Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis.

Uterine leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies, such as selective progesterone receptor modulators, are often used as temporary treatment options to reduce symptoms or to slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported that aberrant expression of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene-expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone-responsive genes in UL ensuing in the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones.

Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression.

BACKGROUND: Breast cancer is the most common malignancy in women, and is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers express normal levels of REST (repressor element 1 silencing transcription factor) mRNA but lack functional REST protein. Loss of REST function is seen in ~ 20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. METHODS: TCGA data was analyzed for the expression of REST target genes in breast cancer patient samples. We then utilized gene knockdown in MCF-7 cells in the presence or absence of steroid hormones estrogen and/ progesterone followed by RNA sequencing, as well as chromatin immunoprecipitation and PCR in an attempt to understand the tumor suppressor role of REST in breast cancer. RESULTS: We show that REST directly regulates CEMIP (cell migration-inducing and hyaluronan-binding protein, KIAA1199) and MMP24 (matrix metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cells leads to significant upregulation of CEMIP and MMP24. In addition, we found REST binds to RE-1 sites (repressor element-1) within the genes and influences their transcription. Furthermore, we found that the estrogen receptor (ESR1) signaling pathway is activated in the absence of REST, regardless of hormone treatment. CONCLUSIONS: We demonstrate a critical role for the loss of REST in aggressive breast cancer pathogenesis and provide evidence for REST as an important diagnostic marker for personalized treatment plans.

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway.

Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase-protein kinase B/AKT-mammalian target of rapamycin (PI3K/AKT-mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K-AKT-mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids.

Development of an early biomarker for the ovarian liability of selective estrogen receptor modulators in rats.

Selective estrogen receptor modulators (SERMs) have the potential to treat estrogen sensitive diseases such as uterine leiomyoma and endometriosis, which are prevalent in reproductive age women. However, SERMs also increase the risk of developing ovarian cysts in this population, a phenomenon that is not seen in postmenopausal women. It is believed that current SERMs partially block estradiol's ability to downregulate gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus thereby interfering with estradiol's negative feedback, leading to increased ovarian stimulation by gonadotropins, and cyst formation. It has been postulated that a SERM with poor brain exposure would have less negative effect on the HPO axis, therefore reducing the risk of developing ovarian cysts. In order to test this hypothesis, we identified an early marker of SERM-dependent ovarian effects: upregulation of Cyp17a1 mRNA. SERMs known to cause ovarian cysts upregulate Cyp17a1 after only 4 days of dosing and suppression of the HPO axis prevented this regulation, indicating that ovarian expression of Cyp17a1 was secondary to SERM's effect on the brain. We then characterized three SERMs with similar binding affinity and antagonist effects on the uterus for their relative brain/plasma exposure and ovarian effects. We found that the degree of brain exposure correlated very well with Cyp17a1 expression.

A study of 17beta-estradiol-regulated genes in the vagina of postmenopausal women with vaginal atrophy.

BACKGROUND: Vaginal atrophy (VA) is a prevalent disorder in postmenopausal women that is characterized by decreased epithelial thickness, reduced vaginal maturation index (VMI) and increased vaginal pH. Current medical therapy consists of local or systemic replacement of estrogens. OBJECTIVE: The goal of this study was to understand, at a molecular level, the effect of estradiol (E2) on the vaginal epithelium. METHODS: Nineteen women were treated with E2 delivered through a skin patch at a dose of 0.05mg/day for 12 weeks. The diagnosis of VA was confirmed by a VMI with < or =5% superficial cells and vaginal pH>5.0. Vaginal biopsy samples were collected at baseline and after treatment. Differentially expressed mRNA transcripts in these biopsies were determined by microarray analysis. RESULTS: All 19 subjects had increased VMI (>5%) and/or reduced pH (< or =5) following treatment. Most subjects also had increased serum E2 levels and reduced serum FSH levels. Transcriptional profiling of vaginal biopsies identified over 3000 E2-regulated genes, including those involved in several key pathways known to regulate cell growth and proliferation, barrier function and pathogen defense. CONCLUSIONS: E2 controls a plethora of cellular pathways that are concordant with its profound effect on vaginal physiology. The data presented here are a useful step toward understanding the role of E2 in vaginal tissue and the development of novel therapeutics for the treatment of VA.

Gene expression profiling and its practice in drug development.

The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.

Recent Advances in Uterine Fibroid Etiology.

Uterine fibroids, also known as uterine leiomyoma (UL), are monoclonal tumors of the smooth muscle tissue layer (myometrium) of the uterus. Although ULs are considered benign, uterine fibroids are the source of major quality-of-life issues for approximately 25% of all women, who suffer from clinically significant symptoms of UL. Despite the prevalence of UL, there is no treatment option for UL which is long term, cost-effective, and leaves fertility intact. The lack of understanding about the etiology of UL contributes to the scarcity of medical therapies available. Studies have identified an important role for sex steroid hormones in the pathogenesis of UL, and have driven the use of hormonal treatment for fibroids, with mixed results. Dysregulation of cell signaling pathways, miRNA expression, and cytogenetic abnormalities have also been implicated in UL etiology. Recent discoveries on the etiology of UL and the development of relevant genetically modified rodent models of UL have started to revitalize UL research. This review outlines the major characteristics of fibroids; major contributors to UL etiology, including steroid hormones; and available preclinical animal models for UL.

Trace LC/MS/MS quantitation of 17beta-estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain.

A sensitive LC/MS/MS method has been developed by derivatization of 17beta-estradiol (E2) with dansyl chloride to quantitate 17beta-E2 in female rat serum. The use of E2-d(5) minimized interferences from endogenous 17beta-E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 microl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17beta-E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic-pituitary-ovarian (HPO) axis. Variations in 17beta-E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing.

Identification and characterization of cDNAs encoding four novel proteins that interact with translin associated factor-X.

Translin-associated factor X (TRAX) is the predominantly cytoplasmic binding partner of TB-RBP/translin in mouse testis. Four mouse testis cDNAs encoding specific TRAX-interacting proteins were isolated from a yeast two-hybrid library screen. One novel cDNA designated Tsnaxip1 (TRAX-interacting protein-1) encodes 709 amino acids. We isolated a cDNA encoding the 427 carboxy-terminal amino acids of MEA-2, a Golgi-associated, maleenhanced autoantigen; a cDNA encoding 429 amino acids with 73% homology to centrosomal Akap9; and a cDNA encoding 346 amino acids with 75% homology to SUN1, a predicted human protein that contains a SUN domain (which is present in some perinuclear proteins). Interactions were verified using in vitro synthesized fusion proteins. All four genes were expressed in the testis and enriched in germ cells. Confocal microscopy studies using green fluorescent protein fusion proteins determined that these TRAX-interacting proteins colocalize with TRAX. The data suggest that TRAX may have a function associated with perinuclear organelles during spermatogenesis.

KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX).

Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin. A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins. KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells. The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins. Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo. Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location. KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells. The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis.

The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells.

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.

Translin-associated factor X is post-transcriptionally regulated by its partner protein TB-RBP, and both are essential for normal cell proliferation.

To determine the functions of the DNA/RNA-binding protein TB-RBP in somatic cells, we examined cultured primary mouse embryonic fibroblasts (MEFs) derived from TB-RBP-deficient mice. The TB-RBP-deficient MEFs exhibit a reduced growth rate compared with MEFs from littermates. Reintroduction of TB-RBP remedies this defect. A partner protein of TB-RBP, Translin-associated factor X (TRAX), was absent in TB-RBP-deficient MEFs, despite normal TRAX mRNA levels. TRAX is dependent upon the presence of TB-RBP and is removed from null MEFs following ubiquitination. Re-introduction of TB-RBP, but not TB-RBP lacking an oligomerization domain, into null MEFs stabilized TRAX protein without changing TRAX mRNA levels. The coordinated expression of TB-RBP and TRAX is also seen in synchronized cells, where the amount of TRAX protein but not TRAX RNA closely parallels TB-RBP levels throughout the cell cycle. In transgenic mice overexpressing TRAX in testis, total TB-RBP and TRAX levels are constant with reductions of endogenous TRAX compensating for exogenous TRAX. Using RNA interference, reductions of either TB-RBP or TRAX (without affecting TB-RBP) slow cell growth rates. We conclude that TRAX is post-transcriptionally stabilized by TB-RBP and both proteins are needed for normal cell proliferation.