 1, C and D). Next, the effect of MK-2206 on cell viability and proliferation were assessed in OSIS and ESC grown in culture (Figure 2). Cell viability decreased in response to increasing concentrations of MK-2206 (0M–10M) for both OSIS and ESC in a similar manner (Figure 2A). Cell pro-
Statistical analysis Statistical analyses were performed with Graphpad Prism. Unpaired two-tailed t tests between ESC and OSIS groups, or paired, two-tailed t tests between control and MK-2206 groups were used to analyze the differences between 2 groups. Western blots were quantitated using Image J. Data were transformed, and a linear regression was done. IC50 was calculated, and the slopes of the curves for ESC and OSIS were compared. P values ⬍ .05 were considered statistically significant.
Results MK-2206 inhibits the AKT pathway in human OSIS cells Stromal cells from human ovarian endometriomas (OSIS) as well as the endometrium from disease-free women (ESC) exhibit increased levels of p(Ser473)-AKT in the presence of growth medium in vitro. Addition of the
Figure 2. Effect of MK-2206 on viability and proliferation of ESC and OSIS. ESC or OSIS cells were treated with increasing concentrations of MK-2206 and viability using the WST assay (A) or proliferation using the BrdU assay (B) were measured. Data are expressed as fold change relative to vehicle and are the mean ⫾ SEM of experimental replicates from 4 patients. *, P ⬍ .05 for comparison between ESC and OSIS at each concentration of MK-2206.
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liferation as measured by the BrdU assay was significantly reduced by MK-2206 in OSIS, whereas effects on ESC were more modest (Figure 2B). Decreases were also dosedependent (0M–1M) for OSIS cells. These data suggest that the AKT pathway plays an important role in OSIS cell proliferation. Induction of endometriosis in PRcre/ⴙPtenf/ⴙ mice Although the above studies demonstrate that the AKT pathway plays an important role in OSIS cell proliferation in culture, it remains unknown whether increased activation of the AKT pathway observed in endometriosis contributes to the pathogenesis of the disease. To address this question, we used the bigenic PRcre/⫹Ptenf/⫹ mice in which Pten is deleted in 1 allele in only cells expressing PR. PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway, and its absence due to deletions or mutations
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result in hyperactivation of the AKT pathway (17). Specifically, a line of mice in which cre recombinase is under the control of the PR promoter (PRcre) was crossed with a mouse line containing the floxed Pten allele. This allowed tissue-specific Pten heterozygotes in all PR-expressing cells, which includes cells of the uterus (14). These heterozygous mice are distinct from the PRCre/⫹Ptenf/f homozygotes, which have been shown to develop endometrial carcinoma by 1 month of age (14). In contrast, the PRcre/⫹Ptenf/⫹ heterozygotes have a normal endometrium even at 2 months of age (Figure 3A). Immunohistochemical analysis for p(Ser473)-AKT in the endometrium of intact mice at 2 months of age showed very little staining (Figure 3B). Staining of a few stromal cells was evident at the higher magnification. Interestingly, the endometrium from mice that were ovariectomized
Figure 3. Histological analysis of the uterus from PRcre/⫹Ptenf/⫹ mice. A, Gross morphology of a uterus from a 2-month-old PRcre/⫹Ptenf/⫹ mouse is shown. A cross-section of uterine tissue is visualized with hematoxylin and eosin staining. B, Immunohistochemical staining was performed for p(Ser473)-AKT of uterine tissue from 2-month-old PRcre/⫹Ptenf/⫹ mice that are intact or ovariectomized (OVX) and treated with E2.
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and treated with exogenous E2 stained for p(Ser473)AKT in a number of glandular cells, implicating estradiol in increasing activation of AKT in these heterozygous Pten mice. Autologous implantation of uterine tissues has been used to demonstrate establishment of ectopic lesions in the peritoneal cavity in the mouse strains ICR, C57BL/6, and BALB/c (18 –21). Following similar methods, 1 horn from PRcre/⫹Ptenf/⫹ or the control, Ptenf/⫹ was minced and placed back into the peritoneal cavity of the same mouse. Mice were given E2 2 days before tissue implantation (Figure 4A). After 4 weeks, the peritoneum was examined and
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found to contain an average of 4.33 ⫾ 0.33 lesions per Ptenf/⫹ mouse compared with 7.50 ⫾ 0.43 lesions in the PRcre/⫹Ptenf/⫹ mice (Figure 4B). Hematoxylin and eosin staining of ectopic lesions showed endometrial tissues resembling that of the eutopic endometrium (Figure 4C). These data imply that increased activation of the AKT pathway is promoting the establishment of ectopic lesions. To determine that increased AKT was the primary mechanism by which ectopic lesions were established in the PRcre/⫹Ptenf/⫹ mice, the allosteric AKT inhibitor MK2206 was given to both Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice on the same day as tissue injection into the peritoneum. After 4 weeks of MK-2206 treatment, the number of lesions counted was on average 2.50 ⫾ 0.22 and 2.60 ⫾ 0.51 for both Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice, respectively, which is significantly lower than the 4.50 ⫾ 0.34 or 7.25 ⫾ 0.48 lesions, respectively, in the untreated mice (Figure 5A and B) . Histological analysis of ectopic tissues did not show any significant differences between vehicle- and MK2206 –treated Ptenf/⫹ or PRcre/⫹Ptenf/⫹ mice (Figure 5B). The eutopic endometrium did not show any uncharacteristic histological changes either. Despite no obvious differences in tissue histology, immunohistochemical staining of the ectopic tissues revealed differences between vehicle- and MK-2206 –treated mice. Levels of p(Ser473)-AKT were lower in ectopic tissues of MK-2206 –treated mice compared with vehicle-treated mice (Figure 5C). Levels of FOXO1, a downstream target of AKT, were higher with MK-2206 mainly in the stroma. Ki-67 staining was modest in the vehicle-treated tissues, yet levels were even lower in ectopic tissues in MK-2206 – treated mice. Finally, we previously reported that inhibition of AKT using MK-2206 of human endometriotic stromal cells increased protein levels of PR (13). In the PRcre/⫹ Ptenf/⫹ mice treated with MK-2206, levels of PR were higher in the stroma compared with ectopic tissues in vehicle-treated mice. Thus, although uterine tissues from PRcre/⫹Ptenf/⫹ mice that did attach to peritoneal tissues were indistinguishable morphologically between vehicle and MK-2206 treatment, biochemically, they expressed different levels of proteins, signifying that the AKT inhibitor was affecting the ectopic uterine tissues.
Discussion Figure 4. Established endometriosis lesions in PRcre/⫹Ptenf/⫹ mice. A, Experimental schematic of induction of endometriosis in control Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice. B, After 4 weeks of tissue injection, the peritoneal cavity was examined for lesions and counted. Data are mean ⫾ SEM of 6 mice per genotype. ***, P ⬍ .001. C, Hematoxylin and eosin staining of ectopic lesions and eutopic endometrium was done for Ptenf/⫹ and PRcre/⫹Ptenf/⫹ mice.
In this study, we have demonstrated that the AKT inhibitor MK-2206 decreased AKT activity and preferentially inhibited proliferation of human endometriotic stromal cells in culture compared with disease-free endometrial stromal cells. Furthermore, mice with Pten deleted in 1 allele in uterine cells (PRcre/⫹Ptenf/⫹) formed more ectopic lesions after
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Figure 5. Effect of AKT inhibition on establishment of endometriotic lesions. A, Mice were treated as depicted in Figure 4A with the administration of vehicle or 360 mg/kg MK-2206 at the time of tissue injection and then once a week for 4 weeks. Lesions in the peritoneal cavity were counted. Data are mean ⫾ SEM of 4 to 6 mice per group (vehicle Ptenf/⫹, n ⫽ 6; vehicle PRcre/⫹Ptenf/⫹, n ⫽ 4; MK-2206 Ptenf/⫹, n ⫽ 6; MK2206 PRcre/⫹Ptenf/⫹, n ⫽ 5). B, Hematoxylin and eosin staining of ectopic lesions and eutopic endometrium was done for Ptenf/⫹ and PRcre/⫹ Ptenf/⫹ mice treated with vehicle or MK-2206. C, Immunohistochemical staining of ectopic lesions was done for p(Ser-473)-AKT, FOXO1, Ki-67, and PR for vehicle- or MK-2206 –treated PRcre/⫹Ptenf/⫹ mice.
autologous implantation of uterine tissues compared with Pten intact mice. Treatment of the PRcre/⫹Ptenf/⫹ mice with MK-2206 significantly decreased the number of ectopic lesions formed in these mice, demonstrating the major role that AKT plays in establishment of endometriosis. Although in vitro studies of primary cells from endometriosis and xenografted human tissues in mice are essential to study cellular and molecular mechanisms associated with its pathobiology, mimicking establishment of disease outside of a woman’s body, let alone deciphering the role of a pathway such as AKT in the establishment of disease, has remained a challenge. Animal models of endometriosis range from stitching uterine tissues to the peritoneal wall (22–25) to injecting endometrial fragments or menstrual tissue into the peritoneal cavity (21, 26) to study the early events of disease establishment and progression. Syngeneic and autologous models in mouse strains, ICR, C57BL/6, and BALB/c, where endometrial fragments are autotransplanted into the peritoneal cavity, have been used to test various compounds (18 –22). The major advantage of using such models is the presence of an intact immune system, which is lost in xenotransplant models. These models do not, however, allow for the study of how a particular gene or pathway influences endometriosis,
unless the endometrial tissues are manipulated before implantation. For example, overexpression or knockdown of genes or even labeling of the cells in the tissues can be done to determine how a gene influences establishment of disease. Efficient delivery of gene constructs to all cells remains a limitation to this type of manipulation. Transgenic mice have been used to study pathogenic progression of endometriosis (22). Endometriosis was induced in steroid receptor coactivator-1 (SRC-1)–null mice as well as Tnf⫺/⫺ and Mmp9⫺/⫺ mice by stitching uterine tissues to the peritoneum (22). This group demonstrated that a 70kDa SRC-1 proteolytic isoform forms from activation of TNF␣-induced matrix metallopeptidase 9 (MMP9) activity in endometriotic mouse tissue. The endometriotic 70kDa SRC-1 C-terminal fragment prevented TNF␣-mediated apoptosis in human endometrial epithelial cells and caused epithelial-mesenchymal transition and the invasion of human endometrial cells. In another study, estrogen receptor-␣ (ER␣)-knockout and ER-knockout mice were used to study the role of ER and E2 in endometriosis (27). They induced endometriosis in these immunocompetent mice using the donor/recipient transplantation strategy and demonstrated the importance of ER␣ for establishment of endometriosis lesions. To our knowledge,
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Figure 5. Continued.
ours is the first study using a bigenic mouse to determine the role of a signaling pathway in the establishment of endometriosis. Specifically, only cells expressing PR have Pten deleted in 1 allele, which means the uterus and not the peritoneal tissues have Pten deletion. This bypasses the need for gene manipulation in cells before induction of endometriosis, does not require surgical stitching of tissues, and does not require transplantation of donor transgenic tissues into wild-type recipients. With our autologous implantation of uterine tissues from PRcre/⫹Ptenf/⫹ mice, we demonstrate that the AKT pathway promotes establishment of lesions, specifically, that aberrant signaling within the eutopic endometrium causes ectopic lesion formation. Improvements to this model can be made in terms of detecting and quantitating ectopic lesions. As only those visible by dissecting scopes were identified, there could have been smaller lesions that formed that were not visible. Labeling of uterine tissues before transplantation into the peritoneum, whether it be through flu-
orescent labels such as calcein AM fluorescent dye or by crossing mice with mice harboring green fluorescent protein, would improve detection of lesions. The decreased number of lesions found in both Ptenf/⫹ or PRcre/⫹Ptenf/⫹ mice in response to MK-2206 treatment could be due to either the inability to attach or rapid regression after attachment. In other words, increased AKT activity could promote attachment of tissues through aberrant expression of adhesion molecules, or inactivation of this pathway compromises its survival and causes attached lesions to regress. Additional studies will be required to understand how inhibiting AKT results in fewer ectopic lesions. We have demonstrated recently that levels of PR increase in human endometriotic stromal cells in response to MK-2206 (13). Interestingly, we see an increase in PR protein in the stroma of ectopic tissues in the PRcre/⫹ Ptenf/⫹ mice treated with MK-2206. The consequence of this is unknown at this time, because the mice in this study
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doi: 10.1210/en.2013-1951
were ovariectomized and only E2 was given during the duration of the experiment. Unliganded PR activity could contribute to the observations in this study. This requires further experimentation potentially with PR antagonists to better understand the role of unliganded PR in AKTmediated attachment and survival of lesions. If this occurs in the eutopic endometrium, modulation of progesterone function could affect fertility. Given our longstanding interest in AKT/PR crosstalk in the endometrium (13, 28 – 32), this mouse model is ideal to study progesterone sensitivity in the eutopic endometrium in the PRcre/⫹Ptenf/⫹ mice and its effects on fertility. Whether the presence of ectopic lesions can affect fertility in this mouse model, whether inhibition of AKT sensitizes these mice to progesterone, and identification of the endometrial markers involved remain to be investigated. The use of mouse models such as the one reported here provides invaluable information regarding the functional role of a specific gene or pathway in a disease process. Here we have demonstrated that deletion of Pten in 1 allele in PR-expressing cells promotes the establishment of endometriosis-like lesions in an autologous implantation model of endometriosis. The ability of a specific AKT inhibitor to decrease the number of lesions established indicates that the AKT pathway is critical for the establishment of this disease in this mouse model. As with any mouse model for human disease, data should be properly interpreted within the boundaries of its context. As such, questions remain as to whether women with hyperactivated AKT signaling in their eutopic endometrium are more prone to developing endometriosis, because retrograde menstruation occurs in most premenopausal women. Transgenic models such as the one presented in this study provide evidence that if there is an inherent defect in the eutopic endometrium that is placed in the peritoneum, this increases the likelihood of establishment of lesions. This is in contrast to the animal models where normal endometrial tissue or menstrual effluent are placed in the peritoneal cavity. Although there is no perfect model for human endometriosis aside from the human model, studies such as these provide the proof-of-principle evidence to explore and consider potential targets of therapy for endometriosis.
Acknowledgments We are grateful to the Mouse Histology and Phenotyping Core facilities at the Robert Lurie Cancer Center at Northwestern University. The MK-2206 was generously provided by Merck Sharp and Dohme Corp and the National Cancer Institute, National Institutes of Health (NIH).
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Address all correspondence and requests for reprints to: J. Julie Kim, PhD, Department of Obstetrics and Gynecology, Robert H. Lurie Comprehensive Cancer Center, 303 East Superior, Room 4 –117, Northwestern University, Chicago, IL 60611. Email: [email protected]. This work was supported by NIH Grants R01HD044715 (to J.J.K.), U54 HD07495 (to J.P.L.), and R01HD057873 (to J.W.J.); the Friends of Prentice Grants Initiative (to J.J.K.), and the Medical Student Summer Research Program (to L.L.). Disclosure Summary: The authors have nothing to disclose.
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