ResearchPad - steroid-hormones-and-receptors https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SUN-736 Knockout of Membrane Androgen Receptor ZIP9 Results in Reduced Female Fecundity and Abnormal Egg Activation in Zebrafish]]> https://www.researchpad.co/article/elastic_article_8746 Recently, our research group cloned and characterized a putative membrane androgen receptor from teleost ovarian tissue that was found to be homologous with the zinc transporter protein ZIP9 (Slc39a9). To date, ZIP9 is the only zinc transporter that is known to be ligand activated or possess steroid receptor activity. Since the discovery of its androgen receptor activity, ZIP9 has been found to mediate androgen actions in a variety of tissues including teleost ovarian follicle cells, human cancer cell lines, and murine Sertoli cells. However, ZIP9 has not been examined in an in vivo model so the precise physiological functions of this receptor remain unclear. A ZIP9-mutant strain of zebrafish was developed using a CRISPR-Cas9 system in order to examine the role of the protein in teleost reproduction. While ZIP9-mutant males had similar breeding occurrence and fertilization rates to wild-type fish, mutant females exhibited severe reductions in fecundity compared to wild-type fish. ZIP9-mutant females spawn significantly fewer eggs of which a high proportion failed to undergo chorion elevation, a characteristic of normal egg activation. Eggs that showed this failed chorion elevation phenotype had significantly lower fertilization rates and produced larvae that exhibit a high incidence of pericardial/yolk sac edema and reduced growth compared to larvae hatched from wild-type eggs. However, no differences were observed in the proportions of oocytes at later stages of development between ZIP9-mutant and wild-type fish, suggesting the observed phenotypes are not related to abnormal oogenesis. We observed that mature wild-type eggs have numerous cortically located vesicles that are autofluorescent under ultraviolet light and decrease in number when the eggs undergo activation, suggesting they undergo exocytosis during the cortical reaction. While zinc is known to be stored in vesicles that undergo exocytosis in mammalian eggs, the role of zinc in teleost egg activation is currently unknown. In eggs from wild-type fish, we observed an increase in extracellular zinc levels upon egg activation and treatment with a zinc ionophore (zinc pyrithione) significantly reduced the number of eggs that undergo normal chorion elevation when activated. This suggests a role for zinc in zebrafish egg activation similar to that observed in mammals. Of interest, ZIP9-mutant eggs that did not undergo chorion elevation had significantly smaller vesicles than those found in wild-type fish eggs. This abnormal vesicle morphology and failure to undergo chorion elevation suggest a role of ZIP9 in egg activation. Additional insight into the role of zinc in zebrafish egg activation and the mechanism by which ZIP9 disruption leads to abnormal cortical vesicles and egg activation will help determine if ZIP9 plays a role in zinc transport and flux in zebrafish eggs during activation.

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<![CDATA[OR09-03 Brain Aromatase Is Essential for Regulation of Sexual Activity in Male Mice]]> https://www.researchpad.co/article/elastic_article_8737 Introduction: The biologically active form of estrogen, estradiol (E2), has important organizational roles in brain development and activational roles in adult brain physiology and behavior. It has been proposed that E2 formation in the brain might regulate sexual activity in various species. The mechanisms that link estrogen formation in the brain and sexual behavior, however, remain unclear. Aromatase is the key enzyme that catalyzes the conversion of testosterone (T) to E2 in the testis and brain of male mice. To determine the role of brain aromatase in male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse model. Additionally, a newly generated total aromatase knockout (tArKO) mouse model served as a positive control. Methods: We generated the floxed aromatase mice (Aromfl/fl), which flanked the transcription and translation start sites and the common splice acceptor site for the upstream brain promoter I.f of the aromatase gene. We then crossed Nestin-Cre mice with Aromfl/fl mice to generate bArKO mice. Using the same Aromfl/fl mice, we bred tArKO via crossing with ZP3-Cre mice. Circulating and tissue (brain and testis) E2 levels were measured using liquid chromatography-tandem mass spectrometry. We assessed sexual activity in 12-14 week-old bArKO, tArKO and littermate control males over two 30-minute trials. The interactions were monitored and videotaped, and the videotape was scored for the sexual activity. To investigate whether the lack of estrogen production in the brain was causative for altered sexual behavior, 20 bArKO and 20 control mice were castrated at ~nine weeks of age and supplemented with exogenous sex hormone via 60-day time release pellet implantation. Results: E2 levels are significantly decreased in the brain but not the testis of bArKO mice as compared to control mice (P < 0.05, n=6-12). As expected, E2 levels in the brain and testis are significantly lower in tArKO mice compared with their WT littermates (n=6-9). Furthermore, we demonstrate that local aromatase expression and estrogen production in the brain is required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited significantly decreased sexual activity in the presence of strikingly elevated circulating T (n=5). In castrated adult bArKO mice, administration of E2 together with T restored maximum sexual behavior (n=5). Thus, aromatase in the brain is necessary for T-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating T of testicular origin. Conclusion: Our findings suggest T activates male sexual behavior in part via conversion to E2 in the brain and provide the foundation for inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators in modifying sexual behavior.

DCB and HZ contributed equally to this work.

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<![CDATA[SUN-LB136 A Comparison of Androgen Receptor Splice Variant, AR-V7, and Glucocorticoid Receptor Activity in Prostate Cancer]]> https://www.researchpad.co/article/elastic_article_8702 Prostate Cancer (PCa) is an androgen dependent disease and patients with metastatic PCa are treated with androgen deprivation therapy (ADT). Although most tumors respond initially, tumors become resistant and are termed castration resistant prostate cancer (CRPC). There is compelling evidence that most of these tumors retain androgen receptor (AR) dependence and some data to suggest that, in some cases, the glucocorticoid receptor (GR) substitutes for AR. AR, itself, is re-activated through a variety of mechanisms including the expression of constitutively active AR splice variants that lack the ligand binding domain (LBD) of AR. Expression of one variant, AR-V7, which contains the amino-terminal domain and DNA binding domain of AR and 16 unique amino-acids, has been correlated with resistance to second line ADT. Although there has been some debate regarding the role of AR-V7, whether it is only a partial substitute for AR or has unique activities, our studies of engineered cell lines treated to express levels of AR-V7 equivalent to AR, clearly show that while the AR isoforms have common targets, they each also have unique targets. Consistent with this, the cistromes of the two show many unique sites as well as common sites. AR-V7 binding is enriched near the transcription start site (TSS) and we have identified a novel de novo binding motif. These findings suggest the possibility of developing a gene signature unique to AR-V7.Because GR activity in PCa has also been suggested as an escape mechanism in response to ADT, and GR binds to the same consensus response elements, we sought to identify a GR signature in PCa, to compare it with the AR and AR-V7 signatures, and to ask whether the AR-V7 and/or GR signatures are enriched in CRPC. Because much of the gene signatures are cell line dependent, we sought to compare GR and AR-V7 action in cells that express both. LN-95 cells express both AR-V7 and GR. MDA-PCa-2b cells, a cell line derived from an African American patient, expresses GR, but not AR-V7. The parental line was infected with a lentivirus that expresses AR-V7 in response to doxycycline. Transcriptomes were determined for AR, GR, and AR-V7 in these lines using RNA-Seq. Overall the magnitude of regulation of gene expression was generally lower than in the LNCaP AR-V7 and VCaP-AR-V7 lines, but there was good overlap of the MDA-PCa-2b AR-V7 regulated with the LNCaP AR-V7 regulated genes. Genes induced by GR overlapped with AR and isoform common genes, but did not overlap with AR-V7 specific genes. A comparison of AR-V7 specific genes common to the LNCaP and VCaP models as well as to publicly available data sets for LN-95 and 22RV1 AR-V7 signatures, show a strong correlation with CRPC compared to primary tumors when analyzed in the Grasso data set.

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<![CDATA[SUN-739 Next Generation AR Antagonists Increase Systemic Active Glucocorticoid Exposure by Altering Glucocorticoid Metabolism]]> https://www.researchpad.co/article/elastic_article_8693 Enzalutamide and apalutamide are potent next-generation androgen receptor (AR) antagonists used in metastatic and non-metastatic prostate cancer. Despite the increased survival benefits of these agents, resistance normally occurs and the disease transitions to its lethal form. We hypothesized that enzalutamide and apalutamide suppress 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which normally converts cortisol to cortisone, leading to elevated cortisol concentrations and increased ratio of active to inactive glucocorticoids. We measured cortisol and cortisol/cortisone ratio (substrate/product of 11β-HSD2) in serum using mass spectrometry before and 1 month on-treatment in 3 clinical trials: 1) neoadjuvant apalutamide + leuprolide (n=25) 2) enzalutamide +/- PROSTVAC for metastatic castration-resistant prostate cancer (n=54) and 3) enzalutamide +/- PROSTVAC for non-metastatic castration-sensitive prostate cancer (n=38 patients). Progression-free survival (PFS) was determined in the metastatic CRPC study of enzalutamide +/- PROSTVAC for those with glucocorticoid changes above and below the median. A statistically significant rise in cortisol concentration and cortisol/cortisone ratio with AR antagonist treatment occurred uniformly across all 3 clinical trials. For example, a rise in cortisol/cortisone ratio occurred in 23/25 (92%) patients (p < 0.001), 36/54 (67%) patients (p < 0.001), and 30/38 (79%) patients (p = 0.051), in the 3 respective trials. In the trial of enzalutamide +/- PROSTVAC for metastatic CRPC, high cortisol/cortisone ratio in the enzalutamide arm was associated with significantly improved PSA progression-free survival and radiographic progression-free survival. However, in the enzalutamide + PROSTVAC arm, the opposite trend was observed. In conclusion, treatment with enzalutamide or apalutamide increases systemic exposure to active glucocorticoids. These findings have potential consequences for immune suppression and the efficacy of treatment combinations using next-generation AR antagonists. On-treatment, glucocorticoid changes might serve as a pharmacodynamic biomarker.

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<![CDATA[OR12-06 Nuclear Receptor CAR Protects Female Mice from the Development of Diet-Induced Nonalcoholic Fatty Liver Disease]]> https://www.researchpad.co/article/elastic_article_8689 NAFLD (Non Alcoholic Fatty Liver Disease) has become the most common cause of chronic liver disease in many developed countries worldwide and represents a major health concern. The prevalence of NAFLD is sexually dimorphic with men suspected to be more susceptible to the development of hepatic steatosis than women. Women are mostly protected until hormonal imbalance induced by menopause. Nuclear receptor CAR (Constitutive Androstan Receptor) is at the crossroads between endocrine and metabolic regulations and could therefore represent an interesting therapeutic target. It is primarily expressed in the liver and involved in the catabolism of hormones such as thyroid hormones, corticosteroids and estrogens. In addition, several studies reveal a metabolic role of CAR through regulation of major hepatic pathways such as neoglucogenesis, beta-oxidation and de novo lipogenesis. Our research is aimed at better understanding the role of CAR using a mouse model genetically deficient for CAR. To explore the metabolic functions of CAR, knock-out male and female mice were subjected to a high fat diet (HFD) for 16 weeks. Concomitant CAR deletion and high fat diet induces sexually dimorphic metabolic disorders. Knock-out of CAR in males exacerbates HFD-induced fasted hyperglycemia whereas in females, it aggravates body weight gain and adipose tissue accumulation. In accordance with epidemiological studies revealing a protection of women from the development of hepatic steatosis, HFD-fed WT female mice present less important hepatic steatosis than HFD-fed WT male mice. However, following CAR deletion, HFD-fed female mice develop a severe steatosis along with important hepatic injury. Ongoing studies aim to understand the transcriptomic and endocrine dysregulations that may explain these phenotypes. These results reveal a previously unrecognized dimorphic role of CAR in energy homeostasis and highlights its involvement in the protection of female mice towards the development of hepatic steatosis. Overall, this research provides further insights in the pathogenesis of NAFLD and its dimorphic prevalence.

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<![CDATA[SUN-740 Low-Dose Dihydrotestosterone Lowers Lipogenic Master Regulator in Liver and Adipose Tissue from Female Mice]]> https://www.researchpad.co/article/elastic_article_8687 Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects 1 in 10 women. These hallmarks are also integral elements of non-alcoholic liver disease (NALFD), a disorder that is common in women with PCOS. Administering low dose dihydrotestosterone (DHT) induced a lean female mouse model with a PCOS-like phenotype, displaying IR and NAFLD. The molecular mechanism of HA-induced NAFLD has not been determined. We hypothesized that low dose DHT would interrupt hepatic lipid metabolism leading to NAFLD. To investigate the role of androgens on the master regulator of lipogenesis, sterol regulatory element-binding protein 1 (SREBP1), we extracted white adipose tissue (WAT), liver, and skeletal muscle from wild-type, control and low dose DHT female mice; and performed Western blot and real-time quantitative PCR (qRT-PCR) analysis of lipogenic intermediates of the tissue homogenates. Low-dose DHT lowered the active form of cytosolic SREBP1 in the liver and WAT compared to controls. Additionally, low dose DHT lowered inactive SREBP1 in the liver. However, the condition did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT, though the active form was lowered in skeletal muscle. Further, p-ACC levels were unaltered in liver and WAT. FAS levels were unchanged in WAT and skeletal muscle. Taken together, our findings support the hypothesis that cytosolic SREBP1 decreased due to its translocation to the nucleus, where it regulates lipogenic protein levels. We speculate that low-dose DHT promotes the translocation of SREBP1 from the cytosol to the nucleus to influence lipogenic gene expression leading to increased lipogenesis contributing to NAFLD.

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<![CDATA[SUN-735 Functional Analysis of Testis-Specific Noncoding Genes in Estrogen-Dependent Transcription]]> https://www.researchpad.co/article/elastic_article_8635 Emerging studies have shown that germ cell (GC)-specific genes play critical roles in several cancers. The expression of these genes is tightly regulated and restricted to testis; however, many of them escape regulation and become aberrantly expressed in tumors. Interestingly, our genomic analysis suggests that several of these genes are long noncoding RNAs (lncRNAs) and are located at regions previously considered to be gene deserts in the human genome. In this regard, we used an integrated genomic approach to identify GC-lncRNA genes that are overexpressed in breast cancer. Further, by incorporating gene expression analysis from RNA-seq data from MCF-7 and T47D breast cancer cells, we generated a comprehensive list of estrogen-regulated GC-lncRNA genes. We hypothesize that GC-lncRNA genes regulate estrogen-dependent signaling in breast cancer. The selected genes: (a) CAERRC (Chromatin Associated Estrogen-Regulated RNA in Cancer, (b) LncRNA568, (c) LncRNA16 are primate-specific, and exclusively expressed in testis. All of them are regulated by estrogen, and their expression predicts poor outcome in ERα+ breast cancer patients. They have now been fully annotated (transcription start and stop site, 5’ cap, polyA tail, and exon/intron structure), and cloned. Further, we have created gene-specific KO MCF-7 cell lines using CRISPR to study their molecular roles. Our data suggest that these genes regulate estrogen-dependent gene expression and tumor growth in breast cancer cells. Genome-wide analysis of ERα binding and gene expression data indicate that they play a critical role in the estrogen-dependent transcription. Collectively, our results suggest that GC-genes, including CAERRC, LncRNA568, and LncRNA16, are excellent targets with prognostic and therapeutic potential in ER+ breast cancers.

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<![CDATA[OR12-03 Mineralocorticoid and Glucocorticoid Receptors Adopt Distinct Quaternary Structures and Can Form Heteromultimers That Affect Chromatin-Binding Profiles]]> https://www.researchpad.co/article/elastic_article_8612 The mineralocorticoid and glucocorticoid receptors (MR and GR) are evolutionarily related nuclear receptors with high sequence conservation and a shared hormone response element (HRE). Both receptors are activated by glucocorticoids, but MR can be selectively activated by aldosterone. Using the imaging technique Number & Brightness (N&B) it has recently been proposed that liganded GR dimers form tetramers upon binding to HREs in live cells. We now show that agonist-bound MR adopts a tetrameric organization in the nucleoplasm and forms complexes with an average of 7 receptor units upon binding an HRE. Interestingly, MR antagonists eplerenone and spironolactone induced intermediate oligomerization arrangements, strongly suggesting that higher order oligomerization is essential for receptor activity. Site-directed mutagenesis and deletion analysis suggest that the N-terminus of MR is a main determinant of higher order oligomerization. Both with corticosterone and aldosterone, GR can incorporate into MR complexes partially displacing MR monomers. Genome-wide chromatin binding studies suggest that the presence of GR in the same cells profoundly change MR interaction with distinct sets of enhancers in a ligand-dependent way, contributing to receptor-specific signaling. Certain genes respond to only one receptor while others respond to both receptors. The interaction of these two closely related receptors has important implications for the mechanisms for glucocorticoid signaling and transcription factors in general.

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<![CDATA[SAT-737 Low-Dose Testosterone Augmentation for Treatment-Resistant Depression in Women: An 8-Week, Two-Site, Randomized, Placebo-Controlled Study]]> https://www.researchpad.co/article/elastic_article_7156 Objective: Nonresponse to selective serotonin reuptake inhibitor and serotonin norepinephrine reuptake inhibitor treatment is common in patients with major depressive disorder (MDD), particularly in women, occurring in about 70% of patients despite adequate dosing. Well-tolerated augmentation strategies are needed, particularly ones that do not cause or exacerbate symptoms such as fatigue and sexual dysfunction. Low-dose testosterone has been shown to improve depression symptom severity, fatigue and sexual function in small studies of women not formally diagnosed with MDD. We sought to determine whether adjunctive low-dose transdermal testosterone improves depression symptom severity, fatigue, and sexual function in women with treatment-resistant MDD. A functional MRI (fMRI) substudy examined effects of testosterone on activity in the anterior cingulate cortex (ACC), a brain region important in mood regulation.

Methods: Randomized, double-blind, placebo-controlled, 8-week trial of adjunctive testosterone cream (AndroFeme® 1, Lawley Pharmaceuticals, Australia) in 101 women, ages 21–70, with treatment-resistant MDD. Testosterone was titrated to achieve blood levels near the upper normal reference limit. Primary outcome measure was depression severity by Montgomery-Asberg Depression Rating Scale (MADRS). Secondary endpoints included fatigue, sexual function, and safety measures. fMRI substudy (n=20) primary outcome was change in ACC activity.

Results: Mean age was 47±14 (SD) years and mean baseline MADRS score was 26.6±5.9. Eighty-seven (86%) participants completed 8 weeks of treatment. MADRS depression scores decreased in both arms [testosterone: 26.8±6.3 to 15.3±9.6; placebo: 26.3±5.4 to 14.4±9.3 (baseline to 8 weeks, respectively)], with no difference between groups (p=0.91). Fatigue and sexual function improved without differences between groups. There were no group differences in side effects. fMRI results demonstrated a relationship between ACC activation and androgen levels pretreatment but no difference in ACC activation with treatment.

Conclusions: This rigorously designed, double-blinded clinical trial did not find significant group differences between adjunctive low dose transdermal testosterone and placebo for antidepressant augmentation in women with treatment-resistant MDD and had a high placebo response rate. Low-dose testosterone was well tolerated but failed to differentially impact overall depressive symptom severity, fatigue, or sexual dysfunction. Testosterone did not result in greater activity in a brain region (ACC) implicated in MDD etiopathology compared to placebo. Thus, the addition of low-dose testosterone to ineffective antidepressant treatment should not be recommended for women with MDD. Further studies using strategies designed to reduce placebo effects may be warranted.

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<![CDATA[SAT-736 Dissecting the Relative Role of Estrogen and Androgen in Fibrosis, Skeletal Muscle Atrophy, and Inguinal Hernia Formation]]> https://www.researchpad.co/article/elastic_article_6965 Introduction: More than one in four men develop symptomatic inguinal hernia, and hernia repair is the most commonly performed general surgical procedure in the US. Despite its prevalence, the molecular mechanisms causing inguinal hernia remain unclear. Aromatase, the key enzyme for the conversion of testosterone (T) to estradiol (E2), is present in human but not mouse skeletal muscle tissue. We recently demonstrated that robustly increased local E2 levels in lower abdominal muscle (LAM) tissue and decreased circulating T levels were associated with fibrosis and myocyte atrophy in LAM tissue, leading to severe scrotal (inguinal) hernia formation in a humanized aromatase transgenic mouse model (Aromhum) with a high LAM human aromatase expression. To further determine the relative role of estrogen and androgen in the development of inguinal hernia, we generated a novel mild Aromhum mouse model with lower LAM aromatase expression compared with the severe model. Methods: Mild Aromhum mice were followed for 6 months to determine hernia incidence and measure hernia size (n=30). We treated mild Aromhum mice with the aromatase inhibitor, letrozole (n=12) for 12 weeks. Circulating and LAM E2 levels in mice were measured using mass spectrometry. LAM tissue fibrosis and myocyte size were determined by Masson’s trichrome staining and H&E staining, respectively. Results: The mild Aromhum mice contain a single copy of the human aromatase genomic fragment with a truncated regulatory region, giving rise to significant but mildly elevated LAM E2 levels (2.5-fold) at 15 weeks of age. Interestingly, these mice maintain normal circulating T levels. Furthermore, we show that mildly increased LAM E2 without decreased circulating T levels cause hernia formation in about 88% of mild Aromhum mice in contrast to 100% hernia formation in mice containing the full-length human aromatase regulatory region (severe Aromhum model), suggesting that higher LAM estrogen and low serum T levels contribute to this severe phenotype. Treatment with an aromatase inhibitor restores LAM E2 levels to normal levels and completely prevents inguinal hernia formation in the mild Aromhum mice. In LAM fibroblasts of mild Aromhum mice, we find very high levels of estrogen receptor-α expression, which possibly mediates estrogen-induced hernia formation. Conclusion: Taken together, our findings from the mild Aromhum mouse model suggest that lower levels of estrogen excess in LAM are the primary driver of muscle atrophy and hernia formation because this mouse model do not exhibit circulating T deficiency. Our findings will constitute a starting point for dissecting the relative roles of estrogen and androgen action in inguinal hernia development. This has the potential to facilitate drug development to prevent and treat hernias, especially recurrent hernias after primary hernia repairs in vulnerable populations such as elderly men.

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<![CDATA[SUN-LB134 Androgen Receptor Phosphorylated at Serine 815 in Mouse and Human Prostates]]> https://www.researchpad.co/article/elastic_article_6854 Androgen receptor (AR) regulates male sexual development and maintenance. AR forms a homodimer in the cytoplasm and monomerizes following hormonal activation, translocating to the nucleus in Cos-1 cells (Shizu et al. Scientific reports. 2019). Utilizing Ser815 of AR, the conserved phosphorylation residue within the ligand binding domains of steroid hormone receptors (NR3C), whether and how this phosphorylation regulates AR functions was investigated. While, like AR WT, a phosphomimic AR S815D mutant formed a homodimer in the cytoplasm, unlike the WT, this mutant remained as a homodimer in the cytoplasm even after hormone treatment. Apparently, Ser815 phosphorylation disabled AR’s capability to monomerize and nuclear translocate in Cos-1 cells. A phospho-Ser815 peptide antibody was used to detect phosphorylation of endogenous AR in mouse as well as human prostates. Immunohistochemistry showed phosphorylation present in both the cytoplasm and nucleus. Mouse prostates were cell fractionated in cell membrane, mitochondria, endoplasmic reticulum (ER) and cytosolic fractions for subsequent Western blot analysis. While AR was found in all of these fractions, phosphorylated AR was only detected in the ER and cytosolic fractions. A cDNA microarray analysis of PC-3 cells with ectopic expression of AR S815D suggested that phosphorylated AR may regulate ER stress.

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<![CDATA[OR12-04 Regulatory Sharing between Estrogen Receptor Alpha Bound Enhancers]]> https://www.researchpad.co/article/elastic_article_6850 Mammalian genomes encode an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by combinations of enhancers. We previously found that neighboring estrogen-responsive enhancers exhibit cooperative/synergistic contributions to an estrogenic transcriptional response1. However, when the same combinations of enhancers are targeted with synthetic activators in the absence of estrogens, then the regulatory regions exhibit independent effects on gene expression2. Taken together, these findings indicate that estrogen receptor alpha (ER) bound enhancers cooperate with each other in cis but influence target gene promoters independently. To determine the molecular underpinnings of enhancer cooperativity, we generated genetic deletions of individual ER bound enhancers. We discovered “regulatory sharing” between enhancers in which loci containing full estrogen response elements (EREs) contribute ER binding to neighboring sites, while enhancers with pre-existing histone acetylation/accessibility contribute this permissible chromatin environment to the neighboring enhancers upon estrogen induction. Genome engineering revealed that a cluster of two half ERE enhancers could not compensate for a full ERE site loss within the cluster. However, two full ERE enhancers produced a transcriptional response greater than the wild-type locus, suggesting that combinations of enhancers are not necessarily configured for a maximal response. By swapping genomic sequences, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is critical for ER recruitment, but the presence of pre-existing histone acetylation and accessibility within an enhancer cluster is also needed in order for estrogen-induced gene regulation to occur.

References 1. Carleton JB, Berrett KC, Gertz J (2017). Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor α-Bound Enhancers. Cell Syst, 5(4), 333-344.e5.2. 2. Ginley-Hidinger M, Carleton JB, Rodriguez AC, Berrett KC, Gertz J. Sufficiency analysis of estrogen responsive enhancers using synthetic activators. Life Sci Alliance, 2(5).

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<![CDATA[SAT-LB135 A Novel Cytoplasmic Membrane Estrogen-Mediated Biogenic Signaling Pathway]]> https://www.researchpad.co/article/elastic_article_6664 The estrogen receptor α (ERα) is known to convey both genomic and extra-genomic activities. The extra-nuclear estrogen signaling pathway is thought to involve a membrane-associated estrogen receptor (ERα), which activates PI3-kinase and Akt signaling. Maximal activation of Akt requires S473 phosphorylation. The essential G1-cyclin, CCND1, is a collaborative nuclear oncogene that is frequently overexpressed in cancer. D-type cyclins bind and activate CDK4/6, contributing to G1-S cell-cycle progression. Herein, cyclin D1 was shown to be located in the cytoplasmic membrane of patients with inflammatory breast cancer, human diploid fibroblasts and cancer cell lines (breast, prostate). The extra-nuclear vs. nuclear E2-induced signaling pathways can be distinguished using 17β-estradiol linked to a dendrimer conjugate (EDC), which excludes estradiol from the nucleus. In contrast with the nuclear-localized form of cyclin D1 (cyclin D1NL), the cytoplasmic membrane-localized form of cyclin D1 (cyclin D1CML) was sufficient to induce phosphorylation of the serine threonine kinase Akt (Ser473) and augmented extra-nuclear localized 17β-estradiol dendrimer conjugate (EDC)-mediated phosphorylation of Akt (Ser473). Cyclin D1CML was sufficient to induce G1-S cell-cycle progression, cellular proliferation, colony formation. In contrast with cyclin D1NL, the cyclin D1CML induced transwell migration and the velocity of cellular migration. Together these studies suggest distinct subcellular compartments of cell cycle proteins may convey distinct functions. The major adjuvant therapy for the ~70% of ERα expressing human breast cancer involves anti-estrogen therapy and the ERα/PI3K/Akt complex pathway is hyperactivated in aggressive breast tumors. The non-genomic actions of E2/ERα, mediated via cyclin D1CML may provide an important additional target. References. 1. 2. Casimiro MC et al Mol Endocrinol. 2013;27(9):1415-28. Di Sante, G, Expert Rev Anticancer Ther. 2019 Jun 20:1-19.

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<![CDATA[SUN-746 Thyroid Hormone Receptor Alpha Sumoylation Is Important for the Development of Adipose Tissue]]> https://www.researchpad.co/article/elastic_article_6632 Thyroid hormone (TH) plays an essential role in normal development. TH action requires binding to its receptor. There are two types of thyroid hormone receptor, alpha (THRA) and beta (THRB). THRB is important for regulation of the Hypothalamic-Pituitary-Thyroid axis and regulating cholesterol metabolism. THRA is involved in the development and the function of brain, adipose tissue, small intestine, bone and heart. Both THRA and THRB are post-translationally modified by small ubiquitin-like modifier (SUMO). Previously, we showed in an in vitro model that mutation of a THRA sumoylation motif impairs proliferation and differentiation of human primary white adipocytes. This is due to interference of the desumolyated THRA in the cell cycle at G1/S phase and disruption of PPAR signaling. To determine the in vivo effects of a desumoylated THRA, we generated a mouse model carrying a mutation of the THRA gene, which eliminates SUMO conjugation at lysine (K) 283. At 12 weeks of age, there was no difference in body composition (body weight, fat mass, and lean mass) in THRA mutant mice compared to wild-type (WT). From 12 weeks onward mutant mice and WT did not have significant change body weight compared to Wt mice. However, THRA sumoylation mutant mice had much lower percent body fat. Dissected WAT fat pads from mutant mice were significantly smaller compared to WT mice. Histological analysis showed that the cell size of white adipose tissue in mutant mice was significantly smaller than that in Wt mice as well. Serum leptin levels in the mutant mice were significantly lower than that in mice, consistent with reduced fat mass. We isolated fat stem cells from stromal vesicular fraction of 6 week old mice and found that the number of fat stem cells was significant less in mutant mice compared to those in WT mice. This data suggest that de-sumoylated THRA is associated with reduced fat stem cells at a young age, resulting in reduced adipose tissue mass in adult. THRA sumoylation is important for thyroid hormone modulation of fat mass.

Reference: (1) Yan-Yun Liu et al, JBC 2012 (2) Yan-Yun Liu et al., JBC 2015

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<![CDATA[SUN-743 Understanding the Role of Pancreas and Testis Specific lncRNA86 in Estrogen-Dependent Signaling in Breast Cancer]]> https://www.researchpad.co/article/elastic_article_6596 Long noncoding RNAs (lncRNAs) are emerging as key regulators of diverse cellular processes, but their roles in breast cancer biology are just beginning to be elucidated. In this study, integration of powerful techniques, RNA-seq data from subcellular fractionated RNA with GRO-seq data has yielded a comprehensive catalog of estrogen-regulated lncRNAs in MCF-7 cells. Analysis of RNA-seq data from samples representing molecular subtypes of breast cancer and normal tissue types, revealed that many lncRNAs (such as lincRNA86) show distinct expression patterns. LincRNA86 shows highest normal expression in pancreas followed by testis in normal human tissues. The hypothesis is lincRNA86 regulate estrogen-dependent signaling in breast cancer. In functional assays, knockdown of lncRNA86 inhibits the growth of ER-positive breast cancer cells. Amplified expression of lncRNA86 in breast cancer correlates with clinical outcome. LncRNA86 have now been fully annotated (transcription start and stop site, 5’ cap, polyA tail, and exon/intron structure), and cloned. We are now performing detailed molecular analyses to better understand the underlying mechanisms of action of the lncRNA. We are also currently have experiments underway to view cancer phenotypes: estrogen-dependent tumor growth. Collectively, our preliminary results suggest that lincRNA86 plays a critical role in ERα-dependent pathways.

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<![CDATA[SAT-749 Defining The Role Of Androgens In Hernia Associated Skeletal Muscle Fibrosis]]> https://www.researchpad.co/article/elastic_article_6534 Introduction: Inguinal hernia is a highly prevalent condition occurring in 27% of adult men in their lifetime. The recurrence rate of hernia is 5-20%, resulting in a substantial cost burden in surgical repair procedures. Until recently, the mechanisms leading to the lower abdominal muscle (LAM) weakening characteristic of hernia were unknown. Our group developed the first mouse model of inguinal hernia through expression of the human aromatase enzyme in male mice (AromHum). Aromatase converts androgens to estrogens, and is expressed in the skeletal muscle in humans, but not mice. We found that locally formed estrogen from aromatase activity in LAM and decreased circulating testosterone levels are associated with muscle atrophy and fibrosis resulting in hernia. However, it is unclear how decreasing androgen levels might affect muscle fibrosis, and defining this potential mechanism could impact hernia treatment. We hypothesized that low androgen levels promote muscle fibroblast proliferation and fibrosis, and that androgen treatment would prevent hernia progression in AromHum mice.

Methods: Arom Hum mice (3 weeks old) were treated with high-dose dihydrotestosterone (DHT) via injection for 7.5 weeks with hernia volume continuously recorded (n=5/group). Primary fibroblasts were isolated from LAM from WT and AromHum mice (n=5/genotype). Cells were treated for 24 hours with increasing doses (0.001, 0.01, 0.1, 1, 5, 10 and 100 nM) of R1881, a synthetic androgen, and compared to untreated cells by western blot.

Results: Hernia volume was significantly decreased in AromHum mice treated with DHT compared to vehicle-treated mice, and volume remained consistently suppressed after DHT treatment (p < 0.005). In both primary fibroblast lines, R1881 treatment increased AR levels in a dose dependent manner, indicating that the treatment was effective. Preliminary data indicated that low doses of R1881 (0.001 and 0.01 nM) increased PCNA levels in LAM WT and LAM AromHum fibroblasts. Densitometry normalized to GAPDH showed 80% and 60% increases for 0.001 nM and 0.01 nM respectively in LAM WT fibroblasts, and 20% and 30% increases at these doses in LAM AromHum fibroblasts. Higher doses of R1881 decreased PCNA levels in LAM AromHum fibroblasts by 40% (10 nM) and 30% (100 nM), whereas a 25% decrease was detected in LAM WT fibroblasts at 100 nM.

Conclusion: These data suggest that low androgen doses increase LAM fibroblast proliferation, which possibly contributes to hernia formation. Androgen treatment at higher doses can partially block the progression of hernia in vivo. However, it is unclear whether and how androgen deficiency in combination with excess estrogen affects fibroblast proliferation and hernia formation. Additional research is required to determine if androgen supplementation in sufficient doses is a potential therapeutic for inguinal hernia and other muscle weakness diseases.

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<![CDATA[SAT-733 Improving the Diagnosis, Treatment, and Prevention of Endocrine Diseases Through Accurate and Reliable Laboratory Measurements with CDC’s Clinical Standardization Programs]]> https://www.researchpad.co/article/elastic_article_6419 Laboratory measurements are critical for the correct diagnosis and treatment of patients as well as in the investigation of chronic diseases such as hypogonadism, PCOS, and bone-and kidney-related diseases. Inaccurate measurements can lead to misclassification of patients and incorrect treatment. Furthermore, the effective use of research findings in patient care is prevented. The CDC Clinical Standardization Programs (CDC CSP) assess the analytical performance of assays against performance goals defined by clinical and medical organizations. The CDC CSP assist with assay calibration, the certification of analytical performance, and the monitoring of analytical performance during the measurement of patient and/or study samples. CDC CSP have programs in place for the calibration and certification of commercial assays and laboratory developed tests (LDTs) for total testosterone (TT), estradiol (E2), vitamin D (VD), free thyroxine (FT4), total cholesterol (TC), total glycerides (TG), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C). The programs available for monitoring analytical performance during routine testing include TT, VD, TC, TG, HDL-C, apolipoprotein AI and B. CDC CSP also support accuracy-based external quality assurance surveys such as those offered by the College of American Pathologists. Enrollment of assays and LDTs in CDC’s certification programs has resulted in improvements in calibration accuracy; i.e. the absolute mean bias of assays participating in the CDC Vitamin D Standardization Certification Program was well below the allowable bias of 5% each year. Assays standardized in CDC’s certification programs also demonstrated higher accuracy in routine patient testing; i.e. CDC VD certified assays have a lower bias compared to non-certified assays. Similar observations were made with assays certified in the CDC’s program for TT. Monitoring data over the past 10 years from the CDC Lipid Standardization Program indicated that the majority of TC measurements performed in routine testing were consistently within the recommended bias limits of ±3%. CDC CSP continue to improve the analytical performance of assays by addressing measurement bias caused by factors other than incorrect calibration such as interfering compounds. The programs are responding to new clinical and public health needs with the addition of new analytes such as PTH and glucose. The CDC CSP support projects aiming at establishing reference intervals and other research studies. The CDC CSP work with stakeholders, such as the Partnership for the Accurate Testing of Hormones and the Endocrine Society, to educate the clinical and laboratory communities about the importance of using standardized assays in patient care, research, and public health. References: Partnership for Accurate Hormone Testing (PATH). www.hormoneassays.org. College of American Pathologists (CAP). www.cap.org.

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<![CDATA[OR09-06 HNRNPA2B1 Mediates Endocrine-Sensitivity and Alters PSAT1 in Breast Cancer Cells]]> https://www.researchpad.co/article/elastic_article_6162 Higher expression of the RNA binding protein HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), a reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is found in endocrine-resistant, estrogen receptor (ERα)+ LCC9 and LY2 breast cancer cells derived from MCF-7 endocrine-sensitive luminal A cells (1). HNRNPA2B1 interacts with DGCR8 in the DROSHA complex to promote processing of pri-miRNAs to pre-miRNAs. We identified HNRNPA2B1-regulated miRNAs by RNA seq and target pathways, including serine family amino acid metabolic processes, TGFβ signaling, response to estrogen, and cell junction by MetaCore enrichment pathway analysis (1). Stable 4.5-fold overexpression of HNRNPA2B1 in MCF-7 cells (MCF-7-A2B1 cells) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased ~ 1.6-fold. Conversely, transient knockdown of HNRNPA2B1 expression in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant, without changing ESR1 expression. MCF-7-A2B1 cells showed increased migration, reduced E-cadherin and increased vimentin, suggestive of EMT; however, they exhibited reduced clonogenic survival. Follow-up on the identification of HNRNPA2B1-miRNA regulation of the serine pathway revealed higher expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) transcripts and proteins in LCC9, LY2, and MCF-7-A2B1 compared to MCF-7 cells. We identified two miRNAs, miR-424-5p and miR-145-5p downregulated in MCF-7-A2B1 cells that directly targeted the PSAT1 3’UTR in dual luciferase assays. Lower miR-424-5p and miR-145-5p in endocrine-resistant LCC9 and LY2 correlate with increased PSAT1 and higher PSAT1 is associated with reduced overall and metastasis-free survival in breast cancer patients. Overall, our data suggest a role for increased HNRNPA2B1 in tamoxifen-resistance. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Sci. Rep. 2019; 9:9430.

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<![CDATA[SAT-741 Salivary Cortisol and Cortisone Measurement Provide a Novel and Non-Invasive Method of Monitoring Medical Therapy in Cushing’s Syndrome]]> https://www.researchpad.co/article/elastic_article_6122 Introduction:

Salivary glucocorticoids (cortisol [SalF], cortisone [SalE]) are collected non-invasively, unaffected by variation in cortisol binding globulin (CBG) and an established tool in the investigation of Cushing’s syndrome (CS). We have previously shown a strong correlation between salivary glucocorticoids and circulating free serum cortisol, better than that with total serum cortisol. Measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS) permits lower limits of quantification, eliminates cross-reactivity both between cortisol and cortisone, and by metyrapone-induced elevations in 11-deoxycortisol.

Previous studies in CS have demonstrated that a mean (based on 5 samples during a single day) serum cortisol (serumF) of 150-300 nmol/l equates to a normal isotopically calculated cortisol production rate. We report the use of salivary glucocorticoid measurement in metyrapone-treated CS patients undergoing dose titration to achieve a mean serumF of 150-300 nmol/L.

Methods:

Seventeen (11 females; age-range 24-74 years) patients with CS undergoing dose titration with metyrapone were studied on 44 occasions: 15 ACTH-dependent (5 ectopic) and 2 adrenal.

24 healthy male volunteers (HV) were also studied.

Both cohorts had paired serumF and SalE and SalF samples collected at 5 time-points (10:00; 11:30; 13:00; 14:30; 16:00).

Serum and salivary glucocorticoids were measured using LC-MS/MS.

The TR for salivary glucocorticoids was determined from the mean SalE and SalF in HV whose mean serumF (n=20) was in the desired range (150-300 nmol/L). In CS patients the metyrapone dose had been titrated to achieve a mean serumF of 150-300 nmol/L on 14 (out of 44) occasions.

Results:

Mean SalE (r=0.70; p<0.001) and mean SalF (r=0.51; p<0.001) showed a significant correlation with serumF. The TR from HV for mean SalE and mean SalF were 5.8–24.7 nmol/L and 0.6-5.4 nmol/L respectively.

In CS patients, SalE had greater sensitivity (79% vs. 75%) and specificity (80% vs. 57%) in predicting a mean serumF in the TR than SalF.

Conclusion:

We have demonstrated a close correlation between mean SalE and mean serumF in metyrapone treated CS patients. Salivary glucocorticoids within the derived TR were highly predictive for a target serumF. It is unsurprising, due to the effect of CBG on serumF measurements, that the sensitivity and specificity of SalE and SalF are not greater than reported. Consequently, SalE and SalF, as surrogates for free serum F, have the potential to be superior than serumF when assessing adequacy of medical therapy in CS and may permit out-of-hospital monitoring. Further work is required to validate these findings.

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<![CDATA[SUN-747 Steroid Hormone Metabolism Mediated Racial Disparity in Men with Benign Prostatic Hyperplasia]]> https://www.researchpad.co/article/elastic_article_5940 Introduction and Objective: Racial disparity in prostate cancer has been well established, with African American (AA) men having higher rates of diagnoses and death from the disease compared to Caucasian American (CA) men. AA men also have a high incidence of benign prostatic hyperplasia (BPH), a disease associated with lower urinary tract symptoms (LUTS) that affect >210 million men worldwide. Furthermore, AA men with BPH have an increased incidence of non-surgical treatment failure, larger prostates at time of surgery, and surgery occurring at a younger age. The use of selective estrogen receptor modulators (SERMs) in the treatment of BPH has been proposed, as an increase in ERα has been associated with disease progression. AA men have higher levels of circulating estrogens as compared to CA leading to an increased prenatal exposure to estrogens. Estrogen exposure has been shown to alter the epigenetic landscape of genes, and this prenatal exposure to estrogens could sensitize the AA men to altered steroid homeostasis leading to an increase susceptibility to BPH and an altered response to treatment. In this study, we examine the prostate expression and localization changes in estrogen receptors (ERα, ERβ) as well as steroid metabolism genes in AA and CA with or without BPH. Methods: To examine the impact of race on BPH, we examined prostate tissue from 66 men. We utilized 21 normal transition zone controls from radical prostatectomies, 8 normal transition zone controls from organ donors, and 37 BPH samples divided between CA and AA men. Using multispectral quantitative multiplex IHC, we examined the steroid hormone related protein expression of ERα, ERβ, CYP7B1, and AKR1C1 on each FFPE tissue section. We quantified the optical density of each protein of interest as well as examined colocalization and coexpression through cell and tissue segmentation. Results: In CA men, there is a dysregulation of ERα:ERβ homeostasis with BPH relative to normal as an increase in ERα and a decrease in ERβ expression was observed. Furthermore, an increase in CYP7B1, an enzyme that degrades ERβ ligands, was also observed. In AA men, we observed no difference between normal and BPH states, however in both normal and BPH prostate tissues, ERα and ERβ were increased relative to CA men. In addition, there is a decrease in AKR1C1, the enzyme that metabolizes DHT to an ERβ ligand. Conclusions: Our study supports the concept that differences in hormone pathways exist between AA and CA men. Understanding how these racial difference in steroid metabolism enzymes as well as ERs between CA and AA men with BPH could enhance treatment strategies for men with BPH.

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