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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.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor (TF) that controls the tissue- and gene-specific transactivation and transrepression of thousands of target genes. Distinct GR DNA binding sequences with activating or repressive activities have been identified, but how they modulate transcription in opposite ways is not known. We show that GR forms phase-separated condensates that specifically concentrate known co-regulators via their intrinsically disordered regions (IDRs)in vitro.A combination of dynamic, multivalent (between IDRs) and specific, stable interactions (between LxxLL motifs and the GR ligand binding domain) control the degree of recruitment. Importantly, GR DNA-binding directs the selective partitioning of co-regulators within GR condensates such that activating DNAs cause enhanced recruitment of co-activators. Our work shows that condensation controls GR function by modulating co-regulator recruitment and provides a mechanism for the up- and down-regulation of GR target genes controlled by distinct DNA recognition elements.

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.

Long noncoding RNAs (lncRNAs) have been demonstrated to be involved in diverse cellular processes as important regulators, such as in cancer. However, their roles in breast cancer biology are greatly unknown so far. In our study, integrated analysis of subcellular fractionation RNA-seq with gene expression profile from human reproductive tissues yielded a comprehensive catalog of estrogen-regulated reproductive tissue-specific lncRNAs. We selected long intergenic noncoding RNA 16 (LINC16) for further study as it was the top upregulated lncRNA by estrogen and associates with clinical outcome. Analysis of RNA-seq data from different human tissues, we found that LINC16 is highly expressed in testis and followed by other reproductive organs, cervix and uterus. Interestingly, interrogation of expression data from human cancer tissues showed LINC16 is highly expressed in breast cancer compared to other cancers. We have determined the 5’ and 3’ ends of LINC16 and its exon/intron structure, and cloned LINC16 to study its function in molecular and cell-based assays. Our preliminary results suggest that LINC16 plays a critical role in ERα-dependent pathways.

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

Objective: Nonresponse to selective serotonin reuptake inhibitor and serotonin norepinephrine reuptake inhibitor treatment is common in patients with major depressive disorder (MDD), particularly in women, occurring in about 70% of patients despite adequate dosing. Well-tolerated augmentation strategies are needed, particularly ones that do not cause or exacerbate symptoms such as fatigue and sexual dysfunction. Low-dose testosterone has been shown to improve depression symptom severity, fatigue and sexual function in small studies of women not formally diagnosed with MDD. We sought to determine whether adjunctive low-dose transdermal testosterone improves depression symptom severity, fatigue, and sexual function in women with treatment-resistant MDD. A functional MRI (fMRI) substudy examined effects of testosterone on activity in the anterior cingulate cortex (ACC), a brain region important in mood regulation. Methods: Randomized, double-blind, placebo-controlled, 8-week trial of adjunctive testosterone cream (AndroFeme® 1, Lawley Pharmaceuticals, Australia) in 101 women, ages 21–70, with treatment-resistant MDD. Testosterone was titrated to achieve blood levels near the upper normal reference limit. Primary outcome measure was depression severity by Montgomery-Asberg Depression Rating Scale (MADRS). Secondary endpoints included fatigue, sexual function, and safety measures. fMRI substudy (n=20) primary outcome was change in ACC activity. Results: Mean age was 47±14 (SD) years and mean baseline MADRS score was 26.6±5.9. Eighty-seven (86%) participants completed 8 weeks of treatment. MADRS depression scores decreased in both arms [testosterone: 26.8±6.3 to 15.3±9.6; placebo: 26.3±5.4 to 14.4±9.3 (baseline to 8 weeks, respectively)], with no difference between groups (p=0.91). Fatigue and sexual function improved without differences between groups. There were no group differences in side effects. fMRI results demonstrated a relationship between ACC activation and androgen levels pretreatment but no difference in ACC activation with treatment. Conclusions: This rigorously designed, double-blinded clinical trial did not find significant group differences between adjunctive low dose transdermal testosterone and placebo for antidepressant augmentation in women with treatment-resistant MDD and had a high placebo response rate. Low-dose testosterone was well tolerated but failed to differentially impact overall depressive symptom severity, fatigue, or sexual dysfunction. Testosterone did not result in greater activity in a brain region (ACC) implicated in MDD etiopathology compared to placebo. Thus, the addition of low-dose testosterone to ineffective antidepressant treatment should not be recommended for women with MDD. Further studies using strategies designed to reduce placebo effects may be warranted.

Introduction: Aromatization of exogenous testosterone might result in increased estradiol levels. Concerns have been raised about undesired estrogenic effects in assigned female at birth (AFAB) transgender people. How serum estradiol levels change after initiation of testosterone therapy and if these levels should be monitored, remains unclear. Methods: This prospective cohort study was part of the European Network for the Investigation of Gender Incongruence (ENIGI). Serum levels of sex steroids were assessed in 746 AFAB transgender people during a three-year follow-up period, starting at the initiation of hormone treatment. Results: Estradiol levels decreased from median [P25-P75] 45.5[24.0-102.2]pg/mL to 36.5[25.0-46.2]pg/mL over three years (P

Lung cancer is a leading cause of cancer mortality worldwide. Premenopausal women often has worse survival with advanced stages of the disease compared to postmenopausal women, suggesting an involvement of sex steroids and their receptors in the progression of non-small cell lung cancer (NSCLC). Progesterone receptor (PR) was reported to be involved in an inhibition of NSCLC cell proliferation and correlated with better clinical outcome. In addition, PRB suppressed epidermal growth factor (EGF)-induced NSCLC cell proliferation and activation of ERK1/2, in the absence of progestin. However, clinical and biological significance of PRB in NSCLC patients has remained virtually unknown. Therefore, we performed immunohistochemistry using monoclonal antibody specific to the N-terminus of PRB (250H11 mAb) and 1294mAb which could detect both PRA and PRB in 124 NSCLC cases: 94 adenocarcinoma and 30 squamous cell carcinoma (SCC). Overall survival (OS) was analyzed using the Kaplan-Meier plotter (KM plotter) database, examining the correlation between the status of PRs and survival rate of the patients. 19 cases were immunohistochemically positive for PRB and 23 PRA/B positive NSCLC cases, and all of four cases harboring abundant PRs were also positive for PRB. Therefore, PRB positivity was considered to be significantly correlated with the whole PR ( Our data demonstrated that not only PR but also PRB could be a good prognostic factor and have an important role on tumor progressing in NSCLC patients. In order to further elucidate the molecular mechanisms of PRB signaling in NSCLC, we are now performing further in vitro studies. Results of our present study could contribute to the development of novel therapeutic strategies targeting PR and/or PRB in NSCLC patients.

The growth arrest-specific 5 (gas5) gene encodes a long non-coding RNA (lncRNA) that is required for normal growth arrest, slows down the cell cycle, controls apoptosis, and is required for the inhibition of cell growth by mTOR inhibitors such as rapamycin. In agreement with this role in regulating cell proliferation, Gas5 expression is reduced and acts as a tumor suppressor in numerous cancers, including B-cell lymphoma and leukemia. At its 3’ terminal end (nucleotides 546-566) Gas5 contains a predicted stem-loop structure that specifically interacts with steroid receptors (SRs) and blocks DNA-dependent steroid signalling. In steroid-sensitive cancer cells such as prostate cancers this SR binding motif is responsible for Gas5 effects on cell growth. This is not true in other cell types, however, where proliferation is not strongly dependent on SR signaling (e.g. leukemic T cells). Therefore, other regions in Gas5 must be active and use different mechanisms to regulate cell survival. We have used SHAPE chemical probing to analyze the secondary structure of Gas5 in vitro and in cellulo. We find that the secondary structure of endogenous Gas5 resembles that of in vitro transcribed Gas5 RNA. The molecule contains three separate structural modules: a 5’ module with low secondary structure content, a highly structured core module, and the SR binding module, which forms separate from the rest of the molecule close to its 3’ end. Functional studies in leukemic T cells show that the 5’ module mediates Gas5’s role in inhibiting basal cell survival and slowing the cell cycle, whereas the core module is required for mediating the effects of mTOR inhibition. These results confirm that the Gas5 structural modules function independently in cells and each module acts under different cellular conditions, likely using different molecular mechanisms. RNA pull-downs from cell lysates using the identified modules and full-length RNA identified proteins preferentially associated with each module. Proteins preferentially associated with the 5’ terminal region are enriched in splicing and RNA processing factors. The structured central region preferentially interacts with proteins involved in chromosome organization such as the SWI/SNF family of nucleosome remodeling complexes.

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

Background: Szent-Gyorgyi proposed that digoxin wasn’t really drug but was a substitute for an endogenous cardiotonic steroid (ECS). Endogenous ouabain and marinobufagenin have been proposed as ECS. Hypothesis: Ionotropin, our first candidate for the ECS, is unique among steroids because it is the phosphocholine ester of a steroid with 23 carbon atoms. Logically, either there must be a novel mechanism for adding carbon atoms to a pregnenolone-like precursor or a novel mechanism for side-chain cleavage from a cholesterol-like precursor. Experimental design: Serum samples were extracted with acetonitrile, filtered and analyzed by MS-N on an LTQ-XL ion trap mass spectrometer. The instrument permits multiple rounds of fragmentation and identification of the parent ion and each fragment ion. This process permitted recognition of ions that were phosphocholine esters and of the mass of the steroid fragments. The chemical formula of each steroid fragment was determined by trial and error analysis. Although not every mass ion has a unique chemical formula, in fact, each of the steroid ions had a unique formula. Possible isomers were resolved by consideration of knowledge of steroid biosynthetic pathways. Major results: In brief, human serum samples had steroid fragment ions consistent with 23 (354 Da) and 25 (398 Da) carbon atoms. This provides an additional constraint as the synthetic mechanism must account for both products. These mass ions were consistent with condensation of either acetyl-CoA or acetoacetyl-CoA with the phosphocholine ester of pregna-5,7-diene-3β,17α-diol-20-one. After condensation, the steroid adduct would be dehydrated and cyclized to form the corresponding spiral steroid phosphocholine ester. This pathway is similar to the mechanism of addition of 2 carbon fragments to a long chain fatty acid. This is the first explanation for the biosynthesis of endogenous mammalian ECS. Spiral lactones would be expected to cross react with many antibodies specific for digoxin, ouabain or marinobufagenin. Either one of the spiral lactones would satisfy Szent-Gyorgyi’s suggestion as the endogenous digoxin-like material. Conclusions: In summary, we have isolated 2 spiral steroid lactones from mammals and identified the mechanism of their biosynthesis. We propose, as the spiral steroids share structural features with the spironolactone class of potassium sparing diuretics, that they also share functions. Nicholls proposed that a candidate for ECS should not be accepted without [a] isolation, [b] precursors, and [c] a biosynthetic path. As there has been no satisfaction of these requirements for ouabain or marinobufagenin, their existence as ECS in mammals needs to be reconsidered.

The effect of androgen on angiogenesis has been documented. However, its molecular mechanisms underlying has not been well illustrated. Here, we conducted both in vitro migration assay and proliferation assay to investigate whether androgen receptor activation have any impacts on the angiogenesis. Treatment with an androgen receptor (AR) agonist, metribolone (R1881) at a range of concentrations (0.05-5 nM) or dihydrotestosterone (DHT) at a range of concentrations (0.5-2 nM) caused concentration-dependent inhibition of proliferation and migration in human umbilical venous endothelial cells (HUVEC). Blockade of the AR activity by pre-treatment with HF (5 nM), an AR antagonist, or knockdown of AR expression using the lenti-virus shRNA technique abolished the R1881-induced proliferation and migration inhibition in HUVEC, suggesting that AR receptor activation can inhibit endothelial cell proliferation and migration. To further delineate the signaling pathway involved in the AR activation-induced proliferation inhibition, our data indicate that R1881 inhibited proliferation in vascular endothelial cells through activating the AR/cSrc/AKT/p38/ERK/NFκB signaling pathway, which in turn up-regulated the expression of p53, p21 and p27 protein, and finally reduced endothelial cell proliferation. To investigate signaling pathway involved in the AR activation-induced migration inhibition, our data showed that R1881 can reduce the membrane translocation of RhoA and Rac-1, suggesting that inhibition of the RhoA and Rac-1 activity might be involved in the R1881-inhibited endothelial cell migration. Over-expression of RhoA prevented the R1881-inhibited endothelial cell migration and this effect was abolished by pre-treatment with Y27623, a ROCK inhibitor, confirming that inhibiting RhoA activity participated in the R1881-inhibited endothelial cell migration. Using the zebrafish and Matrigel angiogenesis models, we also demonstrated that R1881 inhibited angiogenesis through the AR-mediated pathway in vivo.

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

GPER bears structural and functional characteristics shared by members of the G-protein coupled receptor (GPCR) superfamily, the largest class of cell surface receptors, with more than 800 members encoded in the human genome. GPER is localized predominately in intracellular membranes, in many but not all cell types, and its surface expression is modulated by steroid hormones and during tissue homeostasis. An intracellular staining pattern is not unique among GPCRs, which deploy a diverse array of posttranslational regulatory mechanisms to determine cell surface expression, effectively regulating cognate ligand binding and activity. Here, we show nascent GPER undergoes strict quality control via endoplasmic reticulum associated degradation (ERAD) requiring direct poly-ubiquitinylation of GPER and valosin-containing protein VCP/p97-mediated segregation of misfolded proteins from the ER membrane to the cytoplasm for delivery to the 26S proteasome. Specifically, we find that inhibition of p97 using the pharmacological compound, CB-5083, or by doxycycline-inducible p97 shRNA results in the accumulation of immature glycosylated GPER in the ER. Inhibition of proteasome function facilitates anterograde trafficking with the transport of nonfunctional GPER to the plasma membrane as indicated by no increase in specific estrogen binding using 3H-17β-estradiol in a radioreceptor assay. The forward trafficking of misfolded GPER requires transit through the Golgi as treatment with brefeldin A (BFA) prevents GPER plasma membrane expression. Substitution of all three lysines (K333, K342, and K357) encoded in the cytoplasmic tail of GPER with arginines blunts its polyubiquitinylation and allows GPER to evade degradation by quality control but does not result in increased plasma membrane expression suggesting that additional structural motifs encoded within GPER control its anterograde trafficking. In contrast, functional GPER is recovered at the plasma membrane of human SKBR3 breast cancer cells treated with either 17β-estradiol or the GPER selective antagonist, G15, in the presence of cycloheximide resulting in increased surface GPER. Thus, our findings suggest that estrogens, both natural and synthetic, can function as pharmacochaperones capable of promoting the correct folding of GPER and enhanced expression of functional GPER at the plasma membrane.

The role of long noncoding RNAs (lncRNAs) in cancer biology are just beginning to be elucidated and recent studies have shown that they could be therapeutic targets. In a previous study, combining powerful techniques, Global Run-On sequencing (GRO-seq) and subcellular fractionation RNA-seq in breast cancer cells identified a large number of estrogen-regulated unannotated long noncoding RNAs. Analysis of gene expression data from hundreds of samples representing 13 different tissue types including both cancer and normal tissue, revealed that many lncRNAs are differentially expressed in various cancers. Furthermore, a large number of lncRNAs are divergent transcripts and show distinct expression patterns across molecular subtypes of cancer. In functional assays, knockdown of selected lncRNA, such as lncRNA67, inhibits the growth of breast cancer cells. Amplified expression of lncRNA67 in luminal-subtype of breast cancer correlates with clinical outcome. LncRNA67 has now been fully annotated (transcription start and stop site, 5’ cap, polyA tail, and exon/intron structure), and cloned. Our preliminary molecular analyses indicate that lncRNA67 plays a critical role in ER-dependent and -independent pathways. Collectively, our results suggest that lncRNAs are an integral component of cancer biology.

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

Background: Analytic measurements of a hormone in bodily fluids are a cornerstone of clinical evaluation. However, clinical presentation may be affected also by currently unmeasured modulators of hormonal function. An alternative could be to measure the cumulative effect of all factors present in a bodily fluid on the function of a hormone receptor. Prior studies showed an androgen receptor (AR) BioAssay to accurately measure urine androgens in males undergoing testosterone (T) supplementation (1). The factors integrated by the AR BioAssay include androgens, anti-androgens and, in serum, androgen binding proteins affecting ligand availability. Methods: The AR BioAssay was exposed to male and female serum samples obtained from the CDC’s Hormone Standardization (HoSt) Program, and to female serum samples from the UCSF PCOS Tissue Bank registry. AR activity was quantified against a testosterone standard curve and recorded as ‘T-equivalent’ (‘T-eq’) androgen activity units. Results: In 40 CDC HoSt sera added directly (no extraction) to AR BioAssay cells, androgen activities ranged from 2.57 to 298 ng ‘T-eq’/dl. In the 20 ‘male’ CDC HoSt sera (T>150 ng/dl), the androgen activity measurements were uniformly less (0.35 ± 0.10) that of the T-measurement. By contrast, in the 20 ‘female’ CDC HoSt sera, in which T concentrations are typically lower than the affinity of T for sex hormone binding globulins such that more of the T is available to the AR BioAssay, the measured androgen levels were on average 1.45-fold higher than the T concentration. This androgen to T comparison showed high variability in females with 5 of 20 CDC HoSt samples having androgen concentrations more than double that of T (maximum, 6.1-fold). In female serum samples from the PCOS registry, androgens were higher in patients with a PCOS diagnosis (57.7 +/-17.6 ng ‘T-eq’/dl; n = 23) compared to women not meeting formal PCOS criteria (38.1 +/-10.8 ng ‘T-eq’/dl; n = 4); androgen values again averaged 1.40-fold (maximum, 4.9-fold) that of the T measurements, regardless of PCOS diagnosis. Conclusions: Androgen-binding globulins appear to most influence androgen activity levels in male serum. In females, the lower T concentrations may minimize the impact of androgen-binding proteins and permit the impact of non-T androgens to be more pronounced with possible clinical consequence. Further investigations are needed to determine whether functional androgen measurements may improve clinical diagnosis of certain conditions. Reference: (1) Bailey et al (2016) PLoS One11(3):e0151860

A genome wide meta-analysis by the CORtisol NETwork (CORNET) consortium(1) has identified genetic variants spanning the SERPINA6/SERPINA1 locus on chromosome 14, associated with morning plasma cortisol and predictive of cardiovascular disease (Crawford et al, Unpublished). SERPINA6 encodes Corticosteroid Binding Globulin (CBG), responsible for binding most cortisol in blood and putatively mediating delivery of cortisol to target tissues. We hypothesised that genetic variants in SERPINA6 influence CBG expression in liver and cortisol delivery to extra-hepatic tissues, influencing cortisol-regulated gene expression. The Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task study (STARNET)(2) provides RNA sequencing data in 9 vascular and metabolic tissues from 600 genotyped individuals (mean age 65.8, 70.3% male) undergoing coronary artery bypass grafting. We used STARNET to identify SNPs associated with plasma cortisol at genome wide significance in CORNET as cis-eQTLs for SERPINA6 in liver and as trans-eQTLs for the expression of genes across STARNET tissues. Causal inference methodologies(3) were then employed for the network reconstruction of these trans-genes and their downstream targets. We identified 21 SNPs that both were associated with cortisol at genome wide significance in CORNET (p ≤ 5x10-8) and were cis-eQTLs for SERPINA6 expression in liver (q ≤ 0.05). Moreover, these SNPs were trans-eQTLs for sets of genes in liver, subcutaneous and visceral abdominal adipose tissue, with over-representation of known glucocorticoid-regulated genes in adipose. The highest confidence gene network identified was specific to subcutaneous adipose, with the interferon regulatory trans-gene, IRF2, controlling a putative glucocorticoid-regulated network. Targets in this network include LDB2 and LIPA, both associated with coronary artery disease. We conclude that variants in the SERPINA6/SERPINA1 locus mediate their effect on plasma cortisol through variation in SERPINA6 expression in liver, and in turn affect gene expression in extra-hepatic tissues through modulating cortisol delivery. This supports a dynamic role for CBG in modulating cortisol delivery to tissues. The cortisol-responsive gene networks identified here represent candidate pathways to mediate cardiovascular risk attributable to elevated cortisol. (1) Bolton, et al. (2014) PLOS Genet. 10:e1004474., (2) Franzén et al. (2016). Science 353:827., (3) Wang and Michoel. (2017). PLOS Comput. Biol. 13:e1005703.

Introduction: Salivary glucocorticoids (cortisol [SalF], cortisone [SalE]) are collected non-invasively, unaffected by variation in cortisol binding globulin (CBG) and an established tool in the investigation of Cushing’s syndrome (CS). We have previously shown a strong correlation between salivary glucocorticoids and circulating free serum cortisol, better than that with total serum cortisol. Measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS) permits lower limits of quantification, eliminates cross-reactivity both between cortisol and cortisone, and by metyrapone-induced elevations in 11-deoxycortisol. Previous studies in CS have demonstrated that a mean (based on 5 samples during a single day) serum cortisol (serumF) of 150-300 nmol/l equates to a normal isotopically calculated cortisol production rate. We report the use of salivary glucocorticoid measurement in metyrapone-treated CS patients undergoing dose titration to achieve a mean serumF of 150-300 nmol/L. Methods: Seventeen (11 females; age-range 24-74 years) patients with CS undergoing dose titration with metyrapone were studied on 44 occasions: 15 ACTH-dependent (5 ectopic) and 2 adrenal. 24 healthy male volunteers (HV) were also studied. Both cohorts had paired serumF and SalE and SalF samples collected at 5 time-points (10:00; 11:30; 13:00; 14:30; 16:00). Serum and salivary glucocorticoids were measured using LC-MS/MS. The TR for salivary glucocorticoids was determined from the mean SalE and SalF in HV whose mean serumF (n=20) was in the desired range (150-300 nmol/L). In CS patients the metyrapone dose had been titrated to achieve a mean serumF of 150-300 nmol/L on 14 (out of 44) occasions. Results: Mean SalE (r=0.70; p In CS patients, SalE had greater sensitivity (79% vs. 75%) and specificity (80% vs. 57%) in predicting a mean serumF in the TR than SalF. Conclusion: We have demonstrated a close correlation between mean SalE and mean serumF in metyrapone treated CS patients. Salivary glucocorticoids within the derived TR were highly predictive for a target serumF. It is unsurprising, due to the effect of CBG on serumF measurements, that the sensitivity and specificity of SalE and SalF are not greater than reported. Consequently, SalE and SalF, as surrogates for free serum F, have the potential to be superior than serumF when assessing adequacy of medical therapy in CS and may permit out-of-hospital monitoring. Further work is required to validate these findings.

Glucocorticoids are potent endogenous anti-inflammatory molecules, with its receptor (GR) expressed in nearly all immune cells. Macrophages are heterogeneous cells having a central role in both tissue homeostasis and inflammation. Paradoxically glucocorticoids have a limited efficacy controlling these processes and inflammation resolution of macrophage-related diseases, such as type 2 diabetes, atherosclerosis and rheumatoid arthritis. To address this issue, we explored new glucocorticoid target genes in macrophages with potential clinical and therapeutic implications for these diseases. Analysis of genomic platforms identified the pro-diabetic exopeptidase dipeptidyl peptidase 4 (DPP4) as a novel glucocorticoid-responsive gene. GR directly induces its expression by binding to two glucocorticoid-responsive elements within the DPP4 promoter. Unexpectedly, DPP4 mediated the glucocorticoid-induced spontaneous macrophage migration. These actions were blocked by both GR and DPP4 siRNA knockdowns. Furthermore, two DPP4 inhibitors, Sitagliptin and Linagliptin, used clinically for the treatment of diabetes inhibited glucocorticoid-induced mobility of macrophages. DPP4 induction by glucocorticoids was also observed in murine peritoneal macrophages and pro-inflammatory M1 polarized macrophages and was associated with an increase in their migratory properties. Provocatively, DPP4 has been shown to be involved in the inflammatory macrophage profile associated with type 2 diabetes, obesity and atherosclerosis. Since macrophages require efficient cell movement for all their functions, such as sensing of Pattern Associated Molecular Patterns (PAMPs), phagocytosis and the antigen presentation, the DPP4 induction by glucocorticoids could potentiate the macrophage infiltration and their activation in chronic inflammatory tissues and diabetes.