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Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptor alpha (THRA), plays an essential role in maintaining muscle mass, function, and regeneration. A mouse model of resistance to thyroid hormone carrying a frameshift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury(1,2). We previously demonstrated that the expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA- PV mice. COUP-TFII is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies(3). COUP-TFII physically and functionally interacts with THRA in primary myoblasts isolated from WT and THRA-PV mice, as demonstrated via co-immunoprecipitation and chromatin-immunoprecipitation. We observed that satellite cells from THRA-PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT cells. However, the silencing of COUP-TFII expression using siRNA probes restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 5 days of myogenic induction. Moreover, RNAseq analysis on myoblasts from THRA-PV mice after COUP-TFII knockdown shows that COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during murine post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling during skeletal muscle regeneration, ultimately increasing the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies. References: 1. Milanesi, A., et al., Endocrinology 2016; 2. Kaneshige, M. et al., Proc Natl Acad Sci U S 2001; 3. Lee HJ, et al, Sci Rep. 2017.

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.

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.

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.

Most reports of cortisol half-life in the literature report a range of 90-130 min, which results are based on descriptive model that assumes mono-exponential decay of a single, total cortisol compartment. Free cortisol half-life has been similarly assessed using a descriptive single compartment model (1). However, the descriptive model is not physiologic in view of the rapid exchange between protein-bound and free cortisol compartments and evidence that metabolic elimination is restricted to the free cortisol compartment. In the present study, we sought to explore potential limitations of the descriptive, single-compartment model for cortisol elimination by assessing the influence of CBG concentration ([CBG]) on cortisol half-life estimates obtained using the descriptive model. We studied the influence of [CBG] and other variables on descriptive cortisol half-life using a Monte Carlo simulation of cortisol concentration decay curves developed using data from healthy controls (1). Total cortisol concentration ([TF]) curves were generated on the basis of 4 predictor variables: (i) [CBG], (ii) albumin concentration, (iii) [TF] at time zero following iv bolus (total cortisol at time 0, y-intercept), and (iv) free cortisol half-life central to a mechanistic (dynamic, 3-compartment) model (2). Simulations used a multivariable normal distribution and selected means, SDs, and correlation structure among these 4 variables in healthy controls. After generation of a series of cortisol decay curves (n=1000), half-lives for total and free cortisol were solved using the conventional (descriptive, single-compartment) model. The influence of predictor variables on conventional half-life estimates were assessed using standardized beta (STB) coefficients, which represent change in the SD of the outcome (numerator, i.e. total or free cortisol half-life obtained by descriptive model) for each SD change in a predictor (denominator) in a multivariable context (3). For total cortisol half-life (descriptive model) STBs were 0.91 ([CBG]), 0.73 (free cortisol half-life), -0.37 (y-intercept), and 0.04 ([albumin]) (all P

Invasive Lobular Carcinoma (ILC) is the 2nd most common histotype of breast cancer, but is critically understudied. ~95% of ILC are estrogen receptor (ER) positive, and previous studies demonstrate the importance of estrogen in ILC etiology. However, retrospective studies show that anti-estrogens are substantially less effective in ILC than in ER+ Invasive Ductal Carcinoma (IDC). This strongly suggests that regulation of ER function is unique in ILC, and we hypothesize that this is due to an ILC-specific cohort of ER co-regulators. We performed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) to determine ILC-specific ER-interacting proteins, and identified Mediator of DNA Damage Checkpoint 1 (MDC1) as a novel ER co-regulator in ILC cells. We confirmed ER:MDC1 interaction by co-immunoprecipitation and proximity ligation assays (PLA); interaction was specifically observed in ILC cell lines but not IDC cell lines. Consistent with co-regulator function, we found MDC1 is essential for ER-driven proliferation of ILC cells. MDC1 knockdown dysregulates transcription of ER target genes in ILC cells (e.g. IGFBP4, WNT4). Moreover, RNA-seq analysis showed that in ILC cell line MDA MB 134VI, >50% of ER target genes require MDC1 for their regulation. To understand how MDC1 controls ER transcriptional activity, we performed ChIP-qPCR and found that MDC1 controls ER binding to DNA in ILC cells. Further, MDC1 controls binding of the pioneer factor FOXA1 to DNA, and Dual PLA studies of ER:MDC1 and ER:FOXA1 interaction revealed that MDC1 knockdown decreased ER:FOXA1 interaction. MDC1 canonically functions in DNA damage response, but our preliminary data suggest MDC1 is decoupled from its canonical role in DDR in the context of ER co-regulator activity in ILC cells. Together, these data suggest MDC1, independent of its role in DDR, acts as a novel ER co-regulator in ILC and regulates ER:DNA binding and ER transcriptional function to drive ILC cell proliferation and survival.

Obesity and diabetes are important risk factors for the development of coronary heart disease and stroke. Plasma endocannabinoid (EC) levels are inappropriately elevated in obesity and diabetes, and are hypothesized to play a causal role in central regulation of weight gain. Importantly, it was recently demonstrated that cannabinoid receptor 1 (CNR1) triggers cell stress and induces apoptosis in kidney tubule cells exposed to palmitic acid and high-glucose (HG). HepG2 and human coronary artery endothelial cells (HCAEC) were treated with tunicamycin (TM), thapsigargin (TG), high-glucose (HG), anandamide (AN), and 2-arachondonyl glycerol (2-AG), and endoplasmic reticulum (ER) stress was measured. In cells treated with TM, AM, and 2-AG and the UPR inhibitors 4-phenylbutyrate (4-PB) and taurodeoxycholic acid (TUDCA), both 4-PB and TUDCA prevented AN and 2-AG from promoting ER stress. ER stress in cells treated with AN and 2-AG, but not TM, was inhibited by the CNR1 antagonist rimonabant. Similar results were obtained with HCAEC. Furthermore, treatment with AN and 2-AG induced inositol requiring enzyme 1α and protein kinase R-like endoplasmic reticulum kinase phosphorylation but had no effect on their expression, while activating transcription factor 6 and binding immunoglobulin protein expression were also induced by AN and 2-AG in both HepG2 and HCAEC. Finally, AN and 2-AG treatment induced CNR1 expression in both cell lines. These results strongly suggest that EC’s promote ER stress and potentially induce liver and endothelial cell dysfunction.

Estrogen treatment increases bone mass and reduces fat mass but is associated with adverse effects in postmenopausal women. Knowledge regarding tissue-specific estrogen signaling is important to aid the development of new tissue-specific treatments. We hypothesized that the posttranslational modification phosphorylation in estrogen receptor alpha (ERα) may modulate ERα transcriptional activity in a tissue-dependent manner. Phosphorylation of site S122 in ERα has been shown in vitro to affect ERα activity, but the tissue-specific role in vivo is unknown. We herein developed and phenotyped a novel mouse model with a point mutation at the phosphorylation site 122 in ERα (S122A). Female S122A mice had increased fat mass and serum insulin levels but unchanged serum sex steroid levels, uterus weight, bone mass, thymus weight, and lymphocyte maturation compared to WT mice. In conclusion, phosphorylation of ERα S122 has a tissue-dependent role with an impact specifically on fat mass in female mice. This study is the first to demonstrate in vivo that phosphorylation of a transactivation domain in a nuclear steroid receptor modulates its activity in a tissue-dependent manner.

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.

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.

Lipid and cholesterol metabolism reprogramming is strongly linked to tumorigenesis and therapeutic resistance. Our recent genetic disruptions via CRISPR KO and gene silencing and pharmacological inhibition clearly demonstrated that nuclear receptor RORγ plays a crucial role in control of lipid and cholesterol biosynthesis gene programs specifically in certain types and subtypes of cancer cells and tumors. Indeed, its antagonists display potent tumor inhibitions in patient-derived xenografts (PDX) and in immune-intact tumor models. Interestingly, RORγ inhibition resulted in decreased cholesterol synthesis rate specifically in tumors without significant impact to the host animal cholesterol homeostasis. Our ChIP-seq demonstrated that in a subtype of breast cancer RORγ cistrome is largely overlapping with that of SREBP2, a well-established master regulator of lipid and cholesterol biosynthesis. Our further analyses found that RORγ functions dominantly over that of SREBP2 via its association with SREBP2 and facilitation of its genome-wide recruitment and histone H3K27 acetylation. Inhibition of RORγ completely negates the negative feedback activation of the cholesterol program induced by cholesterol-lowering drug statins and mediated by SREBP2. Treatment of animals with the antagonists in combination with statins showed a highly synergistic anti-tumor effects. Together, our study uncovers RORγ as a novel master regulator of lipid and cholesterol metabolism operating selectively in subtypes of cancer.

BACKGROUND: Inguinal hernias are a widespread public health issue and typically diagnosed in one-fourth of all men. Despite hernia repair being the most commonly performed surgery in the US, the mechanisms causing this disease are currently unknown. We previously developed a mouse model that expresses the human aromatase gene (Aromhum) wherein all male mice develop inguinal hernias. We further showed that high production of estradiol by aromatase in the lower abdominal muscle (LAM) via binding to estrogen receptor caused increased fibroblast proliferation and muscle atrophy which leads to inguinal hernia formation (1). Hypothesis: Disruption of estrogen signaling via ablation of estrogen production using an aromatase inhibitor or inhibition of estrogen receptor by an estradiol antagonist can prevent or reverse the formation of inguinal hernias. Results: We previously demonstrated that aromatase inhibitor, letrozole, completely prevented the formation of inguinal hernias in Aromhum mice (1). Here we show that ER-dependent estradiol antagonist fulvestrant can also prevent LAM tissue fibrosis, muscle atrophy and hernia formation in Aromhum mice (n=4, p=0.0007). WT littermates did not show hernia formation with or without fulvestrant treatment (n=4). Furthermore, we demonstrate that aromatase inhibitor letrozole can reverse mild to moderate size of hernia (150-160 mm2), while placebo-treated mice had progressively enlarged hernias (n=7, p=0.04). We subsequently show a reduction in muscle fibrosis and a restoration of myocyte size in Aromhum mice with letrozole treatment. Conclusion: Estrogen produced as a result of aromatase expression in estrogen-sensitive LAM tissue stimulates the proliferation of estrogen receptor-expressing fibroblasts, fibrosis, muscle atrophy, and hernia formation. Ablation of estrogen production or its signaling not only completely prevents this phenotype but also reverses mild to moderate-sized hernias. Our findings pave the pathway for developing the first potential preventive and therapeutic pharmacological approach for combating recurrent inguinal hernias in elderly men through modulation of estrogen signaling in abdominal muscle tissue. Reference: (1) Zhao H, et al.,PNAS. 2018 Oct 30;115(44):E10427-36.

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent. Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop. In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant. Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.

3β-hydroxysteroid dehydrogenase-1 (3βHSD1), catalyzing conversion of dehydroepiandrosterone (DHEA) to Δ 4-androstenedione, is an essential enzyme in the pathway toward production of biologically active androgens such as dihydrotestosterone from the adrenally produced precursor DHEA sulfate, the most predominant steroid hormone in circulation. We previously identified, in the gene (HSD3B1) that encodes 3βHSD1, a germline gain-of-function missense-encoding variant that has now been validated in several studies as predicting more rapid progression in prostate cancer patients treated with gonadal testosterone deprivation. Production of androgens from adrenal precursors is important not just in the context of prostate cancer but in other physiologic and pathophysiologic processes, which could include asthma. Androgens are associated with better lung function in both asthma and healthy cohorts, and increasing circulating androgen levels in males help explain the switchover in asthma being more common in boys than girls but then more common in women than men. A main treatment for asthma, as well as other inflammatory processes, is administration of glucocorticoids, yet unresponsiveness to glucocorticoids in a subset of patients remains a major problem. Systemic glucocorticoid administration suppresses adrenally produced DHEA and DHEA-S, suggesting a depleted pool for biologically active androgen production as a mechanism for glucocorticoid resistance. Our surprising preliminary data support a link between glucocorticoid responsiveness and the more active HSD3B1 allele: patients homozygous for the adrenal-permissive HSD3B1(1245C) allele exhibit better response to oral glucocorticoids than those homozygous for the adrenal-restrictive HSD3B1(1245A), with heterozygous patients falling in the middle. This suggests a model in which patients with the more active (adrenal-permissive) form of 3βHSD1 produce sufficient androgens despite the depleted pool of precursor hormones whereas patients with the less active (adrenal-restrictive) form cannot. To further elucidate the link between 3βHSD1 activity and immune response, we assayed HSD3B1 expression in different types of white blood cells. Leukocyte subsets from asthma patients and healthy controls were purified using fluorescence-activated cell sorting, and HSD3B1 expression was analyzed using qPCR. White blood cells of several types expressed HSD3B1 at levels comparable to or greater than both prostate cancer and placental choriocarcinoma cell lines with very robust 3βHSD1 activity. Further determining the cell type specific expression and activity of this key enzyme is an important step in unraveling the link between the HSD3B1 polymorphism and asthma along with potentially many other immune processes.

Glucocorticoids used in pharmacological doses for the treatment of a variety of medical conditions, and endogenous glucocorticoid excess - Cushing’s syndrome, may result in several adverse effects, but currently there is no clinically useful biomarker of glucocorticoid activity. We have applied a proteomic approach to the discovery of glucocorticoid-responsive proteins potentially measurable in human serum samples. To minimise the masking by abundant serum proteins, we conducted discovery proteomics on the secretome of ex vivo-stimulated peripheral blood mononuclear cells (PBMC) isolated from 12 volunteers. The PBMC were divided into 4 treatment groups; +/- dexamethasone 100 ng/mL (dex) for 4h, or +/- dex for 24h. In all treatment groups, media was changed to serum free for 3h before collection. Media samples were processed for proteomics, with 561 and 273 proteins analysed by label-free quantification (LFQ) for the 4h and 24h secretome, respectively. Paired statistical analysis at the 2 time points generated a shortlist of 43 candidate biomarker proteins, which was verified using a multiple reaction monitoring (MRM) assay, confirming the differential secretion of 12 proteins at both 4h and 24 h. Five proteins were selected for validation using enzyme linked immunosorbent assay (ELISA) in an independent cohort: β2 microglobulin (B2M), lysozyme C (LYZ), high-mobility group protein 2 (HMG2), nucleophosmin (NPM1) and nucleolin (NCL). Twenty new volunteers (10M and 10F) had venous blood drawn at baseline and 12h after 4 mg oral dex. Four proteins were detectable by ELISA, three of which showed statistically significant change in concentration. Serum LYZ and NPM1 significantly decreased following dex: LYZ - 101 ± 5.5 vs 67 ± 4.4 ng/mL, (P

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.

Background: Sex hormone-binding globulin (SHBG) is a glycoprotein that regulates the bioavailability of sex hormones, may directly affect glucose metabolism, and increases with age in the general population. SHBG concentrations are higher in people with HIV, a population in whom accelerated aging has been hypothesized. It is unclear whether age-related trajectories of SHBG differ by HIV serostatus. Methods: SHBG was measured in 182 men with HIV (MWH) and 267 men without HIV (MW/oH) from the Multicenter AIDS Cohort Study (MACS) in four U.S. cities, who had ≥2 samples over a 10 year period. Outcome measure: SHBG, log2SHBG. Multivariable linear mixed-effects regression models were used to evaluate whether SHBG (log2-transformed) and its rate of change differed by HIV serostatus after adjustment for covariates: age, race, BMI, smoking, education, hepatitis C virus infection, total testosterone concentrations, time of day of blood drawn and comorbidities (history of diabetes, kidney disease, liver disease, cancer, depression, hypertension) Results: At baseline, mean age among MWH was similar to MW/oH (51±5 vs 49±6 years). However, SHBG values were higher in MWH compared to MWo/H (65.6±48.8 nmol/L vs. 45.4±22 nmol/L, p

Adrenal corticosteroids, such as glucocorticoids and mineralocorticoids, are indispensable for mediating response to stress, development, limiting inflammation, and maintaining energy and fluid homeostasis. These hormones exert their actions via binding to two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The GR has low affinity for corticosteroids, but is expressed in nearly every cell. In contrast, the MR shows a higher affinity for corticosteroids and its expression is largely confined to those tissues where electrolyte exchange and fluid balance are required. GR and MR act as ligand-activated transcription factors which, following interaction with co-regulators and DNA responsive elements, either promote or repress gene transcription. The affinity for the same ligands, structural homology, and binding to the same DNA regions suggest GR and MR can compensate for each other’s actions. Yet, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate this interplay, we developed U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (GRMR). Immunofluorescence analysis showed that treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear traslocation of GR and MR. Conversely, treatment with 1 nM of aldosterone (Aldo) promoted nuclear translocation of the MR only. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR dimers were detected in the nucleus of GRMR cells. Surprisingly, nuclear GR-MR dimers were also detected in the presence of Aldo, suggesting that it is necessary to activate at least one receptor to induce nuclear traslocation of the heterocomplex. To decipher the functional contribution of GR-MR dimers in the transcriptional response of GR to Dex and MR to Aldo, we performed RNA-seq in GR, MR, and GRMR cells treated with 1 nM of Dex or Aldo. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a blunted Dex-mediated gene response which affected only 1608 genes, suggesting a functional antagonism of MR. Aldo-activated MR regulated the transcription of 1660 genes. However, co-expression of GR expanded the Aldo-mediated gene response to 3150 genes. Strikingly, 74% of these genes were also regulated by Dex via GR, suggesting that GR-MR dimers in the presence of aldosterone are able to mimic the glucocorticod transcriptional response. Our data suggest that the role of distinct GR and MR homo- and hetero-dimers is relevant for regulating gene expression. Dissecting the mechanism and investigating the cross-talk between GR and MR may be useful to understanding these two receptors in heath and disease.

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.

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.

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.

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.

Background: Crystallographic structures of nuclear receptor ligand binding domains provide a static model of a receptor stably wrapped around an internalized ligand. Understanding the dynamics of a receptor at different stages of ligand binding has been hampered by the paucity of crystal structures for unliganded nuclear receptors. Molecular dynamic models have been constructed for some nuclear receptors to fill that void. Methods: The molecular simulation docking program MORDOR (MOlecular Recognition with a Driven dynamics OptimizeR)(1) was used to study the structural dynamics of the androgen receptor ligand binding domain (AR LBD) modeled from the static structure of the AR LBD bound to testosterone (T) (PDB ID: 2AM9). The goals of the study were to understand a) the dynamic interaction of the T in its binding pocket, b) AR LBD structural flexibilities that permit T entry/exit from the binding pocket and c) a model of the unliganded AR LBD. Results: Modeling AR LBD structure flexibility over time revealed possible alternative dynamic structures, including those without ligand, overlaid against the canonical nuclear receptor structure. The model dynamically tracks the structural changes as a ligand enters into the ligand binding domain and nestles into the ligand binding pocket. The model predicted the appearance of alpha helices within the AR LBD that transiently fold/unfold during the ligand entry phases. Once in the pocket, the ligand itself remains very dynamic in a still flexible pocket. The model predicted also AR LBD amino acids that sequentially interact with the ligand during its dynamic entry into the AR LBD. Intriguingly, those AR amino acids include those mutated in castration-resistant prostate tumors that continue to grow during androgen suppression therapy. Functional studies showed those mutant ARs had a primary consequence of enhancing response to lower level T, and other androgens, consistent with their role in creating a higher affinity AR that can scavenge low-level androgens in an androgen-suppressed patient. Conclusions: The molecular model of T binding to the AR LBD suggests a degree of structural dynamism not evident in the crystallographic structures commonly associated with nuclear receptors. Some AR mutations activating prostate tumor growth may do so by impacting androgen entry/exit, rather than by altering androgen fit into the ligand binding pocket. Reference: (1) Guilbert C, James TL (2008) J Chem Inf Model. 2008 48(6): 1257-1268. doi: 10.1021/ci8000327

Background: Unexplained or idiopathic infertility comprises approximately 30% of couples who present with infertility. This has led to investigations seeking to determine the cause(s) of this important diagnosis of exclusion. Estrogen’s role in reproduction has been well- established. Estrogens bind to two hormone receptors (namely estrogen receptor-alpha and estrogen receptor-beta), which are distributed differentially throughout the body. Specifically, the estrogen receptor-beta, coded by the Estrogen Receptor 2 (ESR2) gene, is highly expressed in granulosa cells and growing follicles. The one female patient reported with an ESR2 mutation presented with hypergonadotropic hypogonadism. However, subfertility with inefficient ovulation and resistance to exogenous ovulatory stimulation is seen in an ESR2 knockout mouse model. We therefore hypothesized that less severe ESR2 variants could lead to a normal female phenotype and pubertal development but could be a cause subfertility. Methods: DNA samples from 200 women with unexplained infertility were obtained from the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) clinical trial, which investigated optimal ovulation induction medications for unexplained infertility. These samples were subjected to targeted next-generation sequencing (NGS) for the ESR2 gene. Likely pathogenic variants that occurred with a minor allele frequency of < 0.01 in the gnomAD database and a Combined Annotation Dependent Depletion (CADD) score of > 20 were selected for confirmation by Sanger sequencing. Results: From the 200 patient samples, five heterozygous missense variants and one heterozygous in-frame deletion identified by targeted NGS were confirmed by Sanger sequencing. Further studies will need to be performed in vitro to confirm the likely pathogenicity of these variants. Conclusion: These studies raise the possibility that If these variants in ESR2 that impair estrogen signaling, they could be a potential newly recognized etiology of unexplained infertility in women with unexplained infertility. Conclusion: These studies raise the possibility that variants in ESR2 that impair estrogen signaling could be a potential newly recognized etiology of unexplained infertility in women.

The metabolism of 11β-hydroxyandrostenedione (11OHA4), a major adrenal C19 steroid, was first characterised in our in vitro prostate models showing that 11OHA4, catalysed by 11βHSDs, 17βHSDs and 5α-reductases, yields potent androgens, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) in the 11OHA4-pathway [1]. Findings have since led to the analysis of C11-oxy steroids in PCOS, CAH and 21OHD. However, the only circulating C11-oxy steroids included to date have been 11OHA4, 11keto-androstenedione (11KA4), 11β-hydroxytestosterone (11OHT) and 11KT, with 11KT reported as the only potent androgen produced from 11OHA4. We have identified higher levels of 11KDHT compared to 11KT in prostate cancer tissue and benign prostatic hyperplasia tissue and serum, with data suggesting impeded glucuronidation of the C11-oxy androgens [2,3]. The assessment of 11KDHT and the inactivation/conjugation of the C11-oxy steroids in clinical conditions is therefore crucial. We investigated the metabolism of testosterone, 11KT, 11OHT, dihydrotestosterone, 11KDHT and 11OHDHT in JEG-3 placenta choriocarcinoma, MCF-7 BUS and T-47D breast cancer cells, focusing on glucuronidation and sulfation. Steroids were assayed at 1 µM and metabolites were quantified using UPC2-MS/MS. Conjugated steroids were not detected in JEG-3 cells with DHT (0.6 µM remaining) metabolised to 5α-androstane-3α,17β-diol and androsterone (AST), and 11KDHT (0.9 µM remaining) to 11OHAST and 11KAST. 11OHA4 was converted to 11KA4 (12%) and 11KT (2.5%); and 11KT to 11KDHT (14%). In MCF-7 BUS cells, DHT was significantly glucuronidated, whereas 11KDHT was not. 11KAST was the only steroid in the MCF-7 BUS and T-47D cells that was significantly sulfated (p As there exists an intricate interplay between steroid production and inactivation, impacting pre- and post-receptor activation, efficient conjugation would limit adverse downstream effects. Our data demonstrates the production and impeded conjugation of active C11-oxy C19 steroids, allowing the prolonged presence of androgenic steroids in the cellular microenvironment. Identified for the first time is the 11OHA4-pathway in placenta and breast cancer cells, and the sulfation of 11KAST. Characterising steroidogenic pathways in in vitro models paves the direction for in vivo studies associated with characterising clinical disorders and disease, which the C11-oxy C19 steroids and their intermediates, including inactivated and conjugated end-products, have highlighted. [1] Bloem, et al. JSBMB 2015, 153; [2] Du Toit & Swart. MCE 2018, 461; [3] Du Toit & Swart, JSBMB 2020, 105497.

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.

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.

Introduction: An oral testosterone (T) replacement therapy (TRT) would be the preferred choice for many hypogonadal men. Until recently, the only oral TRT approved in the US was methyl-T which has been associated with hepatotoxicity. The safety of a novel oral T undecanoate (TU) formulation was evaluated in hypogonadal men for up to 2 years. Subjects and Methods: Two open-label, multicenter, dose-titration trials were conducted in hypogonadal men (serum T ≤ 300 ng/dL) age 18-75 years. Trial I was a randomized, active-controlled, 2-arm, 12-month study. Trial 2 was a long-term extension of those who completed Trial 1. Statistical analyses were only conducted with the subjects who completed Trial 1 and continued treatment in Trial 2, thus providing up to 2 full years of data. Safety was assessed by physical exam, AE reporting, and routine clinical laboratory measurements. Results: Overall, up to 81 subjects were available for evaluation. T concentration increased from 208.3 ± 102.4 ng/dL (Mean ± SD) at baseline (BL) to 470.1 ± 396.5 ng/dL after 24 Mo of therapy with oral TU, and 84% of men achieved T in eugonadal range (300-1000 ng/dL) after 90 days of therapy. Mean T concentrations remained in the eugonadal range throughout Trial 2. There were no clinically significant changes in liver function tests - ALT (28.0 ± 12.3 to 26.6 ± 12.8 U/L), AST (21.8 ± 6.8 to 22.0 ± 8.2 U/L), and bilirubin (0.58 ± 0.22 to 0.52 ± 0.19 mg/dL) throughout the two studies. At Day 270, one subject had an ALT level of 227 U/L, which was > 4x the ULN (ULN for ALT = 45 U/L). Despite continued use of oral TU, ALT was measured again on Day 290, and the level dropped to 87 U/L, < 2x ULN. This was the only instance of an LFT elevation. There was a modest initial increase in prostate-related growth endpoints (i.e. PSA and prostate volume) that stabilized over time. There were not any significant changes in IPSS total score (-0.06 ± 3.9 vs BL). There were significant, yet modest, increases in mean HCT (+2.52 ± 3.7% vs BL, p < 0.001) and cuff systolic BP (+5.6 ± 15.0 mmHg vs BL, p = 0.006). The change in prostate-related growth variables and CV endpoints changed initially and stabilized throughout the 2 trials. For example, systolic BP consistently showed a mean increase from BL between 3 - 6 mmHg. Conclusion: This oral TU formulation is an effective long-term therapy for hypogonadal men and has a safety profile consistent with other approved T products. Notably, no evidence of liver toxicity was observed. The long-term efficacy and safety profile of oral TU may provide a treatment option that avoids issues associated with other TRTs, such as injection site pain or transference to partners and children.

Premature birth leads to a significant increase in adverse clinical outcomes, including Respiratory Distress Syndrome, Bronchopulmonary Dysplasia, Necrotizing Enterocolitis and Intraventricular Hemorrhage. Synthetic Glucocorticoids (sGC) are administered prenatally to pregnant mothers at risk to reduce the chance of these complications. However, there is a correlation between long-term neurological defects in the infant and the clinical use of sGC prenatally. The use of the sGCs have been linked to the development of cerebral palsy and deficits in attention and concentration. To investigate the cellular basis of these abnormalities, we examined the consequences of sGC administration of the developing murine brain. Our studies demonstrated that premature exposure to sGC alters neural stem cell biology and has long term consequences for adult behavior in mice. In humans, site-specific phosphorylation of the Glucocorticoid Receptor (GR) on Serine 211 versus Serine 226 is associated with activated or repressed transcriptional states and clinical studies indicate that the ratio of S220/S226 phosphorylation is associated with increased predisposition to specific psychiatric disease states, including Major Depressive Disorder and Bipolar Disorder. To examine the role of these phosphorylation sites in the development of behavioral abnormalities, we utilized a knock-in mouse model where Serine 220 (equivalent to human Serine 211) was replaced with an alanine (S220A). In-vitro microarray analysis of neural stem cells and QPCR validation were performed to examine the expression changes in individual transcripts in critical pathways that may correlate with long-term neurologic disorders. Our results indicated that changing the phosphorylation status of GR alters the expression of 2570 genes. Ingenuity Pathway Analysis indicated that the major pathways altered include those involved in cellular proliferation, mitochondrial function, Valine degradation and G-coupled protein receptors involved in neurotransmission. Both in-vitro and in-vivo experiments indicated that the S220A mutation alters the cells response to sGC administration by impacting proliferation and differentiation. The long-term goal of these experiments was to demonstrate a role for S220 phosphorylation in the development of neuropsychiatric disorders.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant metastatic disease remain unresolved. HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), an RNA binding protein that functions as reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is upregualted in tamoxifen- and fulvestrant-resistant, estrogen receptor (ERα)+ LCC9 and LY2 cells derived from MCF-7 endocrine-sensitive luminal A breast cancer cells (1). The miRNA-seq transcriptome of MCF-7 cells transiently overexpressing HNRNPA2B1 (A2B1) identified gene ontology (GO) pathways including “cellular response to steroid hormone signaling and estradiol” and “positive regulation of protein ser/thr kinase activity”. Modest (~ 4.5-fold) stable HNRNPA2B1 overexpression in MCF-7 cells (MCF-7-A2B1) 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. Conversely, transient knockdown of HNRNPA2B1 in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant while reducing ERα. MCF-7-A2B1 cells showed increased migration, invasion, clonogenicity, soft agar colony size, and markers of epithelial-to-mesenchymal transition. Like LCC9 cells, MCF-7-A2B1 cells showed activation of AKT and MAPK and high androgen receptor (AR). Treatment of MCF-7-A2B1 cells with either PI3K inhibitor Wortmannin or MEK inhibitor PD98059 inhibited soft agar colony formation and reduced colony size. Knockdown of HNRNPA2B1 in MCF-7-A2B1 reduces clonogenicity, but had no effect on the clonogenicity of either LCC9 or LY2 cells. These data suggest a role for HNRNPA2B1 in promoting the initiation of acquired endocrine-resistance by activating ser/thr kinase growth factor signaling pathways. Selective inhibition of HNRNPA2B1 may be a target to prevent acquistion of endocrine therapy resistance, but not treat established metastatic disease. 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. Scientific reports 2019; 9:9430

Renal sodium reabsorption is important for blood pressure homeostasis and is physiologically regulated by aldosterone; glucocorticoids may also contribute. Abnormal steroid hormone activity within the kidney contributes to hypertension but the mechanisms are not fully defined. Molecular profiling of receptors and metabolising enzymes indicates that steroid hormone action is compartmentalised within the kidney. Ambient steroid concentrations are a critical factor governing bioactivity at a cellular level, but this is largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) was applied recently to localise steroids in brain and testes, and here is applied to kidney. Image reconstruction permits characterisation and co-registration of kidney histological regions based on regional markers detectable by MSI. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of murine kidneys, using an optimised MSI method. This approach has the potential to map steroids within functional zones of the kidney, providing fundamental new information relevant to hormone action in health and in disease. Cryosections of male C57BL6 mouse kidneys (age 12 weeks, n=6) were subject to MSI following derivatisation using Girard T reagent and α-cyano-4-hydroxycinnamic acid matrix application. Images were reconstructed, and methods optimised to enhance signal and limit diffusion of analytes of interest. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry. Ions with m/z 458.3010, 460.3166 and 474.2957 were detected, using MALDI, in renal sections, close to the predicted masses of 458.3013 (Δppm=0.65), 460.3169 (Δppm=0.65), and 474.2962 (Δppm=1.05), for derivatives of 11-dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. The Heat maps generated indicated that corticosterone intensity was higher in the inner cortex area close to the corticomedullary junction than the rest of the kidney. In contrast 11-dehydrocorticosterone was detected mainly in medulla and aldosterone signal was equally strong in medulla and outer cortex. Thus, MSI can be used map the sites where glucocorticoid and mineralocorticoids are most active in regulating renal tubular function. Co-localisation of steroids of interest with zonal markers by MSI permits steroid mapping in functional renal zones of the kidney. This approach provides fundamental new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure. The project was supported and funded by Kidney Research UK.

This presentation has two parts. The 1st section describes processes during pregnancy with unknown, underlying biochemistry. The 2nd section shows the role of spiral steroids (SS) in these processes. 1] Fetal nutrition is provided through the placenta. Plasma electrolytes are 145 mM Na+ and 3-5 mM K+. Fetal K+ requirements reach a maximum during the 3rd trimester and must be pumped into cells against the plasma- intracellular gradient. 2] During the 3rd trimester, aldosterone is present in fetal plasma but the signal for endothelial sodium channel (ENaC) synthesis is blocked, leading to fetal Na+ wasting. The mechanism is unknown. 3] After parturition, infants are fed by nursing. Human milk contains 100 mM K+ and 10 mM Na+. Newborn infants are Na+ wasting, despite normal levels of aldosterone. Na+ wasting ends during the 2nd week post-partum without change in serum aldosterone levels. Infant physiology changes from K+ saving to Na+ saving by an unknown mechanism.4] Pre-eclampsia syndrome (proteinuria and hypertension during the 2nd half of pregnancy) affects 3-5 % of pregnant women. These symptoms usually resolve after parturition. 5] Women who have had pre-eclampsia have long-term, excess risk of cardiac and renal disorders. In 2018, we discovered Ionotropin, a SS. SS are phosphocholine esters of steroids with a lactone E-ring, similar to that of spironolactone. SS compounds function as aldosterone antagonists and regulate the NaK-ATPase. SS are involved in each of the 5 steps.1] SS are present in high levels in cord serum and were probably present in fetal plasma throughout gestation. SS stimulate the NaK-ATPase to pump K+ into cells against the gradient, just as does spironolactone.2] SS interfere with aldosterone signaling, just as does amiloride. This leads to increased fetal Na+ wasting, which becomes amniotic fluid.3] SS disappear from the infant circulation during the 1st week after parturition and decrease to adult levels during the 2nd week post-partum. Simultaneously, Na+ wasting ends and growth resumes.4] Women with pre-eclampsia have excess precursors for SS. These would be converted to SS in the fetal-placental unit and, during the 3rd trimester, diffuse into the maternal circulation and could cause pre-eclampsia.5] Ouabain, a plant toxin with a lactone E ring, causes renal and cardiac disorders in rat models. In women with pre-eclampsia, persistent excess SS may cause long-term damage. During gestation, the fetus requires K+ for growth. Our theory is, if (when) a fetus has inadequate K+ (hypokalemia), [a] the mother is signaled to produce SS precursors, [b] the feto-placental unit converts the precursors to SS, [c] fetal SS increase K+ transfer into tissues, and [d] excess SS transfuse back into the maternal circulation and damage maternal organs. We propose that pre-eclampsia is a side effect of fetal efforts to increase supply of K+. There are many possible origins of fetal hypokalemia.

The cornea is the dome-shaped transparent outermost layer of the eye, forming a physical barrier to protect the internal structures of the eye. Glucocorticoids are a mainstay in the treatment of ophthalmic diseases for their anti-inflammatory and anti-angiogenic properties. However, high doses or chronic use of glucocorticoid therapy can lead to vision-impairing effects such as increase in intraocular pressure and the formation of cataracts. The exact signaling pathways responsible for these undesirable effects of glucocorticoid use is poorly understood. One of the major molecular actions of glucocorticoids is to regulate transcription through its cognate nuclear receptor, the glucocorticoid receptor. We have previously reported the effect of glucocorticoids on global gene expression and their role in wound healing and barrier function in immortalized human corneal epithelial cells (HCE-T). In the current study, we knocked down glucocorticoid receptor using siRNA (GRKD) to determine the function of the glucocorticoid receptor in HCE-T cells. Successful knockdown of glucocorticoid receptor was confirmed by RT-PCR and immunoblot experiments. Genome-wide microarray analysis was performed and an FDR adjusted p value less than 0.01 was considered the cut off to create the list of differentially expressed genes (DEGs). Comparison of GRKD cells to HCE-T cells expressing endogenous glucocorticoid receptor (referred as NTC for No Target Control siRNA) revealed that expression of 2150 genes was altered in HCE-T cells when glucocorticoid receptor was knocked down, indicating that glucocorticoid receptor in corneal epithelial cells regulates a large cohort of genes. Inhibition of matrix metalloproteases, granulocyte adhesion and diapedesis, cyclins and cell cycle regulation were the top canonical pathways predicted by Ingenuity Pathway Analysis (IPA) to be altered in GRKD cells. In a 6-hour treatment with dexamethasone (Dex), a synthetic glucocorticoid, or with vehicle as a control, NTC cells had 2039 Dex-regulated genes, while Dex was still able to regulate 1087 genes in GRKD cells. Of these 1087 genes, 895 genes were uniquely regulated by Dex in GRKD cells suggesting that glucocorticoids might be signaling through another receptor in corneal epithelial cells. The top canonical pathways predicted to be altered by Dex in GRKD cells included PI3K/ATK Signaling, ERK5 Signaling, Prostrate Cancer Signaling, Aldosterone Signaling in Epithelial Cells, and PPAR signaling. These findings suggest that Dex could regulate large cohorts of genes through other nuclear receptors in corneal epithelial cells. Given the wide use of ophthalmic Dex in forms including eyedrops, ointments, gels, and implants, it is of clinical significance to understand the molecular actions of synthetic glucocorticoids since they appear to be ligands for multiple nuclear receptors in ocular cells and tissues.

Introduction: Chronic glucocorticoid administration is necessary in a variety of conditions including but not limited to autoimmune, inflammatory and cancer-related diseases in order to relieve symptoms and sustain disease progression. However, there are adverse effects that include increase in glucose levels and others whose severity depends on the dose and duration of glucocorticoid exposure. It has been described that dexamethasone induces oxidative stress in cells by increasing reactive oxygen species (ROS) and this is one of the causes of insulin resistance at the cellular level. Nrf2 is a transcription factor which co-ordinates the antioxidant response and its activation has been shown to ameliorate insulin resistance in murine models. Hypothesis: We hypothesized that deletion of Nrf2 will lead to a more glucose intolerant insulin resistant phenotype in mice chronically treated with dexamethasone as cells would be exposed to higher ROS levels. Methods: To this end, 3-months old wild-type (WT) and Nrf2 knockout (KO) C57BL6J mice were treated intraperitoneally with 2 mg/kg dexamethasone or saline 3 times per week for 3 months. 5-10 mice were included per genotype per treatment and both male and female mice were used. Weekly measurements of body weights were performed and intraperitoneal glucose tolerance tests were done on the second and third month of treatment. Mice were sacrificed 24 hours after the last dose of dexamethasone and blood, white adipose tissue, soleus muscle and liver were collected for RNA preparation and quantitative RT-PCR analysis. Quantitative analysis of trabecular bone parameters was performed by micro-CT. Results: Both male and female mice treated with dexamethasone gained less weight over time and surprisingly were more glucose tolerant than the control group. Absence of Nrf2 did not seem to considerably affect the body weight but KO mice tended to have lower body weights after dexamethasone treatment in both genders with the effect on male mice being statistically significant (25% lower, p

Glucocorticoids are prescribed in many chemotherapy protocols for neoplasms of lymphoid origin based on their ability to inhibit lymphocyte proliferation and promote cell death (apoptosis). Lymphocytes have been shown to be profoundly sensitive to glucocorticoid-induced apoptosis, however glucocorticoid resistance restricts the efficacy of these stress hormones and reduces their clinical value. Bcl-2 is an anti-apoptotic protein that promotes cell survival and resistance to cell death during glucocorticoid therapy. Predictably, Bcl-2, as well as other anti-apoptotic Bcl-2 family members, have been found to be overexpressed in a variety of human cancers, including in over 90% of human follicular lymphomas and 95% of chronic lymphocytic leukemias. Unfortunately, approaches to overcome Bcl-2 resistance in cancer cells including anti-sense oligonucleotides, drugs that inhibit Bcl-2 function, and BH3 mimics have not been effective. We took a unique approach to overcome Bcl-2 resistance in tumor cells expressing this anti-apoptotic protein by first identifying points along glucocorticoid-induced cell death signaling program that are explicitly inhibited by Bcl-2. We then intervened at this stage that bypasses the point of inhibition. Provocatively, drugs such as CCCP and several microbial toxins which disrupt the mitochondrial membrane potential, when used in combination with glucocorticoids overcome the resistance afforded by Bcl-2 and kills the cells. Our data suggests drugs which alter the mitochondrial membrane potential in combination with glucocorticoids provide an effective approach in killing lymphocytic tumor cells expressing Bcl-2. This combination should be considered in accompanying current chemotherapeutic drug combinations to provide additional therapeutic benefit in overcoming Bcl-2 resistant tumors.

Nuclear receptor genes often contain alternative promoters but the physiological relevance of the different promoters or the proteins they generate is largely unknown. Hepatocyte Nuclear Factor 4α (HNF4α), master regulator of hepatocyte differentiation and the most abundant nuclear receptor in the liver, is regulated by two promoters (P1 and P2). P1-HNF4α, a tumor suppressor, is the major isoform in the adult liver while P2-HNF4α is expressed in fetal liver and liver cancer: a role for P2-HNF4α in normal adult liver has not been identified. Here we show for the first time that P2-HNF4α is expressed at ZT9 and ZT21 in the normal adult liver and use P2-HNF4α-expressing exon swap mice and ‘omics approaches to demonstrate that P2-HNF4α orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions. Cytochrome P450 and sex-specific gene expression is impacted as well as carbohydrate and fatty acid metabolism. The exon swap mice exhibit subtle differences in circadian gene regulation and disruption of the clock increases expression of P2-HNF4α. They also exhibit notable differences in carbohydrate versus fatty acid metabolism with P1-HNF4α driving gluconeogenesis and P2-HNF4α driving ketogenesis. Our results also show that P2-HNF4α is required for the elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure in the Hnfa gene is designed, at least in part, to respond to the availability of food resources and maintain the balance between gluconeogenesis and ketogenesis in a circadian and sex-specific fashion. (Funding: NIH NIDDK, West Cost Metabolomics Center, NIEHS T32, USDA NIFA, CCFA, Superfund Research Program)

Postpartum depression (PPD) affects up to 20% of women and exerts adverse consequences on mother and child. Gestational stress and abnormalities in neuroendocrine function have been implicated in the development of PPD. Here, we measured effects of chronic psychosocial stress during pregnancy on maternal behavior as well as hypothalamic-pituitary-adrenal (HPA) axis function and regulation in the early postpartum period. From gestational day 6.5 to 17.5, pregnant C57Bl/6 female mice were exposed to a novel chronic stress paradigm consisting of variable psychosocial stressors such as foreign object exposure, rat odor exposure, bedding removal and were assessed from postpartum day 2 to 7 for behavioral alterations in maternal care, depression, and anxiety as well as circadian and brief restraint-stress associated corticosterone response. mRNA changes in molecular regulators of the HPA axis were measured in 1mm micropunches of the hypothalamic paraventricular nucleus (PVN) via qPCR. Mice exhibited deficits in maternal care after undergoing chronic psychosocial stress during pregnancy displayed as increased latency to retrieve pups during pup retrieval task (p

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor, making it an excellent target for endocrine therapy. When endogenous and exogenous ligands engage ERα, the receptor binds to regulatory regions in the genome known as enhancers, where it associates with coregulatory proteins, increases histone modifications associated with active enhancers, promotes chromatin looping, stimulates enhancer transcription and the production of enhancer RNAs (eRNAs), and ultimately regulates target gene transcription. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. Current studies on the roles of eRNAs have suggested that eRNAs may 1) recruit regulatory proteins to enhancers; 2) promote RNA polymerase II transition; 3) regulate histone modification; and 4) increase chromosome looping. One challenge facing the field is the lack of accurate eRNA annotations and cloned full-length eRNAs; eRNAs have typically been studied as signals from genomic assays on ‘browser tracks,’ not as physical entities (e.g., cDNAs). We have employed precision nuclear run-on of capped RNA (PRO-cap) to determine the transcription start sites of all expressed eRNAs. In addition, we have also used ultra-deep RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to detect eRNAs and examine for the potential splicing of eRNAs. Combining these genomic data, our annotations provide critical information for the large-scale cloning of eRNAs. We are now testing whether eRNAs originating from ERα enhancers may regulate target gene expression, either in cis or in trans. We cloning specific eRNAs based on our genomic annotations and testing them either by (1) tethering them to their cognate ERα enhancers by using CRISPR/dCas9 technology (i.e., cis) or (2) ectopically expressing them (i.e., trans) in MCF-7 breast cancer cells. Ultimately, our genomic annotation and biochemical tools will allow us to determine the mechanisms by which eRNA contribute to ERα enhancer assembly and function. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Women, who carry a germline mutation in the breast cancer-associated gene 1 (BRCA1) have an increased risk (60-85%) of developing breast cancer during their lifetime. While BRCA1 mutated patients usually develop triple-negative, basal like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. Estrogen and progesterone promote carcinogenesis in the breast. Progesterone promotes proliferation of both benign epithelial cells and cancer cells and can expand the mammary stem cell pool. It is unclear how PR functions in the background of BRCA1 mutation in normal non-cancerous mammary epithelial cells before breast cancer develops. In order to determine whether BRCA1 mutation affects hormone response in benign mammary cells, human mammospheres from patients with wild type (WT; n=6) or BRCA1 mutation (n=6) were treated in vitro with a stepwise menstrual cycle hormone regimen of estrogen (E2) and progesterone (P4) over the course of 28 days. During the last 14 days of treatment, the SPRM, telapristal acetate (TPA) was added to the mammospheres. The mammospheres grown from benign breast tissues retained the expression of hormone receptors (ER and PR) even after the 28 day hormone treatment in vitro. Human mammary epithelial cells expressed aldehyde dehydrogenese-1 ALDH-1 with increasing levels observed in the BRCA1 mutated mammospheres compared to the WT mammospheres. RNA sequencing (RNA-seq) analysis revealed distinct differentially expressed genes between the WT and BRCA1 groups. Specifically, genes associated with extracellular matrix (ECM) organization were differentially regulated by TPA in the BRCA1 mutation group but not in the WT group. In fact, these ECM genes were differentially expressed in response to E2+P4 in WT versus BRCA1 mammospheres. In addition, cell cycle genes were responsive to TPA but in the WT group only. Taken together, these data show that BRCA1 mutation influences the hormone response and in particular to TPA compared to WT cells. Our physiologically relevant model systems have provided important insights into the role of progesterone in BRCA1 mutated breast cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

Nuclear receptors are transcriptional factors controlling critical gene expression networks essential to the developmental and homeostatic regulation of several physiological processes. Transcriptional control of nuclear receptors is achieved by interaction with specific ligands and in response to cellular signalling pathways, which often results in post-translational modifications of receptors. Among such modifications, SUMOylation has emerged as a key process regulating nuclear receptor function. Our recent work has revealed an atypical SUMOylation process that requires phosphorylation, thereby defining a novel SUMO conjugating motif termed pSuM. Functional pSuM has been described for estrogen nuclear receptor ERβ and bile acid receptor FXR, and sequence analysis has indicated a conserved pSuM in the orphan nuclear receptor Nor-1. This study aims to characterize the function of Nor-1 pSuM-mediated SUMOylation. Nor-1 has various functions in the nervous system, including a role in neuronal survival and hippocampal development. In line with an implication of the SUMOylation process in neurodegenerative diseases, we speculate that pSuM SUMOylation might regulate Nor-1 activity and participate in the maintenance of neuronal integrity. The SUMOylation potential of Nor-1 and the mechanism regulating this process were investigated using immunoprecipitation and Western blot analysis. Our results indicate that Nor-1 was SUMOylated at the pSuM motif as well as at another consensus SUMOylation motif, thereby conjugating SUMO-1 and SUMO-2. We also found that the phosphorylation of the pSuM motif was essential for pSuM activation and SUMO conjugation. Furthermore, phosphorylation of adjacent residues to the pSuM appeared to potentiate the SUMOylation of Nor-1, suggesting a role for pSuM extension. This demonstrates the implication of specific kinase pathways required to promote pSuM activation. Using luciferase reporter assays and qPCR analysis, we also showed that SUMOylation regulates Nor-1 transcriptional activity and responsive gene expression in neuronal cells. Our findings provide new insights on the mechanistic regulation of Nor-1 function by phosphorylation and SUMOylation. We also identify a new atypical and highly regulated SUMOylation process as an additional targeting possibility for SUMO conjugation to coordinate transcriptional competence. This study contributes to expand the intricate dynamics of the SUMOylation process used by incoming signals to govern gene networks by nuclear receptors and possibly other transcription factors. Sources of Research Support: This work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institute of Health Research (CIHR) award, and Fonds de la recherche en santé du Québec (FRQS) award.

Over 70% of breast tumours express estrogen receptor alpha (ERα), a ligand-inducible transcription factor. Stimulation with estradiol leads to receptor binding to estrogen response elements (EREs) in target genes regulatory sequences in association with transcriptional cofactors. This results in altered gene expression, increased cell proliferation and accelerated tumour growth. To systematically identify genes contributing to ERα expression and signalling in breast cancer, we performed a genome-wide screen in human ERα+ luminal breast cancer cell lines stably expressing estrogen-induced luciferase reporters using an arrayed format. A primary screen (Sigma Mission lentiviral library, 16083 genes, three shRNA/gene) and secondary screens (615 genes identified as hits by at least two shRNAs with concordant impact on reporter gene expression without major effects on cell survival) were carried out using estrogen-sensitive cells (T47D-ERE3-Luc, MCF-7-ERE-Luc) and insensitive controls (antioxidant response reporter T47D-ARE-Luc). We ultimately characterized by RNASeq the impact of suppressing expression of each of 30 prioritized hits (2-3 shRNAs with >50% reduction of RNA levels per hit) on gene expression in ERα+ breast cancer cells. Knockdown of several of these hits had extensive impacts on expression of direct estrogen target genes and on ERα+ breast cancer cell proliferation. Some hits regulated ERα gene expression and several interacted with ERα in co-IP experiments, indicating roles as upstream regulators and/or transcriptional cofactors of ERα.

Polycystic ovary syndrome (PCOS) affects 10% of women and is hallmarked by hyperandrogenemia (HA), which is also a component of non-alcoholic fatty liver disease (NAFLD). In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, sterol regulatory element-binding protein 1 (SREBP1). To this end, skeletal muscle from lean, female wild type mice and mice administered dihydrotestosterone (DHT) for one month were extracted and analyzed to determine the effect of low dose DHT on lipogenic cytosolic protein and gene expression. We performed Western blot and real-time quantitative polymerase chain reaction (qRT-PCR) analysis of lipogenic intermediates in homogenates of an energy storage tissue. Skeletal muscle was used as a control. Low-dose DHT lowered the active form of cytosolic SREBP-2 in skeletal muscle. However, FAS and p-ACC protein and mRNA expression levels were unchanged in skeletal muscle. The results indicate that SREBP-2 is the primary SREBP isoform in skeletal muscle but that it is not playing a role in DHT-induced lipogenic gene expression or NAFLD. Thank you to HUCM, JHUSOM, and Georgetown University Medical Center (GUMC) for supporting this work.

Estetrol (E4) acts as a typical estrogen (E) agonist in stimulating the endometrium, and when administered to postmenopausal women in various doses, reverses vaginal atrophy and decreases vasomotor symptoms. However, in contrast to other Es administered orally, E4 demonstrates a minimal hepatic effect and does not stimulate breast proliferation. Data in pre and postmenopausal women show minimal stimulatory effects on triglycerides, sex hormone binding globulin, corticosteroid-binding globulin, and angiotensinogen. Similarly, E4 exerts minimal effects on coagulation markers, and when combined with drospirenone as a combined oral contraceptive does not increase the generation of thrombin as measured by the activated protein C resistance ratio, unlike the use of ethinylestradiol. Because of the tissue selectivity of E4, this study sought to better delineate the molecular mode of action of E4. Selective E Receptor Modulators (SERMs) exhibit specific cell and tissue estrogenic, neutral or anti-estrogenic activities. SERMs interact with the ligand binding domain of the E Receptor alpha (ERα) in a way that is distinct from that of E. By preventing the kinking of helix 12 in ERα, SERMs recruit other co-receptor activators and inhibitors that are responsible for SERM action. We compared in replicates the binding pattern and affinities of 154 motifs of ERα co-regulator activators and repressors to complexes of ERα with Estradiol (E2), Estriol (E3) or E4 in a specific Pam Gene assay (1).The pattern of co-receptor binding in the presence of E4 was identical to that elicited by E2 and E3, but with a 50-fold lower potency. This pattern is strikingly distinct from that observed with 4-hydroxytamoxifen or raloxifen. These data together with previously published studies of the crystalline structures of ERα with E2, E3 or E4 complexes confirm that E4 interacts with nuclear ERα in a manner identical to that of the other Es (2). Previous studies have shown that E4 inhibits the activity of the membrane ERα, and blocks the stimulatory action of E2 in this membrane ERα subpopulation. In conclusion, E4 is a unique E with specific actions in tissues, demonstrating absence of specific membrane receptor effects, and no interaction with ERα in the way characteristic of SERMs. The unique profile of E4 activity with lower hepatic effects (minimal or no effects on triglycerides and coagulation) may ultimately translate into safer clinical use. 1) Broekema et al. Endocrinology. 2018; 159:2397. 2) Abot et al. EMBO Mol Med. 2014; 6:1328 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Introduction Metastatic castration-resistant prostate cancer (CRPC) remains a clinical challenge despite antitumoral therapies such as taxane chemotherapeutics, enzalutamide, and abiraterone acetate. Clinical studies have highlighted the benefits of these therapies on overall and progression-free survival, but the changes these treatments impose on the steroid metabolome have not yet been fully mapped. In this study, we investigated the steroid metabolomes of CRPC patients on second-line therapies before and during treatment and after clinical progression was observed. Method Plasma samples from 30 patients from the ongoing CIRCUS trial were selected. Patients were included if progression on androgen-deprivation therapy was confirmed and patients were scheduled to receive additional secondary treatment with antiandrogens (enzalutamide (n=10) or apalutamide (n=1)), abiraterone + prednisone (n=2), docetaxel + prednisone (DP, n=10) or cabazitaxel + prednisone (CP, n=14). Seven patients completed two treatments during their enrollment in CIRCUS. Following liquid-liquid extraction of steroids from plasma, multi-steroid profiling by liquid chromatography-tandem mass spectrometry was carried out targeting 16 steroid hormones from classic and 11-oxygenated androgen pathways as well as mineralocorticoid and glucocorticoid biosynthetic pathways. Results Accurate quantification was achieved for all targeted steroids with the exception of dihydrotestosterone and androstenedione. The most abundant active androgen in CRPC patients was 11-ketotestosterone (11KT) with a median concentration of 0.46 nM (range 0.03 - 2.39 nM), whereas the median testosterone concentration was 0.14 nM (range 0.03 - 0.76 nM). 11KT and its precursors were suppressed by CP treatment. Analysis of all patients who started treatment with glucocorticoids revealed a potent suppression of 11KT by 88.5% (range 17.9 - 97.5%) and testosterone by 70.1% (range 24.1 - 95%), as well as suppression of 11-oxygenated androgen precursor steroids. Increased 11KT levels were observed in the DP-treated patients at the time of progressive disease. Discussion In this study, we present in-depth characterization of the previously unexplored steroid metabolomes in CRPC patients revealing 11KT as the most abundant androgen. 11KT has been shown to bind and activate the AR with similar potency to testosterone and our findings suggest that the 11-oxygenated androgens may be the dominant AR agonists in the CRPC setting. Routine quantification of testosterone alone may therefore underestimate the androgenic potential in CRPC patients. Suppression of adrenal androgens, including 11KT, was achieved by glucocorticoid treatment, which may explain the beneficial effects of glucocorticoids in CRPC patients. Funding This project was funded by the Daniel den Hoed foundation. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

High grade serous ovarian carcinoma (HGSC), the most prevalent and aggressive form of ovarian cancer, contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors are known to contribute to breast and reproductive cancer development. Our understanding, however, of the mechanisms of PR isoform action in the initiation and progression of ovarian cancer is limited. HGSC is thought to originate from the fallopian tube epithelium (FTE), and accumulating evidence suggests that serous tubal intraepithelial carcinomas (STICs) are precursor lesions to most HGSCs. Our IHC analysis of normal FTE and STICs showed expression of both total PR and activated phopho-Ser294 PR (p-PR). Interestingly, STIC lesions had greater intensity of focal nuclear p-PR, indicative of highly active transcriptional complexes. To investigate PR isoform signaling in early stage HGSC, we utilized p53-dominant negative mutant FTE cells to generate stable cell lines expressing either the PR-A or PR-B isoform. Progestin (progesterone; R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent p-PR as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g. HEF1), cell cycle regulators (e.g. FOXO1), and glucocorticoid signaling proteins (e.g. CRISPLD2, NDRG1). Progestin treatment also dramatically inhibited proliferation which was recovered following growth factor stimulus, suggesting that PR signaling may promote cell quiescence (G0). To mimic dissemination of FTE cells from STICs, a 3D spheroid model was established. Interestingly, we observed that PR expression and activation greatly increased the formation, size and number of total spheroids compared to PR negative controls. Cells within these spheroids are predominantly Ki67-negative indicating that they are likely non-proliferative and potentially G0-arrested cells. When the spheroids were embedded into collagen, to mimic tumor cell invasion into the peritoneum, only PR+ spheroids exhibited invasive behavior. Additionally, PR-B+ spheroids were more invasive than PR-A+ spheroids; progestin treatment during 3D formation dramatically increased spheroid invasion. Notably, this is the opposite of our results in 2D cultures, where PR-A+ FTE cells display greater proliferative and migratory phenotypes relative to PR-B+ FTE cells. Taken together, our data suggest that activation of PR signaling promotes and enhances the formation and survival of non-adherent 3D FTE structures. Such effects of progesterone are predicted to influence the shedding, aggregation and dissemination of early STIC lesions that form ovarian and peritoneal metastases. Our findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early ovarian cancer development.