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Abstract High‐risk endometrial cancer has poor prognosis and is increasing in incidence. However, understanding of the molecular mechanisms which drive this disease is limited. We used genetically engineered mouse models (GEMM) to determine the functional consequences of missense and loss of function mutations in Fbxw7, Pten and Tp53, which collectively occur in nearly 90% of high‐risk endometrial cancers. We show that Trp53 deletion and missense mutation cause different phenotypes, with the latter a substantially stronger driver of endometrial carcinogenesis. We also show that Fbxw7 missense mutation does not cause endometrial neoplasia on its own, but potently accelerates carcinogenesis caused by Pten loss or Trp53 missense mutation. By transcriptomic analysis, we identify LEF1 signalling as upregulated in Fbxw7/FBXW7‐mutant mouse and human endometrial cancers, and in human isogenic cell lines carrying FBXW7 mutation, and validate LEF1 and the additional Wnt pathway effector TCF7L2 as novel FBXW7 substrates. Our study provides new insights into the biology of high‐risk endometrial cancer and suggests that targeting LEF1 may be worthy of investigation in this treatment‐resistant cancer subgroup.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Uterine gland development, also known as adenogenesis, is a key uterine morphogenic process indispensable for normal uterine function and fertility. Our earlier studies have reported that overactivation of TGFB receptor 1 (TGFBR1) in the mouse uterus using progesterone receptor (Pgr)-Cre recombinase causes female infertility, defective decidualization, and reduced uterine gland formation, a developmental milestone of postnatal uterus. To understand mechanisms that underpin the disrupted uterine gland formation in mice with sustained activation of TGFBR1, we raised the question of whether early postnatal adenogenesis was compromised in these mice. Experiments were designed using mice with constitutive activation of TGFBR1 driven by Pgr-Cre to determine the timing of adenogenic defects and potential mechanisms associated with dysregulation of adenogenic genes, luminal epithelial cell proliferation and endometrial fibrotic changes. Uterine tissues from mice with constitutive activation of TGFBR1 were collected during the critical time window of adenogenesis and analyzed together with age-matched controls. Multiple approaches including immunohistochemistry, immunofluorescence, Trichrome staining, quantitative real-time PCR, western blot, conditional knockout and human endometrial cell culture were utilized. TGFBR1 activation in the mouse uterus suppressed adenogenesis during postnatal uterine development, concomitant with the aberrant differentiation of uterine stromal cells. Analysis of transcript expression of WNT pathway components revealed dysregulation of adenogenesis-associated genes. Notably, the adenogenic defects occurred in spite of the increased proliferation of uterine luminal epithelial cells, accompanied by increased expression of genes associated with fibrotic changes. Moreover, the adenogenic defects were alleviated in mice where TGFBR1 was activated in presumably half of the complement of uterine cells. Our results suggest that altered differentiation of endometrial stromal cells and formation of stromal compartment promote adenogenic defects.

In the mammary gland, genetic circuits controlled by estrogen, progesterone, and prolactin, act in concert with pathways regulated by members of the epidermal growth factor family to orchestrate growth and morphogenesis during puberty, pregnancy and lactation. However, the precise mechanisms underlying the crosstalk between the hormonal and growth factor pathways remain poorly understood. We have identified the CUB and zona pellucida-like domain-containing protein 1 (CUZD1), expressed in mammary ductal and alveolar epithelium, as a novel mediator of mammary gland proliferation and differentiation during pregnancy and lactation. Cuzd1-null mice exhibited a striking impairment in mammary ductal branching and alveolar development during pregnancy, resulting in a subsequent defect in lactation. Gene expression profiling of mammary epithelium revealed that CUZD1 regulates the expression of a subset of the EGF family growth factors, epiregulin, neuregulin-1, and epigen, which act in an autocrine fashion to activate ErbB1 and ErbB4 receptors. Proteomic studies further revealed that CUZD1 interacts with a complex containing JAK1/JAK2 and STAT5, downstream transducers of prolactin signaling in the mammary gland. In the absence of CUZD1, STAT5 phosphorylation in the mammary epithelium during alveologenesis was abolished. Conversely, elevated expression of Cuzd1 in mammary epithelial cells stimulated prolactin-induced phosphorylation and nuclear translocation of STAT5. Chromatin immunoprecipitation confirmed co-occupancy of phosphorylated STAT5 and CUZD1 in the regulatory regions of epiregulin, a potential regulator of epithelial proliferation, and whey acidic protein, a marker of epithelial differentiation. Collectively, these findings suggest that CUZD1 plays a critical role in prolactin-induced JAK/STAT5 signaling that controls the expression of key STAT5 target genes involved in mammary epithelial proliferation and differentiation during alveolar development.

This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse-when crossed with the appropriate effector transgenic-will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues.

Ultrasonography is a powerful tool to non-invasively monitor in real time the development of the human fetus in utero. Although genetically engineered mice have served as valuable in vivo models to study both embryo implantation and pregnancy progression, such studies usually require sacrifice of parous mice for subsequent phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction in silico of high-frequency ultrasound (HFUS) imaging data for early detection and characterization of murine embryo implantation sites and their development in utero. With HFUS imaging followed by 3-D reconstruction, we were able to precisely quantify embryo implantation site number and embryonic developmental progression in pregnant C57BL6J/129S mice from as early as 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. using a VisualSonics Vevo 2100 (MS550S) transducer. In addition to measurements of implantation site number, location, volume and spacing, embryo viability via cardiac activity monitoring was also achieved. A total of 12 dams were imaged with HFUS with approximately 100 embryos examined per embryonic day. For the post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in mesh or solid overlay format enabled visual representation in silico of implantation site location, number, spacing distances, and site volume within each uterine horn. Therefore, this short technical report describes the feasibility of using 3-D HFUS imaging for early detection and analysis of post-implantation events in the pregnant mouse with the ability to longitudinally monitor the development of these early pregnancy events in a non-invasive manner. As genetically engineered mice continue to be used to characterize female reproductive phenotypes, we believe this reliable and non-invasive method to detect, quantify, and characterize early implantation events will prove to be an invaluable investigative tool for the study of female infertility and subfertility phenotypes based on a defective uterus.

Progesterone, via the progesterone receptor (PGR), is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights into the molecular mechanisms by which progesterone drives hESC decidualization, our findings provide a new conceptual framework that could lead to new avenues for diagnosis and/or treatment of adverse reproductive outcomes associated with a dysfunctional uterus.

Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the Pgr gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the Pgr deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.

AT-rich interactive domain 1A gene (ARID1A) loss is a frequent event in endometriosis-associated ovarian carcinomas. Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus, and 50% of women with endometriosis are infertile. ARID1A protein levels were significantly lower in the eutopic endometrium of women with endometriosis compared to women without endometriosis. However, an understanding of the physiological effects of ARID1A loss remains quite poor, and the function of Arid1a in the female reproductive tract has remained elusive. In order to understand the role of Arid1a in the uterus, we have generated mice with conditional ablation of Arid1a in the PGR positive cells (Pgrcre/+Arid1af/f; Arid1ad/d). Ovarian function and uterine development of Arid1ad/d mice were normal. However, Arid1ad/d mice were sterile due to defective embryo implantation and decidualization. The epithelial proliferation was significantly increased in Arid1ad/d mice compared to control mice. Enhanced epithelial estrogen activity and reduced epithelial PGR expression, which impedes maturation of the receptive uterus, was observed in Arid1ad/d mice at the peri-implantation period. The microarray analysis revealed that ARID1A represses the genes related to cell cycle and DNA replication. We showed that ARID1A positively regulates Klf15 expression with PGR to inhibit epithelial proliferation at peri-implantation. Our results suggest that Arid1a has a critical role in modulating epithelial proliferation which is a critical requisite for fertility. This finding provides a new signaling pathway for steroid hormone regulation in female reproductive biology and furthers our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in human reproductive disorders such as endometriosis.

Unlike cancers of related exocrine tissues such as the mammary and prostate gland, diagnosis and treatment of aggressive salivary gland malignancies have not markedly advanced in decades. Effective clinical management of malignant salivary gland cancers is undercut by our limited knowledge concerning the key molecular signals that underpin the etiopathogenesis of this rare and heterogeneous head and neck cancer. Without knowledge of the critical signals that drive salivary gland tumorigenesis, tumor vulnerabilities cannot be exploited that allow for targeted molecular therapies. This knowledge insufficiency is further exacerbated by a paucity of preclinical mouse models (as compared to other cancer fields) with which to both study salivary gland pathobiology and test novel intervention strategies. Using a mouse transgenic approach, we demonstrate that deregulation of the Receptor Activator of NFkB Ligand (RANKL)/RANK signaling axis results in rapid tumor development in all three major salivary glands. In line with its established role in other exocrine gland cancers (i.e., breast cancer), the RANKL/RANK signaling axis elicits an aggressive salivary gland tumor phenotype both at the histologic and molecular level. Despite the ability of this cytokine signaling axis to drive advanced stage disease within a short latency period, early blockade of RANKL/RANK signaling markedly attenuates the development of malignant salivary gland neoplasms. Together, our findings have uncovered a tumorigenic role for RANKL/RANK in the salivary gland and suggest that targeting this pathway may represent a novel therapeutic intervention approach in the prevention and/or treatment of this understudied head and neck cancer.

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.

As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.

Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

Early embryo miscarriage is linked to inadequate endometrial decidualization, a cellular transformation process that enables deep blastocyst invasion into the maternal compartment. Although much of the cellular events that underpin endometrial stromal cell (ESC) decidualization are well recognized, the individual gene(s) and molecular pathways that drive the initiation and progression of this process remain elusive. Using a genetic mouse model and a primary human ESC culture model, we demonstrate that steroid receptor coactivator-2 (SRC-2) is indispensable for rapid steroid hormone-dependent proliferation of ESCs, a critical cell-division step which precedes ESC terminal differentiation into decidual cells. We reveal that SRC-2 is required for increasing the glycolytic flux in human ESCs, which enables rapid proliferation to occur during the early stages of the decidualization program. Specifically, SRC-2 increases the glycolytic flux through induction of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3), a major rate-limiting glycolytic enzyme. Similarly, acute treatment of mice with a small molecule inhibitor of PFKFB3 significantly suppressed the ability of these animals to exhibit an endometrial decidual response. Together, these data strongly support a conserved mechanism of action by which SRC-2 accelerates the glycolytic flux through PFKFB3 induction to provide the necessary bioenergy and biomass to meet the demands of a high proliferation rate observed in ESCs prior to their differentiation into decidual cells. Because deregulation of endometrial SRC-2 expression has been associated with common gynecological disorders of reproductive-age women, this signaling pathway, involving SRC-2 and PFKFB3, promises to offer new clinical approaches in the diagnosis and/or treatment of a non-receptive uterus in patients presenting idiopathic infertility, recurrent early pregnancy loss, or increased time to pregnancy.

Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone-Indian hedgehog-Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.