WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers.

Wnt ligand WNT4 is critical in female reproductive tissue development, with WNT4 dysregulation linked to related pathologies including breast cancer (invasive lobular carcinoma, ILC) and gynecologic cancers. WNT4 signaling in these contexts is distinct from canonical Wnt signaling yet inadequately understood. We previously identified atypical intracellular activity of WNT4 (independent of Wnt secretion) regulating mitochondrial function, and herein examine intracellular functions of WNT4. We further examine how convergent mechanisms of WNT4 dysregulation impact cancer metabolism. In ILC, WNT4 is co-opted by estrogen receptor α (ER) via genomic binding in WNT4 intron 1, while in gynecologic cancers, a common genetic polymorphism (rs3820282) at this ER binding site alters WNT4 regulation. Using proximity biotinylation (BioID), we show canonical Wnt ligand WNT3A is trafficked for secretion, but WNT4 is localized to the cytosol and mitochondria. We identified DHRS2, mTOR, and STAT1 as putative WNT4 cytosolic/mitochondrial signaling partners. Whole metabolite profiling, and integrated transcriptomic data, support that WNT4 mediates metabolic reprogramming via fatty acid and amino acid metabolism. Furthermore, ovarian cancer cell lines with rs3820282 variant genotype are WNT4 dependent and have active WNT4 metabolic signaling. In protein array analyses of a cohort of 103 human gynecologic tumors enriched for patient diversity, germline rs3820282 genotype is associated with metabolic remodeling. Variant genotype tumors show increased AMPK activation and downstream signaling, with the highest AMPK signaling activity in variant genotype tumors from non-White patients. Taken together, atypical intracellular WNT4 signaling, in part via genetic dysregulation, regulates the distinct metabolic phenotypes of ILC and gynecologic cancers. SIGNIFICANCE: WNT4 regulates breast and gynecologic cancer metabolism via a previously unappreciated intracellular signaling mechanism at the mitochondria, with WNT4 mediating metabolic remodeling. Understanding WNT4 dysregulation by estrogen and genetic polymorphism offers new opportunities for defining tumor biology, precision therapeutics, and personalized cancer risk assessment.

Developmental reprogramming of myometrial stem cells by endocrine disruptor linking to risk of uterine fibroids.

BACKGROUND: The stage, when tissues and organs are growing, is very vulnerable to environmental influences, but it's not clear how exposure during this time causes changes to the epigenome and increases the risk of hormone-related illnesses like uterine fibroids (UFs). METHODS: Developmental reprogramming of myometrial stem cells (MMSCs), the putative origin from which UFs originate, was investigated in vitro and in the Eker rat model by RNA-seq, ChIP-seq, RRBS, gain/loss of function analysis, and luciferase activity assays. RESULTS: When exposed to the endocrine-disrupting chemical (EDC) diethylstilbestrol during Eker rat development, MMSCs undergo a reprogramming of their estrogen-responsive transcriptome. The reprogrammed genes in MMSCs are known as estrogen-responsive genes (ERGs) and are activated by mixed lineage leukemia protein-1 (MLL1) and DNA hypo-methylation mechanisms. Additionally, we observed a notable elevation in the expression of ERGs in MMSCs from Eker rats exposed to natural steroids after developmental exposure to EDC, thereby augmenting estrogen activity. CONCLUSION: Our studies identify epigenetic mechanisms of MLL1/DNA hypo-methylation-mediated MMSC reprogramming. EDC exposure epigenetically targets MMSCs and leads to persistent changes in the expression of a subset of ERGs, imparting a hormonal imprint on the ERGs, resulting in a "hyper-estrogenic" phenotype, and increasing the hormone-dependent risk of UFs.

Parabens Promote Protumorigenic Effects in Luminal Breast Cancer Cell Lines With Diverse Genetic Ancestry.

Context: One in 8 women will develop breast cancer in their lifetime. Yet, the burden of disease is greater in Black women. Black women have a 40% higher mortality rate than White women, and a higher incidence of breast cancer at age 40 and younger. While the underlying cause of this disparity is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and other personal care products has been associated with an increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products, and Black women are disproportionately exposed to products containing parabens. Objective: Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine the effects of parabens on breast cancer progression. Like breast cancer cell lines with European ancestry, we hypothesize that parabens promote protumorigenic effects in breast cancer cell lines of West African ancestry. Methods: Luminal breast cancer cell lines with West African ancestry (HCC1500) and European ancestry (MCF-7) were treated with biologically relevant doses of methylparaben, propylparaben, and butylparaben. Results: Following treatment, estrogen receptor target gene expression and cell viability were examined. We observed altered estrogen receptor target gene expression and cell viability that was paraben and cell line specific. Conclusion: This study provides greater insight into the tumorigenic role of parabens in the progression of breast cancer in Black women.

Epigenetic Modulation of Inflammatory Pathways in Myometrial Stem Cells and Risk of Uterine Fibroids.

The period during which tissue and organ development occurs is particularly vulnerable to the influence of environmental exposures. However, the specific mechanisms through which biological pathways are disrupted in response to developmental insults, consequently elevating the risk of hormone-dependent diseases, such as uterine fibroids (UFs), remain poorly understood. Here, we show that developmental exposure to the endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), activates the inflammatory pathways in myometrial stem cells (MMSCs), which are the origin of UFs. Significantly, the secretome of reprogrammed MMSCs enhances the expression of critical inflammation-related genes in differentiated myometrial cells through the paracrine mechanism, which amplifies pro-inflammatory and immune suppression signaling in the myometrium. The expression of reprogrammed inflammatory responsive genes (IRGs) is driven by activated mixed-lineage leukemia protein-1 (MLL1) in MMSCs. The deactivation of MLL reverses the reprogramming of IRG expression. In addition, the inhibition of histone deacetylases (HDACs) also reversed the reprogrammed IRG expression induced by EDC exposure. This work identifies the epigenetic mechanisms of MLL1/HDAC-mediated MMSC reprogramming, and EDC exposure epigenetically targets MMSCs and imparts an IRG expression pattern, which may result in a "hyper-inflammatory phenotype" and an increased hormone-dependent risk of UFs later in life.

Longitudinal analysis of a dietary mouse model of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD), and resultant non-alcoholic steatohepatitis (NASH), incidence and prevalence are rising globally due to increasing rates of obesity and diabetes. Currently, there are no approved pharmacological treatments for NAFLD, highlighting a need for additional mechanistic studies to develop prevention and/or therapeutic strategies. Diet-induced preclinical models of NAFLD can be used to examine the dynamic changes that occur during NAFLD development and progression throughout the lifespan. To date, most studies utilizing such models have focused exclusively on terminal time points and have likely missed critical early and late changes that are important for NAFLD progression (i.e, worsening). We performed a longitudinal analysis of histopathological, biochemical, transcriptomic, and microbiome changes that occurred in adult male mice fed either a control diet or a NASH-promoting diet (high in fat, fructose, and cholesterol) for up to 30 weeks. We observed progressive development of NAFLD in mice fed the NASH diet compared to the control diet. Differential expression of immune-related genes was observed at an early stage of diet-induced NAFLD development (10 weeks) and persisted into the later stages of the disease (20 and 30 weeks). Differential expression of xenobiotic metabolism related genes was observed at the late stage of diet-induced NAFLD development (30 weeks). Microbiome analysis revealed an increased abundance of Bacteroides at an early stage (10 weeks) that persisted into the later stages of the disease (20 and 30 weeks). These data provide insight into the progressive changes that occur during NAFLD/NASH development and progression in the context of a typical Western diet. Furthermore, these data are consistent with what has been reported in patients with NAFLD/NASH, supporting the preclinical use of this diet-induced model for development of strategies to prevent or treat the disease.

The Interface of Nuclear and Membrane Steroid Signaling.

Steroid hormones bind receptors in the cell nucleus and in the cell membrane. The most widely studied class of steroid hormone receptors are the nuclear receptors, named for their function as ligand-dependent transcription factors in the cell nucleus. Nuclear receptors, such as estrogen receptor alpha, can also be anchored to the plasma membrane, where they respond to steroids by activating signaling pathways independent of their function as transcription factors. Steroids can also bind integral membrane proteins, such as the G protein-coupled estrogen receptor. Membrane estrogen and progestin receptors have been cloned and characterized in vitro and influence the development and function of many organ systems. Membrane androgen receptors were cloned and characterized in vitro, but their function as androgen receptors in vivo is unresolved. We review the identity and function of membrane proteins that bind estrogens, progestins, and androgens. We discuss evidence that membrane glucocorticoid and mineralocorticoid receptors exist, and whether glucocorticoid and mineralocorticoid nuclear receptors act at the cell membrane. In many cases, integral membrane steroid receptors act independently of nuclear steroid receptors, even though they may share a ligand.

Epigenome environment interactions accelerate epigenomic aging and unlock metabolically restricted epigenetic reprogramming in adulthood.

Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.

Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin.

BACKGROUND: Tributyltin (TBT) is a persistent and bioaccumulative environmental toxicant. Developmental exposure to TBT has been shown to cause fatty liver disease (steatosis), as well as increased adiposity in many species, leading to its characterization as an obesogen. OBJECTIVE: We aimed to determine the long-term effects of developmental TBT exposure on the liver. METHODS: C57BL/6J mice were exposed to a dose of TBT (0.5mg/kg body weight per day; 3.07µM) below the current developmental no observed adverse effect level (NOAEL) via drinking water, or drinking water alone, provided to the dam from preconception through lactation. Sires were exposed during breeding and lactation. Pups from two parity cycles were included in this study. Animals were followed longitudinally, and livers of offspring were analyzed by pathological evaluation, immunohistochemistry, immunoblotting, and RNA sequencing. RESULTS: Developmental exposure to TBT led to increased adiposity and hepatic steatosis at 14 and 20 weeks of age and increased liver adenomas at 45 weeks of age in male offspring. Female offspring displayed increased adiposity as compared with males, but TBT did not lead to an increase in fatty liver or tumor development in female offspring. Liver tumors in male mice were enriched in pathways and gene signatures associated with human and rodent nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). This includes down-regulation of growth hormone receptor (GHR) and of STAT5 signaling, which occurred in response to TBT exposure and preceded liver tumor development. CONCLUSIONS: These data reveal a previously unappreciated ability of TBT to increase risk for liver tumorigenesis in mice in a sex-specific manner. Taken together, these findings provide new insights into how early life environmental exposures contribute to liver disease in adulthood. https://doi.org/10.1289/EHP5414.

The Black identity, hair product use, and breast cancer scale.

INTRODUCTION: Across the African Diaspora, hair is synonymous with identity. As such, Black women use a variety of hair products, which often contain more endocrine-disrupting chemicals than products used by women of other races. An emerging body of research is linking chemicals in hair products to breast cancer, but there is no validated instrument that measures constructs related to hair, identity, and breast health. The objective of this study was to develop and validate the Black Identity, Hair Product Use, and Breast Cancer Scale (BHBS) in a diverse sample of Black women to measure the social and cultural constructs associated with Black women's hair product use and perceived breast cancer risk. METHODS: Participants completed a 27-item scale that queried perceptions of identity, hair products, and breast cancer risk. Principal Component Analyses (PCA) were conducted to establish the underlying component structures, and confirmatory factor analysis (CFA) was used to determine model fit. RESULTS: Participants (n = 185) were African American (73%), African, and Caribbean Black women (27%) aged 29 to 64. PCA yielded two components that accounted for 61% of total variance. Five items measuring sociocultural perspectives about hair and identity loaded on subscale 1 and accounted for 32% of total variance (α = 0.82; 95% CI = 0.77-0.86). Six items assessing perceived breast cancer risk related to hair product use loaded on subscale 2 and accounted for 29% of total variance (α = 0.82 (95% CI = 0.74-0.86). CFA confirmed the two-component structure (Root Mean Square Error of Approximation = 0.03; Comparative Fit Index = 0.91; Tucker Lewis Index = 0.88). CONCLUSIONS: The BHBS is a valid measure of social and cultural constructs associated with Black women's hair product use and perceived breast cancer risk. This scale is useful for studies that assess cultural norms in the context of breast cancer risk for Black women.

CARM1 methylates MED12 to regulate its RNA-binding ability.

The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.

Endocrine Disruptors and Developmental Origins of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a growing epidemic worldwide, particularly in countries that consume a Western diet, and can lead to life-threatening conditions such as cirrhosis and hepatocellular carcinoma. With increasing prevalence of NAFLD in both children and adults, an understanding of the factors that promote NAFLD development and progression is crucial. Environmental agents, including endocrine-disrupting chemicals (EDCs), which have been linked to other diseases, may play a role in NAFLD development. Increasing evidence supports a developmental origin of liver disease, and early-life exposure to EDCs could represent one risk factor for the development of NAFLD later in life. Rodent studies provide the strongest evidence for this link, but further studies are needed to define whether there is a causal link between early-life EDC exposure and NAFLD development in humans. Elucidating the molecular mechanisms underlying development of NAFLD in the context of developmental EDC exposures may identify biomarkers for people at risk, as well as potential intervention and/or therapeutic opportunities for the disease.

Endocrine-disrupting chemicals and fatty liver disease.

A growing epidemic of nonalcoholic fatty liver disease (NAFLD) is paralleling the increase in the incidence of obesity and diabetes mellitus in countries that consume a Western diet. As NAFLD can lead to life-threatening conditions such as cirrhosis and hepatocellular carcinoma, an understanding of the factors that trigger its development and pathological progression is needed. Although by definition this disease is not associated with alcohol consumption, exposure to environmental agents that have been linked to other diseases might have a role in the development of NAFLD. Here, we focus on one class of these agents, endocrine-disrupting chemicals (EDCs), and their potential to influence the initiation and progression of a cascade of pathological conditions associated with hepatic steatosis (fatty liver). Experimental studies have revealed several potential mechanisms by which EDC exposure might contribute to disease pathogenesis, including the modulation of nuclear hormone receptor function and the alteration of the epigenome. However, many questions remain to be addressed about the causal link between acute and chronic EDC exposure and the development of NAFLD in humans. Future studies that address these questions hold promise not only for understanding the linkage between EDC exposure and liver disease but also for elucidating the molecular mechanisms that underpin NAFLD, which in turn could facilitate the development of new prevention and treatment opportunities.

Endocrine-disrupting chemicals and uterine fibroids.

Uterine fibroids are the most frequent gynecologic tumor, affecting 70% to 80% of women over their lifetime. Although these tumors are benign, they can cause significant morbidity and may require invasive treatments such as myomectomy and hysterectomy. Many risk factors for these tumors have been identified, including environmental exposures to endocrine-disrupting chemicals (EDCs) such as genistein and diethylstilbestrol. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both rodent models and human epidemiologic studies. The mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, but epigenetic reprogramming of the developing uterus is an emerging hypothesis. Given the remarkably high incidence of uterine fibroids and their significant impact on women's health, understanding more about how prenatal exposures to EDCs (and other environmental agents) may increase fibroid risk could be key to developing prevention and treatment strategies in the future.

Reprogramming of the Epigenome by MLL1 Links Early-Life Environmental Exposures to Prostate Cancer Risk.

Tissue and organ development is a time of exquisite sensitivity to environmental exposures, which can reprogram developing tissues to increase susceptibility to adult diseases, including cancer. In the developing prostate, even brief exposure to endocrine-disrupting chemicals (EDCs) can increase risk for developing cancer in adulthood, with disruption of the epigenome thought to play a key role in this developmental reprogramming. We find that EDC-induced nongenomic phosphoinositide 3-kinase; (PI3K) signaling engages the histone methyltransferase mixed-lineage leukemia 1 (MLL1), responsible for the histone H3 lysine 4 trimethylation (H3K4me3) active epigenetic mark, to increase cleavage and formation of active MLL1 dimers. In the developing prostate, EDC-induced MLL1 activation increased H3K4me3 at genes associated with prostate cancer, with increased H3K4me3 and elevated basal and hormone-induced expression of reprogrammed genes persisting into adulthood. These data identify a mechanism for MLL1 activation that is vulnerable to disruption by environmental exposures, and link MLL1 activation by EDCs to developmental reprogramming of genes involved in prostate cancer.

Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

Phosphorylation of epigenetic "readers, writers and erasers": Implications for developmental reprogramming and the epigenetic basis for health and disease.

Epigenetic reprogramming that occurs during critical periods of development can increase the susceptibility to many diseases in adulthood. Programming of the epigenome during development occurs via the activity of a variety of epigenetic modifiers, including "readers, writers and erasers" of histone methyl marks. Posttranslational modification of these programmers can alter their activity, resulting in global or gene-specific changes in histone methylation and gene transcription. This review summarizes what is currently known about phosphorylation of histone methyltransferases ("writers"), demethylases ("erasers") and effector proteins ("readers) that program the epigenome, and the impact of this posttranslational modification on their activity. Understanding how the activity of these epigenetic programmers is perturbed by environmental exposures via changes in phosphorylation is key to understanding mechanisms of developmental reprogramming and the epigenetic basis of health and disease.

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Hypothesis: Activation of rapid signaling by environmental estrogens and epigenetic reprogramming in breast cancer.

Environmental and lifestyle factors are considered significant components of the increasing breast cancer risk in the last 50 years. Specifically, exposure to environmental endocrine disrupting compounds is correlated with cancer susceptibility in a variety of tissues. In both human and rodent models, the exposure to ubiquitous environmental estrogens during early life has been shown to disrupt normal mammary development and cause permanent adverse effects. Recent studies indicate that environmental estrogens not only have the ability to disrupt estrogen receptor (ER) signaling, but can also reprogram the epigenome by altering DNA and histone methylation through rapid, nongenomic ER actions. We have observed xenoestrogen-mediated activation of several nongenomic signaling pathways and have identified a target for epigenetic reprogramming in MCF-7 breast cancer cells. These observations, in addition to data from the literature, support the hypothesis that activation of rapid signaling by environmental estrogens can lead to epigenetic reprogramming and contribute to the progression of breast cancer.

Phosphorylation: a fundamental regulator of steroid receptor action.

Steroid hormone receptors (SHRs) are hormone-activated transcription factors involved in numerous cellular functions and in health and disease. Their activities depend on the cellular level of the receptor, the presence of coregulator proteins, and the cell signaling pathways that are active in the cell. SHRs and their coregulators are phosphorylated on multiple sites by a wide variety of kinases. Each site may contribute to multiple functions and the net effect of an individual phosphorylation depends on the activating kinase. Here we discuss functions of known SHR phosphorylation sites, kinase regulation, evidence of translational relevance, and crosstalk between SHRs and cell signaling pathways. Understanding how cell signaling pathways regulate SHRs might yield novel therapeutic targets for multiple human diseases.

The requirement for p42/p44 MAPK activity in progesterone receptor-mediated gene regulation is target gene-specific.

Recent studies have suggested that progestins play a role in the etiology of breast cancer; however, the mechanisms by which progestins promote tumor formation/progression have not been defined. Progestin action, in target tissues such as the breast, is mediated by the progesterone receptor (PR). PR signaling is complex and PR regulates transcription of target genes through a variety of mechanisms. Many cell signaling pathways are activated inappropriately in breast cancer cells and these pathways can regulate PR activity. For example, the p42/p44 MAPK pathway can regulate PR function by altering phosphorylation of PR, as well as its coregulators. We found that inhibition of the p42/p44 MAPK signaling pathway with a MEK inhibitor (U0126) impairs PR-mediated gene induction, but not gene repression. In addition, the effects of U0126 on PR-mediated gene transcription are much greater with long-term versus short-term inhibition and are gene-specific. Finally, treatment with U0126 delays phosphorylation of Ser294, but does not block phosphorylation completely, suggesting that p42/p44 MAPK kinase is not the dominant kinase responsible for phosphorylating this site. Collectively, these studies suggest that in addition to the p42/p44 MAPK pathway, other signaling pathways are also important for PR transcriptional activity in breast cancer cells. The integration of PR transcriptional effects and cell signaling pathways has implications for the initiation or progression of breast cancer. Understanding how these pathways interact may aid in the development of prevention and/or treatment strategies for the disease.