Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Contributions of white adipose tissue to energy requirements for female reproduction.

Body composition impacts female fertility and there are established relationships between adipose tissue and the reproductive system. Maintaining functional adipose tissue is vital for meeting the energetic demands during the reproductive process, from ovulation to delivery and lactation. White adipose tissue (WAT) shows plastic responses to daily physiology and secretes diverse adipokines that affect the hypothalamic-pituitary-ovarian axis, but many other interorgan interactions remain to be determined. This review summarizes the current state of research on the dialogue between WAT and the female reproductive system, focusing on the impact of this crosstalk on ovarian and endometrial factors essential for fecundity.

A GREB1-steroid receptor feedforward mechanism governs differential GREB1 action in endometrial function and endometriosis.

Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor's transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.

Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor.

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-ß estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy.

Steroid receptor coactivator-2 drives epithelial reprogramming that enables murine embryo implantation.

Although we have shown that steroid receptor coactivator-2 (SRC-2), a member of the p160/SRC family of transcriptional coregulators, is essential for decidualization of both human and murine endometrial stromal cells, SRC-2's role in the earlier stages of the implantation process have not been adequately addressed. Using a conditional SRC-2 knockout mouse (SRC-2d/d ) in timed natural pregnancy studies, we show that endometrial SRC-2 is required for embryo attachment and adherence to the luminal epithelium. Implantation failure is associated with the persistent expression of Mucin 1 and E-cadherin on the apical surface and basolateral adherens junctions of the SRC-2d/d luminal epithelium, respectively. These findings indicate that the SRC-2d/d luminal epithelium fails to exhibit a plasma membrane transformation (PMT) state known to be required for the development of uterine receptivity. Transcriptomics demonstrated that the expression of genes involved in steroid hormone control of uterine receptivity were significantly disrupted in the SRC-2d/d endometrium as well as genes that control epithelial tight junctional biology and the emergence of the epithelial mesenchymal transition state, with the latter sharing similar biological properties with PMT. Collectively, these findings uncover a new role for endometrial SRC-2 in the induction of the luminal epithelial PMT state, which is a prerequisite for the development of uterine receptivity and early pregnancy establishment.

The NR2F2-HAND2 signaling axis regulates progesterone actions in the uterus at early pregnancy.

Endometrial function is dependent on a tight crosstalk between the epithelial and stromal cells of the endometrium. This communication is critical to ensure a fertile uterus and relies on progesterone and estrogen signaling to prepare a receptive uterus for embryo implantation in early pregnancy. One of the key mediators of this crosstalk is the orphan nuclear receptor NR2F2, which regulates uterine epithelial receptivity and stromal cell differentiation. In order to determine the molecular mechanism regulated by NR2F2, RNAseq analysis was conducted on the uterus of PgrCre;Nr2f2f/f mice at Day 3.5 of pregnancy. This transcriptomic analysis demonstrated Nr2f2 ablation in Pgr-expressing cells leads to a reduction of Hand2 expression, increased levels of Hand2 downstream effectors Fgf1 and Fgf18, and a transcriptome manifesting suppressed progesterone signaling with an altered immune baseline. ChIPseq analysis conducted on the Day 3.5 pregnant mouse uterus for NR2F2 demonstrated the majority of NR2F2 occupies genomic regions that have H3K27ac and H3K4me1 histone modifications, including the loci of major uterine transcription regulators Hand2, Egr1, and Zbtb16. Furthermore, functional analysis of an NR2F2 occupying site that is conserved between human and mouse was capable to enhance endogenous HAND2 mRNA expression with the CRISPR activator in human endometrial stroma cells. These data establish the NR2F2 dependent regulation of Hand2 in the stroma and identify a cis-acting element for this action. In summary, our findings reveal a role of the NR2F2-HAND2 regulatory axis that determines the uterine transcriptomic pattern in preparation for the endometrial receptivity.

TRIM28 modulates nuclear receptor signaling to regulate uterine function.

Estrogen and progesterone, acting through their cognate receptors the estrogen receptor α (ERα) and the progesterone receptor (PR) respectively, regulate uterine biology. Using rapid immunoprecipitation and mass spectrometry (RIME) and co-immunoprecipitation, we identified TRIM28 (Tripartite motif containing 28) as a protein which complexes with ERα and PR in the regulation of uterine function. Impairment of TRIM28 expression results in the inability of the uterus to support early pregnancy through altered PR and ERα action in the uterine epithelium and stroma by suppressing PR and ERα chromatin binding. Furthermore, TRIM28 ablation in PR-expressing uterine cells results in the enrichment of a subset of TRIM28 positive and PR negative pericytes and epithelial cells with progenitor potential. In summary, our study reveals the important roles of TRIM28 in regulating endometrial cell composition and function in women, and also implies its critical functions in other hormone regulated systems.

Functional analysis reveals driver cooperativity and novel mechanisms in endometrial carcinogenesis.

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.

Chromatin architectural factor CTCF is essential for progesterone-dependent uterine maturation.

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.

CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis.

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

Beclin-1-dependent autophagy, but not apoptosis, is critical for stem-cell-mediated endometrial programming and the establishment of pregnancy.

The human endometrium shows a remarkable regenerative capacity that enables cyclical regeneration and remodeling throughout a woman's reproductive life. Although early postnatal uterine developmental cues direct this regeneration, the vital factors that govern early endometrial programming are largely unknown. We report that Beclin-1, an essential autophagy-associated protein, plays an integral role in uterine morphogenesis during the early postnatal period. We show that conditional depletion of Beclin-1 in the uterus triggers apoptosis and causes progressive loss of Lgr5+/Aldh1a1+ endometrial progenitor stem cells, with concomitant loss of Wnt signaling, which is crucial for stem cell renewal and epithelial gland development. Beclin-1 knockin (Becn1 KI) mice with disabled apoptosis exhibit normal uterine development. Importantly, the restoration of Beclin-1-driven autophagy, but not apoptosis, promotes normal uterine adenogenesis and morphogenesis. Together, the data suggest that Beclin-1-mediated autophagy acts as a molecular switch that governs the early uterine morphogenetic program by maintaining the endometrial progenitor stem cells.

A CRISPR/Cas9-engineered mouse carrying a conditional knockout allele for the early growth response-1 transcription factor.

Early growth response 1 (EGR1) mediates transcriptional programs that are indispensable for cell division, differentiation, and apoptosis in numerous physiologies and pathophysiologies. Whole-body EGR1 knockouts in mice (Egr1KO ) have advanced our understanding of EGR1 function in an in vivo context. To extend the utility of the mouse to investigate EGR1 responses in a tissue- and/or cell-type-specific manner, we generated a mouse model in which exon 2 of the mouse Egr1 gene is floxed by CRISPR/Cas9 engineering. The floxed Egr1 alleles (Egr1f/f ) are designed to enable spatiotemporal control of Cre-mediated EGR1 ablation in the mouse. To confirm that the Egr1f/f alleles can be abrogated using a Cre driver, we crossed the Egr1f/f mouse with a global Cre driver to generate the Egr1 conditional knockout (Egr1d/d ) mouse in which EGR1 expression is ablated in all tissues. Genetic and protein analysis confirmed the absence of exon 2 and loss of EGR1 expression in the Egr1d/d mouse, respectively. Moreover, the Egr1d/d female exhibits overt reproductive phenotypes previously reported for the Egr1KO mouse. Therefore, studies described in this short technical report underscore the potential utility of the murine Egr1 floxed allele to further resolve EGR1 function at a tissue- and/or cell-type-specific level.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

Versican provides the provisional matrix for uterine spiral artery dilation and fetal growth.

The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium promotes embryo implantation. Versican is a proteoglycan, a major player in the provisional matrix, and versikine, its N-terminal fragment cleaved by ADAMTS proteinases, serves as a bioactive molecule. Here, since versican expression in the placenta was dynamically altered in humans and mice, we investigated the role of versican in pregnancy using uterine-specific Vcan deletion mice (uKO mice) and ADAMTS-resistant versican expressing mice (V1R mice). uKO mice exhibited insufficient spiral artery dilation, followed by fetal growth restriction and maternal hypertension. Further analysis revealed impaired proliferation of tissue-resident natural killer cells required for spiral artery dilation. V1R mice showed the same results as the control, eliminating the involvement of versikine. Our results provide a new concept that versican, one factor of ECM, contributes to placentation and following fetal growth.

Decidualization of human endometrial stromal cells requires steroid receptor coactivator-3.

Steroid receptor coactivator-3 (SRC-3; also known as NCOA3 or AIB1) is a member of the multifunctional p160/SRC family of coactivators, which also includes SRC-1 and SRC-2. Clinical and cell-based studies as well as investigations on mice have demonstrated pivotal roles for each SRC in numerous physiological and pathophysiological contexts, underscoring their functional pleiotropy. We previously demonstrated the critical involvement of SRC-2 in murine embryo implantation as well as in human endometrial stromal cell (HESC) decidualization, a cellular transformation process required for trophoblast invasion and ultimately placentation. We show here that, like SRC-2, SRC-3 is expressed in the epithelial and stromal cellular compartments of the human endometrium during the proliferative and secretory phase of the menstrual cycle as well as in cultured HESCs. We also found that SRC-3 depletion in cultured HESCs results in a significant attenuation in the induction of a wide-range of established biomarkers of decidualization, despite exposure of these cells to a deciduogenic stimulus and normal progesterone receptor expression. These molecular findings are supported at the cellular level by the inability of HESCs to morphologically transform from a stromal fibroblastoid cell to an epithelioid decidual cell when endogenous SRC-3 levels are markedly reduced. To identify genes, signaling pathways and networks that are controlled by SRC-3 and potentially important for hormone-dependent decidualization, we performed RNA-sequencing on HESCs in which SRC-3 levels were significantly reduced at the time of administering the deciduogenic stimulus. Comparing HESC controls with HESCs deficient in SRC-3, gene enrichment analysis of the differentially expressed gene set revealed an overrepresentation of genes involved in chromatin remodeling, cell proliferation/motility, and programmed cell death. These predictive bioanalytic results were confirmed by the demonstration that SRC-3 is required for the expansion, migratory and invasive activities of the HESC population, cellular properties that are required in vivo in the formation or functioning of the decidua. Collectively, our results support SRC-3 as an important coregulator in HESC decidualization. Since perturbation of normal homeostatic levels of SRC-3 is linked with common gynecological disorders diagnosed in reproductive age women, this endometrial coregulator-along with its new molecular targets described here-may open novel clinical avenues in the diagnosis and/or treatment of a non-receptive endometrium, particularly in patients presenting non-aneuploid early pregnancy loss.

Spatial definition of the human progesterone receptor-B transcriptional complex.

We report the quaternary structure of core transcriptional complex for the full-length human progesterone receptor-B (PR-B) homodimer with primary coactivator steroid receptor coactivator-2 (SRC-2) and the secondary coactivator p300/CREB-binding protein (CBP). The PR-B homodimer engages one SRC-2 mainly through its activation function 1 (AF1) in N-terminus. SRC-2 is positioned between PR-B and p300 leaving space for direct interaction between PR-B and p300 through PR-B's C-terminal AF2 and its unique AF3. Direct AF3/p300 interaction provides long-desired structural insights into the known functional differences between PR-B and the PR-A isoform lacking AF3. We reveal the contributions of each AF and demonstrate their structural basis in forming the PR-B dimer interface and PR-B/coactivator complex. Comparison of the PR-B/coactivator complex with other steroid receptor (estrogen receptor and androgen receptor) complexes also shows that each receptor has its unique mechanism for recruiting coactivators due to the highly variable N-termini among receptors.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

Corrigendum: Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy.

[This corrects the article DOI: 10.3389/fendo.2022.846226.].

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers.

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

Early growth response 1 transcription factor is essential for the pathogenic properties of human endometriotic epithelial cells.

Although a non-malignant gynecological disorder, endometriosis displays some pathogenic features of malignancy, such as cell proliferation, migration, invasion and adaptation to hypoxia. Current treatments of endometriosis include pharmacotherapy and/or surgery, which are of limited efficacy and often associated with adverse side effects. Therefore, to develop more effective therapies to treat this disease, a broader understanding of the underlying molecular mechanisms that underpin endometriosis needs to be attained. Using immortalized human endometriotic epithelial and stromal cell lines, we demonstrate that the early growth response 1 (EGR1) transcription factor is essential for cell proliferation, migration and invasion, which represent some of the pathogenic properties of endometriotic cells. Genome-wide transcriptomics identified an EGR1-dependent transcriptome in human endometriotic epithelial cells that potentially encodes a diverse spectrum of proteins that are known to be involved in tissue pathologies. To underscore the utility of this transcriptomic data set, we demonstrate that carbonic anhydrase 9 (CA9), a homeostatic regulator of intracellular pH, is not only a molecular target of EGR1 but is also important for maintaining many of the cellular properties of human endometriotic epithelial cells that are also ascribed to EGR1. Considering therapeutic intervention strategies are actively being developed for EGR1 and CAIX in the treatment of other pathologies, we believe EGR1 and its transcriptome (which includes CA9) will offer not only a new conceptual framework to advance our understanding of endometriosis but will also furnish new molecular vulnerabilities to be leveraged as potential therapeutic options in the future treatment of endometriosis.