Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease.

Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal endometrial proliferation and, ultimately, endometrial adenocarcinoma, the fourth most common cancer in women. We have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of progesterone receptor (PR) and steroid receptor coactivator (SRC-1) action in the uterus. Here, we demonstrate that absence of Mig-6 in mice results in the inability of P4 to inhibit E2-induced uterine weight gain and E2-responsive target genes expression. At 5 months of age, the absence of Mig-6 results in endometrial hyperplasia. Ovariectomized Mig-6d/d mice exhibit this hyperplastic phenotype in the presence of E2 and P4 but not without ovarian hormone. Ovariectomized Mig-6d/d mice treated with E2 developed invasive endometrioid-type endometrial adenocarcinoma. Importantly, the observation that endometrial carcinomas from women have a significant reduction in MIG-6 expression provides compelling support for an important growth regulatory role for Mig-6 in the uterus of both humans and mice. This demonstrates the Mig-6 is a critical regulator of the response of the endometrium to E2 in regulating tissue homeostasis. Since Mig-6 is regulated by both PR and SRC-1, this identifies a PR, SRC-1, Mig-6 regulatory pathway that is critical in the suppression of endometrial cancer.

estrogen, endometrial cancer, progesterone, progesterone receptor, SRC-1 The ovarian steroid hormones progesterone (P4) and estrogen (E2) are essential regulators of reproductive events associated with all aspects involved in the establishment and maintenance of pregnancy (1, 2). P4 and E2 function both synergistically and antagonistically to regulate appropriate uterine function by acting through their cognate nuclear receptors to coordinate uterine epithelial–stromal communication in the regulation of endometrial cell proliferation and differentiation. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal endometrial proliferation and endometrial adenocarcinoma. Endometrial cancer is the most common gynecological cancer in the United States (3). Endometrioid-type endometrial adenocarcinoma and its precursor lesion, endometrial hyperplasia, are associated with unopposed estrogen exposure (4, 5). Elucidating the molecular mechanisms by which the steroid hormones control uterine physiology is important to understanding the pathology of these diseases. The action of the steroid hormone receptors are modulated in part by members of the p160 family of steroid receptor coactivators (SRCs). The SRCs facilitate steroid hormone receptor regulation of gene transcription by executing a diverse number of processes including chromatin remodeling, RNA processing, and receptor degradation (6). The SRC family is composed of 3 distinct but functionally and structurally related members: SRC-1/NcoA1 (7), SRC-2/TIF2/GRIP1 (8), and SRC-3/RAC3/ACTR/pCIP/AIB1/TRAM1 (9). The SRC family members enhance the transcriptional activity of a variety of nuclear receptors, including estrogen receptor α (ERα, also known as ESR1), estrogen receptor β (ERβ, also known as ESR2), glucocorticoid receptor (GR), and progesterone receptor (PR) (10–12) and are expressed in a variety of hormone-responsive tissues including the uterus, brain, prostate, liver, and breast (7, 13–15). Although female SRC-1−/− mice are fertile, reduced steroid sensitivity in the uterus of SRC-1−/− mice is demonstrated by a reduction in the ability of the endometrial stroma cells to undergo a decidual transformation (7). This phenotype indicates that these coregulators may play a necessary role in coordinating steroid hormone regulation of normal reproductive uterine function. Using high-density DNA microarray technology, we have identified Mig-6 as a gene whose regulation by P4 is dependent upon SRC-1. Mig-6 is an immediate early response gene that can be induced by various mitogens and commonly occurring chronic stress stimuli (16–18). Mig-6 is an adaptor molecule containing a CRIB domain, a src homology 3 (SH3) binding domain, a 14–3-3 binding domain, and an epidermal growth factor receptor (EGFR) binding domain (19, 20). Sustained Mig-6 expression is thought to trigger cells to initiate hypertrophy in chronic pathological conditions, such as diabetes and hypertension (21, 22). Ablation of Mig-6 in mice has led to the development of animals with epithelial hyperplasia, adenoma, and adenocarcinomas in organs, such as the lung, gallbladder, and bile duct (23, 24). Mig-6 is located on human chromosome 1p36, a locus frequently associated with human cancer (25, 26). Decreased expression of Mig-6 is observed in human breast carcinomas that correlate with reduced overall survival of breast cancer patients (27, 28). Mig-6 is mutated in human non-small-cell lung cancer (NSCLC) cell lines NCI-H226 and NCI-H 322M and in one primary human lung cancer (24). Recently, altered Mig-6 expression has been observed in endometrial RNA taken from women with endometriosis (29). These data point to Mig-6 as a tumor suppressor gene in both mice and humans. However, the function of Mig-6 in reproductive biology has remained elusive. In this study, we used conditional ablation of Mig-6 in mice to demonstrate that Mig-6 is an important molecule in uterine physiology in part by regulating the ability of P4 to attenuate E2 signaling. These mice develop endometrial hyperplasia within 5 months, and, if exposed to exogenous E2 for 3 months, develop invasive type I endometrial adenocarcinoma. Analysis of Mig-6 in the human endometrium shows that the expression of Mig-6 is decreased in human endometrial cancers. Thus, these results demonstrate the importance of Mig-6 in steroid hormone regulation and human endometrial cancer.

Identification of P4 and SRC-1 Regulated Genes Using Microarray Analysis. The impact of SRC-1 ablation on uterine mRNA expression profiles in response to P4 was examined by isolating RNA from ovariectomized SRC-1−/− and wild-type mice that were treated with either vehicle (sesame oil) or P4 (1 mg) for 4 h (n = 9 per genotype per treatment). The experimental design not only allowed for the effect of SRC-1 in SRC-1−/− mice to be determined, but also afforded the comparison of the effect of vehicle and P4 on wild-type and SRC-1−/− mice. Using this experimental design, the identification of differentially expressed genes was derived from 3 physiologically relevant comparisons(Fig. S1A). The summary of the number of differentially expressed genes for the 3 comparisons is shown in supporting information (SI) Table S1. Comparison 1 identified genes differentially expressed between wild-type and SRC-1−/− mice in the absence of P4 treatment. Comparison 2 identified genes regulated by SRC-1 in the presence of P4, and comparison 3 identified P4 responsive genes in wild-type mice. A complete list of the increased and decreased genes that are identified as significant for comparisons 1–3 are presented in Table S2. To identify the impact of SRC-1 ablation on P4 induction of gene expression, we identified genes that overlapped between comparisons 2 and 3. (Fig. S1B) graphically shows the overlap in the analysis of SRC-1-dependent P4 responsive genes (comparison 2) and P4 responsive genes (comparison 3). We identified 5 targets of which the mRNA expression was induced by P4 in wild-type mice but not in SRC-1−/− mice (Fig. S1C). These genes were Mig-6, Myc, Il13ra2, and an EST. The EST was not analyzed further because it did not have any known homologous gene and its expression was not PR-dependent, based on our previous microarray data (30). To confirm whether the gene regulatory effects of P4 are mediated through SRC-1 and PR, we ovariectomized SRC-1−/− mice, progesterone receptor knockout (PRKO), and WT mice. After P4 treatment for 4 h, the uteri were collected, RNA was prepared, and total RNA was analyzed by real time RT-PCR. Expression of all 3 genes was highly induced by P4 in the wild-type mice, but induction was significantly decreased in the SRC-1−/− mice (Fig. S1D). The P4 induction was also not detected in the PRKO mice. Therefore, real time RT-PCR confirmed the results of the microarray analysis and further showed that their expression was PR dependent. Ablation of Mig-6 in mice leads to animals with epithelial hyperplasia, adenoma, and adenocarcinoma in organs like the lung, gallbladder, and bile duct (24). This tumor suppressor function in mice (23, 24) is also observed in humans (25, 26). The fact that Mig-6 was found to be a SRC-1-dependent PR-responsive gene prompted us to investigate its function in the murine endometrium. The spatial expression of Mig-6 was examined by in situ hybridization (Fig. S2). Mig-6 transcripts were undetectable in the vehicle-treated uterus. However, Mig-6 mRNAs were strongly expressed in the stroma, luminal epithelium, and glandular epithelium by P4 treatment. In contrast, these mRNAs were significantly decreased in the uteri of ovariectomized SRC-1−/− and PRKO mice receiving P4 treatment. These results demonstrate that Mig-6 is PR and SRC-1 dependently regulated in all compartments of the endometrium including the epithelium and stroma but not myometrium. The Epithelial Hyperplastic Effect in Mice with Conditional Mig-6 Ablation in the Uterus. Since Mig-6 ablation results in numerous pathologies and decreased longevity (23, 24, 31, 32), our ability to investigate the role of Mig-6 in the mouse uterus is severely limited. To effectively investigate the role of Mig-6 in the regulation of uterine function and the response to hormonal stimulation, we generated a Mig-6 conditional null allele, the Mig-6 flox allele (Mig-6f/f) (33). Mig-6f/f mice were bred to PRCre (34) mice to generate conditional Mig-6 ablation (PRcre/+Mig-6f/f; Mig-6d/d) in the reproductive tract. Significant morphological differences in the uteri of Mig-6d/d and Mig-6f/f were not observed at 2 months of age. However, analysis of Mig-6d/d uteri compared to Mig-6f/f mice at 5 months of age (n = 10) showed a significant increase in wet weight (Fig. 1 A and B). Histological analysis of these uteri showed an increase in the number of endometrial glands and in the gland/stroma ratio in the uterus of Mig-6d/d mice (Fig. 1 C–H); however, the myometrium was not enlarged. These histological changes demonstrate that the uterus of the Mig-6d/d mouse displays endometrial hyperplasia, a predisposing factor to endometrial adenocarcinoma in humans. To determine if the endometrial epithelial hyperplasia in the Mig-6d/d mice is caused by alterations of ER signaling, cell proliferation, and/or apoptosis, we performed immunohistochemical staining for ERα, Ser 118 phospho-ERα, phosphohistone H3, and caspase 3. Immunohistochemical staining of caspase 3 was unchanged in the luminal or glandular epithelium of nonpregnant Mig-6d/d mice compared to wild-type control mice (data not shown). However, immunohistochemical staining of phosphohistone H3 showed that the endometrial glandular cells exhibited a significant increase in proliferation in the Mig-6d/d mice (Fig. 1D). Supportive of E2 being the cause of the increase in epithelial proliferation, ERα and phosphorylation of ERα at Ser 118 were also significantly increased in the endometrial glands (Fig. 1 F and H). Steroid Hormone Regulation of Mig-6 in the Murine Uterus. To determine if the uteri of Mig-6d/d exhibited an altered response to steroid hormones, Mig-6d/d and Mig-6f/f mice were ovariectomized and treated with vehicle, E2, P4, or E2 + P4 daily for 3 days and killed 6 h after the last injection (n = 5 per genotype per treatment). Mig-6d/d and Mig-6f/f uteri showed no difference in weight gain or expression of PR, ERα, or their respective target genes when the mice were treated with vehicle, E2, or P4. However, Mig-6d/d mice treated with E2 + P4 showed a significant increase in uterine weight (Fig. 2A), vascularization (Fig. 2B), and expression of endometrial epithelial ERα target genes, Ltf (lactotransferrin), Clca3 (chloride channel calcium activated 3), and C3 (complement component 3), (Fig. 2C) compared to E2 + P4-treated Mig-6f/f uteri. The expression of 2 endometrial epithelial PR target genes, Fst (follistatin) and Areg (amphiregulin), is not changed in the Mig-6d/d mice in response to E2 + P4 treatment (Fig. 2D). However, the expression of PR mRNA is significantly decreased in the Mig-6d/d mice compared to that of Mig-6f/f mice. Immunohistochemical analysis of PR expression in the Mig-6d/d mice shows that epithelial PR expression is normal but the expression of PR in the endometrial stroma cells is significantly reduced (Fig. 2 E and F). Since P4 attenuates E2 regulation of proliferation and gene expression by regulating the expression of a yet-to-be-identified paracrine signal from the stromal cells to the epithelial cells, the regulation of the expression of PR in the endometrial stromal cells by Mig-6 is critical for the ability of P4 to attenuate the E2-regulated uterine weight gain, vascularization, and expression of ER target genes. To assess the role of Mig-6 in uterine function, female Mig-6d/d mice were mated to wild-type male mice for 6 months. Mig-6d/d mice were completely infertile (Table S3). Since the PRCre mouse shows Cre recombinase activity in the pituitary, ovary, uterus, and mammary gland, the cause of infertility in these mice may be the result of a defect in any of these tissues (34). To test for an ovarian phenotype, female Mig-6f/f and Mig-6d/d mice were examined for their ability to ovulate normally in response to a superovulatory regimen of gonadotropins. Mig-6f/f and Mig-6d/d yielded 24.75 ± 6.61 and 24.83 ± 7.88 oocytes, respectively. Also, Mig-6f/f and Mig-6d/d mice did not show any alterations in ovarian morphology and exhibited a normal estrus cycle (data not shown). Finally, cycling female Mig-6f/f and Mig-6d/d mice exhibited normal levels of serum P4 and E2 at 2 and 5 months of age (data not shown). These results show that ovarian morphology, steroidogenesis, and function were not affected in the Mig-6d/d females. These data suggest that the defects observed in the Mig-6d/d mice are inherent to the uterus. Thus, to determine if the infertility was in part because of loss of the ability of the uterus to support implantation, we investigated the ability of the uterus to undergo a decidual reaction. Ovariectomized female Mig-6f/f and Mig-6d/d mice (n = 3) were treated with hormones and the uterus was mechanically stimulated to mimic the signaling of the embryo at implantation and to induce decidualization. Gross anatomy of the decidual and control horn showed an increase in size for Mig-6d/d mice compared to the Mig-6f/f mice (Fig. S3). However, the ratio of stimulated-to-unstimulated horn weight was not changed in the Mig-6f/f and Mig-6d/d mice. These results suggest that ablation of Mig-6 in PR-expressing cells alters murine fertility because of dysregulation of E2 and P4 but not because of defects in ovarian or uterine function. Tumor Suppressor Function of Mig-6 in the Uterus. As endometrial hyperplasia is an immediate precursor to endometrioid-type endometrial carcinoma, the hyperplastic phenotype in the Mig-6d/d mice suggests that Mig-6 has a tumor suppressor role in the tumorigenesis of endometrial cancer. We examined the role of ovarian steroid hormones in the development of the hyperplastic phenotype in Mig-6d/d mice. Six-week-old Mig-6f/f and Mig-6d/d mice were ovariectomized and treated with vehicle, E2, or E2 + P4 and killed at 5 months of age (n = 10 per genotype per treatment). Ovariectomized Mig-6d/d mice did not develop endometrial hyperplasia as observed in intact Mig-6d/d mice (Fig. 3A). This demonstrates that the endometrial hyperplasia phenotype of Mig-6d/d mice is dependent on ovarian hormone stimulation. Mig-6d/d mice treated with E2 for 3 months showed a significant increase in uterine weight compared to Mig-6f/f mice (Fig. 3B). Although the Mig-6f/f mice showed endometrial hyperplasia as expected from chronic E2 treatment, they did not show the pathology observed in the uteri of intact Mig-6d/d mice or Mig-6d/d mice treated with E2. All of the Mig-6d/d mice treated with E2 developed invasive endometrioid-type endometrial adenocarcinoma. The neoplastic endometrial glands in the Mig-6d/d mice invaded through the uterine muscle wall and invaded adjacent structures such as the colon, pancreas, and skeletal muscle. This result demonstrates that Mig-6 may have an estrogen-dependent tumor suppressor function in endometrial cancer. Finally, ovariectomized Mig-6d/d mice treated with E2 + P4 for 3 months showed a significant increase in uterine wet weight and developed endometrial hyperplasia (Fig. 3C) but not the endometrial carcinoma observed in the E2-treated mice. Therefore, P4 treatment was able to attenuate the pathology observed in the Mig-6d/d mice after E2 treatment but not completely block the endometrial hyperplasia. These results demonstrate that Mig-6 is important to regulate the response of the uterus to E2 stimulation in part by mediating the protective action of P4. However, Mig-6 also regulates other pathways independent of P4 that control endometrial cell proliferation. Downregulation of MIG-6 in Human Endometrial Cancer. Mig-6 ablation shows altered uterine function because of the inability of P4 to attenuate E2 action, which is a common characteristic of endometrial dysfunction in humans (35, 36). The expression of Mig-6 in the human endometrium during the menstrual cycle was determined by real-time quantitative PCR and immunohistochemistry. The expression of Mig-6 was highest in the early secretory phase of the cycle (Fig. 4A) and this increase in expression was the result of an increase in the expression of Mig-6 in the endometrial epithelium (Fig. 4B). The increase in expression of Mig-6 during this phase of the cycle correlates with P4 regulation as observed in the mouse. We investigated the expression of MIG-6 in endometrial biopsies from patients with endometrioid carcinoma (n = 10) and normal endometrium (n = 5). The level of MIG-6 mRNA is significantly decreased in patients with endometrioid carcinoma (32.8%) compared to endometrial biopsies taken from normal women during the secretory phase of the cycle. (Fig. 4C). Immunohistochemical analysis also shows a decrease in the protein level of MIG-6 in patients with endometrial cancer compared to normal women (Fig. 4D).

P4, acting through its nuclear receptors, plays important roles in uterine functions associated with the establishment and maintenance of pregnancy (37, 38). The identification of P4-regulated pathways in the uterus is thus crucial for understanding the impairments that underlie disruption of steroid hormone control of uterine cell proliferation and differentiation. In previous studies, we have identified P4-regulated genes using microarray analysis on P4-treated PRKO uteri (30). We have identified Mig-6 as a target of SRC-1 and PR in the uterus. Ablation of Mig-6 in mice results in a 50% reduction of the Mig-6−/− litter size for an unidentified reason (31, 33). The 50% of the Mig-6−/− mice that escape the lethal phenotype, develop joint deformities, and the majority of mice die within 6 months. These mice also develop neoplasias of the lungs and skin (23, 31). The embryonic lethality and multitissue carcinogenesis makes it difficult to investigate the impact of ablation of Mig-6 in uterine biology. To effectively investigate the role of Mig-6 in the uterus, we generated a Mig-6 conditional null allele by introducing LoxP sites (33). The uterine histology of Mig-6d/d mice demonstrate epithelial hyperplasia similar to the 9-month-old survival Mig-6−/− mouse (33). This hyperplastic phenotype of Mig-6d/d and Mig-6−/− supports the tumor suppressor role of Mig-6 in the tumorigenesis of endometrial cancer. Mig-6 mediates the ability of P4 to regulate E2-dependent uterine weight gain. Normally, E2 stimulates uterine growth and epithelial cell proliferation (39). P4 antagonizes E2 actions, such as the stimulation of proliferation of the epithelial cells in the mouse uterus (40). Mig-6d/d and Mig-6f/f mice both respond to E2 treatment with an increase in uterine wet weight. This indicates that ablation of Mig-6 does not enhance the effect of E2 treatment alone. When we examined the ability of P4 to inhibit E2-induced uterine hypertrophy, P4 did not inhibit the E2-induced hypertrophy in Mig-6d/d mice (Fig. 2). In a separate experiment, we treated wild type, PRcre/+, Mig-6f/f, and Mig-6d/d with E2 and P4 for 3 days and measured uterine weight gain. As expected, the response of PRcre/+, Mig-6f/f, and wild-type mice was similar with P4 dampening the E2-induced uterine hypertrophy. The Mig-6d/d mice again demonstrated an increase in uterine weight gain in the presence of P4 and E2. These results demonstrate that Mig-6 mediates the ability of P4 to regulate E2-dependent uterine weight gain. Examination of endometrial epithelial P4 target gene expression showed no change in the ability of PR to regulate the expression of Fst and Areg in the Mig-6d/d mouse. Interestingly, the expression of PR mRNA is significantly decreased in the Mig-6d/d mice compared to that of Mig-6f/f (Fig. 2). Immunohistochemical analysis of PR expression in the Mig-6d/d mice showed that epithelial PR expression is not altered but that PR expression in the endometrial stroma cells is significantly reduced. P4 attenuates E2 regulation of proliferation and gene expression by regulating the expression of a yet-to-be-identified paracrine signal from the stromal cells to the epithelial cells (41, 42). However, the increase in ERα target gene expression at 8 weeks of age was not the result of a change in ERα or coactivator level (data not shown). Interestingly, there is an increase in ERα levels at 5 months of age with the epithelial hyperplastic phenotype, but its impact on gene expression at this time remains unknown. Nonetheless, we have gained valuable insight into steroid hormone regulation in the uterus and Mig-6's role in that regulation. In addition, we have shown that MIG-6 is expressed in the human endometrium in a cycle-dependent manner that correlates with its being under the control of P4 as observed in the mouse. We have also demonstrated that its expression is decreased in endometrioid carcinoma when compared to expression in normal endometrium during the secretory phase. Since the endometriod carcinoma samples assayed were acquired from postmenopausal women, the decrease in MIG-6 expression may be a result of the hormonal status of postmenopausal women. Endometrial diseases such as endometrial cancer and endometriosis are known to be hormone-related malignancies. Exposure to E2 is one of the endocrine risk factors for developing endometrial cancer and endometriosis (35), and a lower incidence of these diseases is noted in women with decreased endogenous E2 production. In contrast, P4 exposure is a negative risk factor for these disease (43), and pregnancy or progestin-based therapies can lead to disease regression in some women (44). Since endometrial cancer is a disease most often found in postmenopausal women, the lower levels of progesterone in these women may result in a lack of induction of MIG-6 that may be required to regulate endometrial epithelial proliferations. Recently, published microarray gene expression profiles of the endometrium of women with or without endometriosis showed that a number of P4 target genes incluing MIG-6 were dysregulated during the window of implantation, at which time the endometrium is exposed to the highest levels of P4 (29, 45). Mig-6d/d mice developed endometrial adenocarcinoma with E2 but not with E2 + P4. Thus, progesterone acting through the PR may be beneficial for controlling endometrial cancer by inducing Mig-6 expression. The Mig-6d/d mice treated with E2 + P4 still develop endometrial hyperplasia, which suggests that Mig-6 is a critical factor involved in P4 protection against the development of endometrial cancer. However, the ability of P4 to prevent E2-induced endometrial adenocarcinoma despite the absence of Mig-6 and the reduction in PR levels in the stroma implicates additional mechanisms of protection. Furthermore, the development of E2-induced endometrial adenocarcinoma in Mig-6d/d mice suggests that Mig-6 has an important role as a negative regulator of E2-induced tumorigenesis. However, it is not only the expression of MIG-6 that has been shown to be altered in cancer. In addition, the MIG-6 gene has been shown to be mutated in the human NSCLC cell lines NCI-H226 and NCI-H 322M, and in 1 primary human lung cancer (24). Thus, when examining cases of P4 resistance in endometrial cancer, aside from examining MIG-6 expression levels, endometrial carcinoma samples should also be assayed for mutations in MIG-6 as these mutations may be as detrimental as loss of MIG-6 expression. Regardless, the molecular mechanism by which loss of MIG-6 function, either by loss of expression or mutations in the MIG-6 gene, regulates endometrial cancer needs to be addressed. Further dissection of the intricacies of these pathways will lend important insight into the mechanisms that regulate endometrial tumorigenesis. In summation, Mig-6 ablation results in increased ERα activity in the presence of P4, which normally antagonizes ER activity. In humans, we have shown that the expression of MIG-6 is decreased in human endometrial endometrioid carcinoma. Our findings demonstrate that Mig-6 is a novel mediator of steroid hormone signaling in the uterus. Endometrial cancer is a uterine disease in which hormonal regulation is perturbed. The altered expression of MIG-6 in endometrial cancer may serve as a possible cause of these pathologies. Mice with conditional ablation of Mig-6 in the uterus provide a more faithful model for human endometrial cancer with respect to pathology and hormone sensitivity than any previous models. This model is useful for finding new targets for the diagnosis and treatment of endometrial cancer. Determining how Mig-6 mediates this action will be critical in understanding the role of steroid hormone signaling in endometrial function and dysfunction and in developing therapy for both uterine diseases.

Animals and Hormone Treatments. Mice were maintained in the designated animal care facility at Baylor College of Medicine according to the institutional guidelines for the care and use of laboratory animals. For microarray analysis, ovariectomized SRC-1−/− and wild-type mice were injected with vehicle (sesame oil) or P4 (1 mg/mouse in 100 μL sesame oil) for 4 h (n = 9 per genotype per treatment). For the steroid hormone treatment, ovariectomized mice Mig-6f/f and Mig-6d/d mice were injected with 1 of the following: vehicle (sesame oil), P4 (1 mg/mouse), E2 (0.1 μg/mouse), P4 plus E2 (n = 5 per genotype per treatment). Hormone injections were repeated every day to prevent the effect of hormone degradation by metabolism. Mice were killed 6 h after the third injection. At the time of dissection, uterine tissues were placed in the appropriate fixative or flash frozen and stored at −80 °C. For the endometrial cancer study, ovariectomized Mig-6d/d and Mig-6f/f mice received a pellet of either vehicle (beeswax), E2 (20 μg/pellet), or E2 + P4 (20 mg/pellet) at 8 weeks of age. Mice were killed at 5 months of age (n = 10 per genotype per treatment). Human Samples. Endometrial samples were obtained from 18 normally cycling women, aged 18–35, after written informed consent, under an approved protocol by the Institutional Review Board at Baylor College of Medicine. The endometrial sample was removed from the uterine fundus with a Pipelle (circle R) biopsy catheter. Tissues were fixed in formalin and embedded in paraffin for histological analysis or snap frozen on dry ice. Histological samples were examined blindly by an independent pathologist, and phases were assigned according to the Noyes criteria (46). Endometrioid carcinoma samples were derived from hysterectomy surgical specimens submitted to the Department of Pathology, M. D. Anderson Cancer Center following the guidelines approved by the M. D. Anderson Cancer Center Committee on Human Research and the Baylor College of Medicine Committee on the Use of Human Subjects in Medical Research. Classification was verified by light microscopic examination of hematoxylin and eosin-stained slides by gynecologic pathologist Russell R. Broaddusd. Normal endometrial samples were obtained from 5 cycling women (4 secretory and 1 atrophic stage) and endometrioid carcinoma samples were obtained from 8 postmenopausal women and 2 cycling women. Endometrioid carcinoma samples from patients with a prior history of hormone use, radiation treatment, or chemotherapy were not used for this study. Microarray Analysis. Microarray analysis was performed by Affymetrix murine genome U74Av2 mouse oligonucleotide arrays (Affymetrix) as previously described (30). All experiments were repeated 3 times. Briefly, we used DNA-Chip analyser dChip version 1.3 (47). We selected differentially expressed genes within each time exposure using 2 sample comparisons according to the following criteria: lower bound of 90% confidence interval of fold change greater than 1.2 and absolute value of difference between group means greater than 80. After excluding expressed sequence tags with no functional annotation, differentially expressed genes were classified according to gene ontology function using Affymetrix annotation, literature search in PubMed and GenMAPP (48). Quantitative Real-Time RT-PCR. Expression levels of regulated genes were validated by real time RT-PCR. Real-time probes and primers were purchased from Applied Biosystems). All real-time RT-PCR was done using RNA samples from 3 separate mice and mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. Statistical analyses used 1-way ANOVA followed by Tukey's post hoc multiple range test with the Instat package from GraphPad. Immunohistochemistry. Uterine sections from paraffin-embedded tissue were cut at 5 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series. Sections were preincubated with 10% normal serum in PBS (pH 7.5) and then incubated with 1:1,000 anti-Mig-6 antibody (Sigma-Aldrich) in 10% normal serum in PBS (pH 7.5). On the following day, sections were washed in PBS and incubated with a secondary antibody (5 μL/mL; Vector Laboratories) for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite ABC kit (Vector Laboratories). Supplementary Material Acknowledgments. We thank Jinghua Li and Bryan Ngo for technical assistance; Heather L. Franco and Janet DeMayo for manuscript preparation. This work was supported by the National Institute of Child Health and Human Development and the National Institutes of Health (NIH) as part of the Cooperative Program on Trophoblast-Maternal Tissue Interactions U01HD042311 and NIH Grant U54HD0077495 (to F.J.D.), SPORE in Uterine Cancer NIH 1P50CA098258–01 (to R.R.B.), NIH Grant R01HD057873 and pilot grant from Specialized Program of Research Excellence in Uterine Cancer NIH 1P50CA098258–01 (to J.W.J.), NIH Grant RO1-CA77530 and the Susan G. Komen Award BCTR0503763 (to J.P.L.), NIH Grant 2U54HD035041–11 (to S.L.Y. and B.A.L.), and by the generosity of the Jay and Betty Van Andel Foundation (to Y.W.Z. and G.V.W.). Footnotes The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/cgi/content/full/0903632106/DCSupplemental.

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