Human Progesterone Receptor Research Articles

Monomethylated trivalent arsenic species disrupt steroid receptor interactions with their DNA response elements at non‐cytotoxic cellular concentrations

Arsenic (As) is considered a top environmental chemical of human health because it has been linked to adverse health effects including cancer, diabetes, cardiovascular disease, and reproductive and developmental problems. In several cell culture and animal models, As acts as an endocrine disruptor, which may underlie many of its health effects. Previous work showed that steroid receptor (SR)-driven gene expression is disrupted in cells treated with inorganic As (arsenite, iAs(+3)). In those studies, low iAs(+3) concentrations (0.1-0.7 μM) stimulated hormone-inducible transcription, whereas somewhat higher but still non-cytotoxic levels (1-3 μM) inhibited transcription. This investigation focuses on the mechanisms underlying these inhibitory effects and evaluates the role of methylated trivalent As metabolites on SR function. Recent evidence suggests that, compared with iAs, methylated forms may have distinct biochemical effects. Here, fluorescence polarization (FP) experiments utilizing purified, hormone-bound human glucocorticoid (GR) and progesterone receptor (PR) have demonstrated that neither inorganic (iAs(+3)) nor dimethylated (DMA(+3)) species of trivalent As affect receptor interactions with glucocorticoid DNA response elements (GREs). However, monomethylated forms (monomethylarsenite, MMA(+3) and monomethylarsonic diglutathione, MADG) strongly inhibit GR-GRE and PR-GRE binding. Additionally, speciation studies of iAs(+3)-treated H4IIE rat hepatoma cells show that, under treatment conditions that cause inhibition of hormone-inducible gene transcription, the intracellular concentration of MADG is sufficient to inhibit GR-GRE and PR-GRE interactions in vivo. These results indicate that arsenic's inhibitory endocrine disruption effects are probably caused in part by methylated metabolites' disruption of SR ability to bind DNA response elements that are crucial to hormone-driven gene transcription.

Selective suppression of endothelial cytokine production by progesterone receptor

Steroid hormones are well-recognized suppressors of the inflammatory response, however, their cell- and tissue-specific effects in the regulation of inflammation are far less understood, particularly for the sex-related steroids. To determine the contribution of progesterone in the endothelium, we have characterized and validated an in vitro culture system in which human umbilical vein endothelial cells constitutively express human progesterone receptor (PR). Using next generation RNA-sequencing, we identified a selective group of cytokines that are suppressed by progesterone both under physiological conditions and during pathological activation by lipopolysaccharide. In particular, IL-6, IL-8, CXCL2/3, and CXCL1 were found to be direct targets of PR, as determined by ChIP-sequencing. Regulation of these cytokines by progesterone was also confirmed by bead-based multiplex cytokine assays and quantitative PCR. These findings provide a novel role for PR in the direct regulation of cytokine levels secreted by the endothelium. They also suggest that progesterone-PR signaling in the endothelium directly impacts leukocyte trafficking in PR-expressing tissues.

Fluorescent Ligand for Human Progesterone Receptor Imaging in Live Cells

We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of 18F into the BODIPY core.

Approaches to the design of selective ligands for membrane progesterone receptor alpha

A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6-C7-double bond. The data suggest that the modes of ligand interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

Progesterone Receptor Isoforms in the Mammary Gland of Cats and Dogs

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

Characterization, Neurosteroid Binding and Brain Distribution of Human Membrane Progesterone Receptors δ and ϵ (mPRδ and mPRϵ) and mPRδ Involvement in Neurosteroid Inhibition of Apoptosis

Three members of the progestin and adipoQ receptor (PAQR) family, PAQR-7, PAQR-8, and PAQR-5 [membrane progesterone (P4) receptor (PR) (mPR)α, mPRβ, and mPRγ], function as plasma mPRs coupled to G proteins in mammalian cells, but the characteristics of two other members, PAQR6 and PAQR9 (mPRδ and mPRε), remain unclear, because they have only been investigated in yeast expression systems. Here, we show that recombinant human mPRδ and mPRε expressed in MDA-MB-231 breast cancer cells display specific, saturable, high-affinity [(3)H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (K(d)s) of 2.71 and 2.85 nm, respectively, and low affinity for R5020, characteristics typical of mPRs. P4 treatment increased cAMP production as well as [(35)S]-guanosine 5'-triphosphate (GTP)γS binding to transfected cell membranes, which was immunoprecipitated with a stimulatory G protein antibody, suggesting both mPRδ and mPRε activate a stimulatory G protein (Gs), unlike other mPRs, which activate an inhibitory G protein (Gi). All five mPR mRNAs were detected in different regions of the human brain, but mPRδ showed greatest expression in many regions, including the forebrain, hypothalamus, amygdala, corpus callosum, and spinal cord, whereas mPRε was abundant in the pituitary gland and hypothalamus. Allopregnanolone and other neurosteroids bound to mPRδ and other mPRs and acted as agonists, activating second messengers and decreased starvation-induced cell death and apoptosis in mPRδ-transfected cells and in hippocampal neuronal cells at low nanomolar concentrations. The results suggest that mPRδ and mPRε function as mPRs coupled to G proteins and are potential intermediaries of nonclassical antiapoptotic actions of neurosteroids in the central nervous system.

Preliminary investigation of the expression and functions of insulin receptor isoforms in endometrial carcinoma

To investigate the expression of insulin receptor isoforms (IR, including IR-A and IR-B) in endometrial carcinoma (EC), and explore the role of IR-A in the growth of endometrial carcinoma cells. The expression of IR isoforms were detected by reverse transcription (RT)-PCR and real-time PCR in 4 different endometrial cancer cell lines (HEC-1-A, Ishikawa, RL95-2 and KLE), with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases, admitted in Peking University People's Hospital from November 2007 to July 2009, with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls. The relationships among the expression of IR, IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis. An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells [RL95-2(IR-A)] for overexpression of IR-A in RL95-2 cells, in which the expression of IR-A originally was low. Non radioactive cell proliferation assay-MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2 (IR-A). (1) There were two isoforms of IR-A and IR-B co-expressed were detected in EC cells and EC tissues. Among four kinds of EC cell lines, the expression level of IR mRNA in RL95-2 cells was the highest, followed by Ishikawa, KLE and HEC-1-A cells, in which the relative IR mRNA expression levels were (26.54 ± 1.82)×10(-4), (15.44 ± 3.29)×10(-4), (10.14 ± 0.10)×10(-4) and (2.63 ± 0.23)×10(-4), respectively (P < 0.01). The expression level of IR-A mRNA was the highest in Ishikawa cells, followed by KLE, RL95-2 and HEC-1-A cells, with the relative expression levels were (12.07 ± 3.31)×10(-4), (4.68 ± 0.63)×10(-4), (3.03 ± 0.22)×10(-4) and(1.46 ± 0.03)×10(-4), respectively (P < 0.01). The relative expression level of IR and IR-A mRNA were 0.017 ± 0.013 and 0.011 ± 0.010 in the EC tissues, respectively, compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls, in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control (P = 0.662, P = 0.780). The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics (FIGO) stage, cell differentiation, depth of myometrial invasion, invasion of lymph-vascular space, lymph nodes metastasis, the expression status of estrogen receptor, human progesterone receptor, and PTEN gene (all P > 0.05). The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus (DM) was significantly higher than that in patients without type 2 DM (P = 0.031), while there were no statistical correlation between the expression of IR mRNA and type 2 DM (P = 0.438). (2) The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio, with the most growth potential in Ishikawa, followed by HEC-1-A, KLE and RL95-2 cells. The overexpression of IR-A in RL95-2 (IR-A) cells showed a significant proliferation-promoting effect than that in control RL95-2 cells (P < 0.01). There are two isoforms of IR-A and IR-B co-expressed in EC. The overexpression of IR-A may promote the proliferation of EC cells.

Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.

Junctional adhesion molecule-A is co-expressed with HER2 in breast tumors and acts as a novel regulator of HER2 protein degradation and signaling

Junctional adhesion molecule-A (JAM-A) is a membranous cell-cell adhesion protein involved in tight-junction formation in epithelial and endothelial cells. Its overexpression in breast tumors has recently been linked with increased risk of metastasis. We sought to identify if JAM-A overexpression was associated with specific subtypes of breast cancer as defined by the expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor. To this end, JAM-A immunohistochemistry was performed in two breast cancer tissue microarrays. In parallel, cross-talk between JAM-A, HER2 and ER was examined in several breast cell lines, using complementary genetic and pharmacological approaches. High JAM-A expression correlated significantly with HER2 protein expression, ER negativity, lower patient age, high-grade breast cancers, and aggressive luminal B, HER2 and basal subtypes of breast cancer. JAM-A and HER2 were co-expressed at high levels in vitro in SKBR3, UACC-812, UACC-893 and MCF7-HER2 cells. Knockdown or functional antagonism of HER2 did not alter JAM-A expression in any cell line tested. Interestingly, however, JAM-A knockdown decreased HER2 and ER-α expression, resulting in reduced levels of phospho-(active) AKT without an effect on the extracellular signal-related kinase phosphorylation. The downstream effects of JAM-A knockdown on HER2 and phospho-AKT were partially reversed upon treatment with the proteasomal inhibitor MG132. We conclude that JAM-A is co-expressed with HER2 and associates with aggressive breast cancer phenotypes. Furthermore, we speculate that JAM-A may regulate HER2 proteasomal degradation and activity, potentially offering a promise as a therapeutic target in HER2-positive breast cancers.

Abstract IA11: Control of gene expression in the nucleus by small RNAs

Abstract Double-stranded RNAs are widely used to control gene expression and are a promising class of therapeutic agent. Most studies have focused on recognition of mRNA in the cytoplasm and inhibition of translation. My presentation focuses on the action of small RNAs in the nucleus to modulate transcription or redirect alternative splicing. In 2005, my laboratory observed that synthetic RNAs complementary to the promoter region at the transcriptional start site of human progesterone receptor (PR) could block PR expression [1]. Inhibition was potent and robust, consistent with the suggestion that the synthetic RNAs were exploiting an endogenous mechanism for controlling gene expression. Promoter-targeted synthetic miRNAs were also capable of robust gene silencing [2], further supporting the existence of endogenous regulatory pathways. Modulation required complete seed sequence complementarity to the PR promoter, consistent with a mechanism that involves RNA-RNA recognition through Watson-Crick base-pairing. In 2007, we observed that small RNAs could also target the PR gene promoter and activate PR expression [3]. Activation was observed in cells that had a low, but detectable, level of PR expression, while gene silencing had been observed in cells with a high level of PR expression. Inactive and active RNAs compete for recognition, demonstrating that small changes in target site location affect activity. These results are reminiscent of protein transcription factors, which bind to specific sequences, are sensitive to target site location or small conformation changes, and can activate gene expression in some contexts but not others. Our promoter-targeted RNAs bind to long noncoding RNA transcripts at the PR promoter [4]. Use of antisense oligonucleotides to reduce transcript levels also reduces RNA-mediated modulation of gene expression. The small RNAs recruit AGO2 to the noncoding transcript, and inhibition of AGO2 expression reverses gene silencing or activation [5]. While AGO2 is normally expected to cleave fully complementary targets in the cytoplasm, our data suggests that AGO2 does not cleave transcripts associated with gene promoters in the nucleus. A noncoding RNA transcript also overlaps the 3′ terminus of the PR gene [6]. Small RNAs complementary to this noncoding transcript modulate PR expression, depending on the cell line used. The PR gene loops, juxtaposing its 3′ and 5′ termini and providing a direct path for a signal transmitting a signal from the 3′ terminus of the gene to the promoter. We have investigated whether RNAs can modulate other nuclear events. We designed small RNAs to be complementary to sequence within exons or introns near splice sites. These RNAs redirect alternative splicing and AGO2 was recruited to pre-mRNA [7]. These data provide more support for the action of small RNA in cell nuclei and for the conclusion that AGO2 may not always cause RNA cleavage when it encounters complementary targets in the nucleus. Our data suggest that small RNAs can bind noncoding transcripts and modulate gene expression. Binding occurs in proximity to the gene promoter, in cis relative to the target gene. The RNA-AGO complex acts like a transcription factor to affect the transcription machinery and tip the balance of inducible genes like PR towards repression or activation. Citation Format: David Corey. Control of gene expression in the nucleus by small RNAs [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA11.

Thermodynamic Dissection of Estrogen Receptor-Promoter Interactions Reveals that Steroid Receptor Family Members Differentially Partition their Binding Energetics

The quantitative principles responsible for steroid receptor-specific gene regulation are poorly understood. This is in part because most studies have measured only apparent binding affinities or used biochemical rather than biophysical approaches. We previously dissected the thermodynamics of human progesterone receptor (PR) isoform assembly at multisite promoter sequences. We found that the two isoforms maintain surprisingly weak dimerization energetics; however, the receptors can achieve full promoter occupancy via strong cooperative interactions. As a step towards assessing whether cooperative binding energetics are a common feature for all steroid receptors, we dissected the thermodynamics of human estrogen receptor-∗ (ER-∗):promoter interactions under conditions identical to our work on PR. Analytical ultracentrifugation and quantitative footprint titrations demonstrate that ER-∗ and PR dimers maintain similar intrinsic binding affinities toward their respective response elements. This is to be expected noting the near identical structures of the ER and PR DNA binding domains. Unexpectedly, however, ER-∗ exhibits negligible cooperativity - orders of magnitude less than predicted based on our studies of PR. This raises the question of how ER-∗ generates full occupancy (and thus full function) at multisite promoters. Our results reveal that the decrease in ER-∗ cooperative free energy is exactly compensated for via an increase in dimerization free energy. Thus homologous transcription factors partition their promoter binding energetics differentially. We speculate that this serves as a mechanism to receptor-specific gene regulation: differences in assembly state and cooperative stabilization allow for preferential occupancy as a function of response element layout.

P5-13-19: Borderline Estrogen and Progesterone Receptor Expression and Efficacy of Anti-Estrogen Therapy Analyzed by Subpopulation Treatment Effect Pattern Plot Analysis.

Abstract Background: The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) recently recommended that estrogen receptor (ER) and progesterone receptor (PR) values be considered positive in the evaluation of breast cancer specimens if there are at least 1% positive tumor nuclei by immunohistochemisty (IHC) scoring. This change constitutes a lower cutoff value than the 5% or 10% widely used in the past and increases the number of patients that can now be treated with anti-estrogen therapy, a therapy which carries significant treatment efficacy and a favorable toxicity profile. Despite the new recommendation however, it remains unclear if patients whose tumors display borderline ER and/or PR expression truly benefit from anti-estrogen therapy. Methods: We examined a total of 608 patient samples from the OHSU Knight Cancer Institute Breast Cancer Tissue Repository for which we have quantitative ER and PR information and their clinical outcome. Of those, 282 patients did not receive any systemic treatment and 165 received anti-estrogen therapy (the majority tamoxifen). Cytosolic steroid hormone receptors were quantitated by conventional steroid binding assay and Scatchard plot analysis or by enzyme-linked immunoassay system. For immunoassays, human estrogen receptor (ER) and progesterone receptor (PR) kits were obtained from Abbott Laboratories and assays were conducted according to manufacturer's specification. We performed subpopulation treatment effect pattern plot (STEPP) analysis to explore the presence of interaction between treatment effects and quantitative ER and PR levels. Clinical outcome used in this study includes 5-year overall survival and 5-year breast cancer free interval. Results: Multi-variate analysis finds neither ER nor PR expression adjusted by treatment status is associated with either overall survival (P= 0.20, median follow up of 63 months) or breast cancer free interval (P= 0.82, median follow up of 60 months). For both ER and PR, there is no interaction between treatment outcome (5 year survival probability) and levels of hormone receptor expression based on Kaplan-Meier estimate (P=0.36 and P= 0.65, respectively). Patients with borderline ER expression appear to benefit from anti-estrogen therapy while patients with borderline PR expression appear to have worse outcome with anti-estrogen therapy. Conclusions: Though descriptive in nature, our STEPP analysis provides support for the recommendation by ASCO and CAP to lower the cutoff for ER positivity in breast cancer specimens. There is overall no interaction between the level of hormone receptor and treatment benefit for all patients from low ER to high ER. Therefore, treatment will benefit all patients including those with low ER levels. Extending hormone therapies to the group of patients whose tumors express borderline PR values, however does not seem to confer therapeutic benefit. More study is needed to determine the benefits of offering anti-estrogen therapy to patients whose tumors express low PR. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-19.

Determination of cardiac histologic injury in a transgenic mouse model of heart failure expressing a novel mitochondrial progesterone receptor (PR-M)

1. To develop an automated histological fibrosis quantification protocol. 2. To determine cardiac histologic injury in a transgenic mouse model of heart failure expressing a human mitochondrial progesterone receptor (PR-M). Transgenic mouse study. PR-M is mitochondrial progesterone receptor isoform that increases cellular respiration. An inducible (TET-On) transgenic mouse model expressing human PR-M under the control of the cardiac myosin heavy chain 6 promoter was developed. PR-M expression was induced with oral doxycycline followed in 2 wks by surgical constant thoracic aortic constriction (cTAC) to increase after-load. Intact male and ovariectomized female mice were treated with progesterone in oil (2.5 mg/day SQ) starting 1 wk prior to cTAC. Mice were sacrificed 8 wks after cTAC and cardiac PR-M expression determined with realtime RT-PCR. Cardiac fibrosis and capillaries were identified with Masson's trichrome and immunostaining with lectin respectively, by an investigator blinded to gene expression and treatment. Histologic images were acquired via digital slide scanning. Morphometrics were computed with ImageJ64 (NIH) and Photoshop CS5 (Adobe Systems). Data from the histologic quantification were analyzed with unpaired Student's t-test. Progesterone treated PR-M positive mice (N = 9) showed greater capillary density (P=0.04), greater capillary density to cardiomyocyte width ratio (P=0.02) and decreased cardiac fibrosis (P=0.03) after cTAC compared to progesterone treated PR-M negative mice (N = 17). The mean myocyte width was not different between the 2 groups (P=0.33). A novel computerized fibrosis quantification method for cardiac tissue was developed. Compared to controls, progesterone treated transgenic mice expressing a human mitochondrial progesterone receptor (PR-M) had fewer histologic signs of cardiac injury. These observations support a role for progesterone via a mitochondrial progesterone receptor in cardiac function.

Progesterone receptor polymorphisms and clinical response to 17-alpha-hydroxyprogesterone caproate

Seventeen-alpha-hydroxyprogesterone caproate (17-OHPC) reduces recurrent preterm birth (PTB). We hypothesized that single nucleotide polymorphisms in the human progesterone receptor (PGR) affect response to 17-OHPC in the prevention of recurrent PTB. We conducted secondary analysis of a study of 17-OHPC vs placebo for recurrent PTB prevention. Twenty PGR gene single nucleotide polymorphisms were studied. Multivariable logistic regression assessed for an interaction between PGR genotype and treatment status in modulating the risk of recurrent PTB. A total of 380 women were included; 253 (66.6%) received 17-OHPC and 127 (33.4%) received placebo. In all, 61.1% of women were African American. Multivariable logistic regression demonstrated significant treatment-genotype interactions (either a beneficial or harmful treatment response) for African Americans delivering<37 weeks' gestation for rs471767 and rs578029, and for Hispanics/Caucasians delivering<37 weeks' gestation for rs500760 and <32 weeks' gestation for rs578029, rs503362, and rs666553. The clinical efficacy and safety of 17-OHPC for recurrent PTB prevention may be altered by PGR gene polymorphisms.

Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters

Synthetic small duplex RNAs that are fully complementary to gene promoters can silence transcription in mammalian cells. microRNAs (miRNAs) are endogenous small regulatory RNAs that sequence specifically regulate gene expression. We have developed a computational method to identify potential miRNA target sites within gene promoters. Ten candidate miRNAs predicted to target the human progesterone receptor (PR) gene promoter were tested for their ability to modulate gene expression. Several miRNA mimics inhibited PR gene expression and miR-423-5p, which targets a highly conserved region of the PR promoter, was chosen for detailed analysis. Chromatin immunoprecipitation revealed that the miR-423-5p mimic decreased RNA polymerase II occupancy and increased histone H3 lysine 9 dimethylation (H3K9me2) at the PR promoter, indicative of chromatin-level silencing. Transcriptional silencing was transient, independent of DNA methylation, and associated with recruitment of Argonaute 2 (AGO2) to a non-coding RNA (ncRNA) transcript that overlaps the PR gene promoter. The miR-423-5p mimic also silenced expression of immunoglobulin superfamily member 1 (IGSF1), an additional gene with a predicted target site within its promoter. While additional investigations of endogenous miRNA function will be necessary, these observations suggest that recognition of gene promoters by miRNAs may be a natural and general mechanism for regulating gene transcription.

Affinity of estrogens for human progesterone receptor A and B monomers and risk of breast cancer: a comparative molecular modeling study

BackgroundThe human progesterone receptor (hPR) belongs to the steroid receptor family. It may be found as monomers (A and B) and or as a dimer (AB). hPR is regarded as the prognostic biomarker for breast cancer. In a cellular dimer system, AB is the dominant species in most cases. However, when a cell coexpresses all three isoforms of hPR, the complexity of the action of this receptor increases. For example, hPR A suppresses the activity of hPR B, and the ratio of hPR A to hPR B may determine the physiology of a breast tumor. Also, persistent exposure of hPRs to nonendogenous ligands is a common risk factor for breast cancer. Hence we aimed to study progesterone and some nonendogenous ligand interactions with hPRs and their molecular docking.Methods and resultsA pool of steroid derivatives, namely, progesterone, cholesterol, testosterone, testolectone, estradiol, estrone, norethindrone, exemestane, and norgestrel, was used for this in silico study. Dockings were performed on AutoDock 4.2. We found that estrogens, including estradiol and estrone, had a higher affinity for hPR A and B monomers in comparison with the dimer, hPR AB, and that of the endogenous progesterone ligand. hPR A had a higher affinity to all the docked ligands than hPR B.ConclusionThis study suggests that the exposure of estrogens to hPR A as well as hPR B, and more particularly to hPR A alone, is a risk factor for breast cancer.

TGF-β1 and BRCA2 Expression are Associated with Clinical Factors in Breast Cancer

The objective of this study was to investigate the possible association between the expression of transforming growth factor beta-1 (TGF-β1) and breast cancer type2 susceptibility protein (BRCA2) with clinical factors in breast cancer. TGF-β1, BRCA2, human epidermal growth factor receptor2 (HER2), estrogen receptor, and progesterone receptor protein levels were measured in 67 samples from breast cancer patients by immunohistochemistry. The expression of these proteins was correlated with various clinical factors including age, pathohistological grade and status of axillary lymph node implication. TGF-β1 and BRCA2 were expressed in breast cancer tissues and expression of HER2 and TGF-β1 was significantly correlated with BRCA2. The authors conclude that elevated expression of BRCA2 correlates with TGF-β1 and HER2 in breast cancer and these three factors act in synergy to promote cancer. Thus, detection of both TGF-β1 and BRCA2 may therefore assist in the prognosis and treatment of breast cancer.

Transcriptional regulation by miRNA mimics that target sequences downstream of gene termini

Transcriptome studies have revealed that protein-coding loci within the human genome are overlapped at their 3'-termini by noncoding RNA (ncRNA) transcripts. Small duplex RNAs designed to be fully complementary to these 3' ncRNAs can modulate transcription of the upstream gene. Robust regulation by designed RNAs suggests that endogenous small RNAs might also recognize 3' ncRNAs and regulate gene expression. A genome-wide evaluation revealed that sequences immediately downstream of protein-coding genes are enriched with miRNA target sites. We experimentally tested miRNA mimics complementary to the well-characterized 3'-terminus of the human progesterone receptor (PR) gene and observed inhibition of PR transcription. These results suggest that recognition of ncRNA transcripts that overlap gene termini may be a natural function of endogenous small RNAs.

A profile of prognostic and molecular factors in European and Māori breast cancer patients

BackgroundNew Zealand Māori have a poorer outcome from breast cancer than non-Māori, yet prognostic data are sparse. The objective of this study was to quantify levels of prognostic factors in a cohort of self-declared Māori and European breast cancer patients from Christchurch, New Zealand.Methods and ResultsClinicopathological and survival data from 337 consecutive breast cancer patients (27 Māori, 310 European) were evaluated. Fewer tumours were high grade in Māori women than European women (p = 0.027). No significant ethnic differences were detected for node status, tumour type, tumour size, human epidermal growth factor receptor, oestrogen and progesterone receptor (ER/PR) status, or survival.In addition, tumour and serum samples from a sub-cohort of 14 Māori matched to 14 NZ European patients were analyzed by immunohistochemistry and enzyme linked immunosorbent assay for molecular prognostic factors. Significant correlations were detected between increased grade and increased levels of hypoxia inducible factor-1 (HIF-1α), glucose transporter-1 (GLUT-1), microvessel density (MVD) and cytokeratins CK5/6 (p < 0.05). High nodal status correlated with reduced carbonic anhydrase IX (CA-IX). Negative ER/PR status correlated with increased GLUT-1, CA-IX and MVD. Within the molecular factors, increased HIF-1α correlated with raised GLUT-1, MVD and CK5/6, and CK5/6 with GLUT-1 and MVD (p < 0.05). The small number of patients in this sub-cohort limited discrimination of ethnic differences.ConclusionsIn this Christchurch cohort of breast cancer patients, Māori women were no more likely than European women to have pathological or molecular factors predictive of poor prognosis. These data contrast with data from the North Island NZ, and suggest potential regional differences.