Glucocorticoid Receptor Levels Research Articles

Alterations of Cortisol and Melatonin Production by the Theca Interna Cells of Porcine Cystic Ovarian Follicles.

Simple SummaryThe mechanism of follicular cyst formation is largely unknown but changes in follicular composition are known to be involved. In particular, there is abnormal hormone secretion in cystic follicles. Here, we found there was disruption of hormone secretion in the fluid of cystic follicles in sows. The glucocorticoid receptor was highly expressed, and the melatonin receptor was weakly expressed in cystic follicles compared with control follicles. Thus, secretion of steroid hormones in cystic follicles is disrupted and disturbances in signaling via cortisol and melatonin are involved in the development of follicular cysts in sows.(1) Background: Cortisol and melatonin (MT) act in regulating follicular development. We hypothesized that abnormal levels of cortisol, MT, and steroids in theca interna cells might be involved in the development of follicular cysts in sows. (2) Methods: To test this hypothesis, we measured the mRNA levels of enzymes involved in steroid hormone synthesis, the glucocorticoid receptor (GR), and melatonin receptors (MTRs) in theca interna cells of cystic and normal porcine follicles. (3) Results: The concentrations of estradiol, progesterone, and cortisol were greater in cystic follicles than in control ones (p = 0.034, p = 0.020, p = 0.000), but the concentration of MT was significantly lower (p = 0.045). The levels of GR, 11β-HSD1, and 11β-HSD2 were higher in cystic follicles than in control l follicles. MT types 1 and 2 were significantly lower in cystic follicles (p < 0.05). The mRNA expression levels of genes encoding the steroid hormone synthesis enzymes, steroidogenic acute regulatory protein (StAR), recombinant cytochrome P45011A1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in theca interna cells of cystic follicles were significantly higher than in control follicles. Thus, there was disruption of hormone secretion in the fluid of cystic follicles in sows. (4) Conclusions: The levels of steroid hormones, cortisol and MT are disrupted in porcine cystic follicles.

Abiraterone switches castration-resistant prostate cancer dependency from adrenal androgens towards androgen receptor variants and glucocorticoid receptor signalling.

IntroductionCastration‐resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, which is largely driven by conversion of adrenal androgen precursors lasting after castration. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1, has been demonstrated to reduce adrenal androgen synthesis and prolong CRPC patient survival. To study mechanisms of resistance to castration and abiraterone, we created coculture models using human prostate and adrenal tumours.Materials and MethodsCastration‐naïve and CRPC clones of VCaP were incubated with steroid substrates or cocultured with human adrenal cells (H295R) and treated with abiraterone or the antiandrogen enzalutamide. Male mice bearing VCaP xenografts with and without concurrent H295R xenografts were castrated and treated with placebo or abiraterone. Response was assessed by tumour growth and PSA release. Plasma and tumour steroid levels were assessed by LC/MS‐MS. Quantitative polymerase chain reaction determined steroidogenic enzyme, nuclear receptor and AR target gene expression.ResultsIn vitro, adrenal androgens induced castration‐naïve and CRPC cell growth, while precursors steroids for de novo synthesis did not. In a coculture system, abiraterone blocked H295R‐induced growth of VCaP cells. In vivo, H295R promoted castration‐resistant VCaP growth. Abiraterone only inhibited VCaP growth or PSA production in the presence of H295R. Plasma steroid levels demonstrated CYP17A1 inhibition by abiraterone, whilst CRPC tumour tissue steroid levels showed no evidence of de novo intratumoural androgen production. Castration‐resistant and abiraterone‐resistant VCaP tumours had increased levels of AR, AR variants and glucocorticoid receptor (GR) resulting in equal AR target gene expression levels compared to noncastrate tumours.ConclusionsIn our model, ligand‐dependent AR‐regulated regrowth of CRPC was predominantly supported via adrenal androgen precursor production while there was no evidence for intratumoural androgen synthesis. Abiraterone‐resistant tumours relied on AR overexpression, expression of ligand‐independent AR variants and GR signalling.

PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.

Environmental enrichment reverses cerebellar impairments caused by prenatal exposure to a synthetic glucocorticoid.

During prenatal life, exposure to synthetic glucocorticoids (SGCs) can alter normal foetal development, resulting in disease later in life. Previously, we have shown alterations in the dendritic cytoarchitecture of Purkinje cells in adolescent rat progeny prenatally exposed to glucocorticoids. However, the molecular mechanisms underlying these alterations remain unclear. A possible molecular candidate whose deregulation may underlie these changes is the glucocorticoid receptor (GR) and neurotrophin 3/ tropomyosin receptor kinase C, neurotrophic complex (NT-3/TrkC), which specifically modulates the development of the neuronal connections in the cerebellar vermis. To date, no evidence has shown that the cerebellar expression levels of this neurotrophic complex are affected by exposure to a synthetic glucocorticoid in utero. Therefore, the first objective of this investigation was to evaluate the expression of GR, NT-3 and TrkC in the cerebellar vermis using immunohistochemistry and western blot techniques by evaluating the progeny during the postnatal stage equivalent to adolescence (postnatal Day 52). Additionally, we evaluated anxiety-like behaviours in progeny using the elevated plus maze and the marble burying test. In addition, an environmental enrichment (EE) can increase the expression of some neurotrophins and has anxiolytic power. Therefore, we wanted to assess whether an EE reversed the long-term alterations induced by prenatal betamethasone exposure. The major findings of this study were as follows: i) prenatal betamethasone (BET) administration decreases GR, NT-3 and TrkC expression in the cerebellar vermis ii) prenatal BET administration decreases GR expression in the cerebellar hemispheres and iii) enhances the anxiety-like behaviours in the same progeny, and iv) exposure to an EE reverses the reduced expression of GR, NT-3 and TrkC in the cerebellar vermis and v) decreases anxiety-like behaviours. In conclusion, an enriched environment applied 18 days post-weaning was able to restabilize GR, NT-3 and TrkC expression levels and reverse anxious behaviours observed in adolescent rats prenatally exposed to betamethasone.

Abstract P015: Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer progressing on enzalutamide

Abstract Background: Preclinical studies have shown that activation of the glucocorticoid receptor (GR) leads to resistance to antiandrogens, which is reversed by GR inhibition. ORIC-101 is a potent, selective, orally bioavailable small molecule GR antagonist undergoing clinical development in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Methods: Ten patients were enrolled in the dose escalation portion of the Phase 1b study during which three dose levels of ORIC-101 given once daily were evaluated in combination with 160 mg enzalutamide. Translational markers were measured both pre-treatment and on study, including PD biomarkers, blood cortisol, GR and AR protein levels, and genomic alterations. PD biomarkers FKBP5, GILZ and PER1 were assessed by RT-qPCR in peripheral blood mononuclear cells (PBMCs), collected on multiple days and times in the first two cycles before and after dosing. PD modulation and target coverage were evaluable in 9 out of 10 patients. In tumor biopsies with &amp;gt;50 tumor cells, GR, AR and PTEN protein status were evaluated by immunohistochemistry (IHC). Liquid biopsies were collected before treatment, at the end of Cycle 2, and/or the end of treatment: nuclear GR and ARv7 protein levels were measured on circulating tumor cells (CTCs) by immunofluorescence (Epic Sciences, San Diego, CA), mutations and copy number alterations in cancer genes were captured in plasma with the 500-gene GuardantOMNI® ctDNA assay (Redwood City, CA). Results: PD suppression after one dose of ORIC-101 was observed in PBMCs of all patients. ORIC-101 reached steady-state target engagement within the first cycle in 6 out of 9 evaluable patients across dose levels, including at the RP2D. Nuclear GR protein was detected by IHC in the 6 pre-treatment biopsies, with H-scores ranging from 11 to 298 (median 95, IQR 55-148). Reduction of GR protein was observed in 3 out of 4 matched on study biopsies (pre-treatment H-scores 110-298), indicating potential elimination of GR positive tumor cells, while the fourth patient had low baseline GR levels (H-score 11). Pre-treatment AR protein levels were consistently high (IHC H-scores 230-300). AR alterations observed in ctDNA or CTCs were L702H (n=1 patient), H875Y (n=1), amplification (n=2), and ARv7 (n=2). PTEN protein aberrations were identified in two patients. The amount of tumor shedding in this cohort ranged from 0.24 to 82% (median 2.2%, IQR 0.54-12.1%), comparable to a clinical cohort of ~10,000 prostate tumors profiled with Guardant360® (Guardant Health). Prevalent genomic alterations at time of enrollment included TP53, RB1, BRCA2, ATM and SPOP, with minimal new genomic changes occurring on treatment. Conclusions: Biomarker data from patients enrolled in the Phase 1b study provide evidence of PD modulation, steady-state target coverage, and on-target tumor cell eradication at all dose levels. Tumor characterization in association with clinical outcome provides an opportunity to refine the patient population during the ongoing dose expansion portion of the study. Citation Format: Anneleen Daemen, Shravani Barkund, Ann Johnson, Aleksandr Pankov, Amber W. Wang, Haiying Zhou, Pratik S. Multani, Edna Chow Maneval, Lori S. Friedman, Rupal Patel. Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer progressing on enzalutamide [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P015.

Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress in Rats Is Associated with Expression of the Glucocorticoid Receptor, Nurr1, and IL-1β in the Hippocampus.

Individual differences in coping with stress may determine either a vulnerable or resilient phenotype. Therefore, it is important to better understand the biology underlying the behavioral phenotype. We assessed whether individual behavioral phenotype to acute stress is related with the hippocampal expression of glucocorticoid receptor (GR), Nurr1, interleukin-1 beta (IL-1β) or brain-derived neurotrophic factor (BDNF). Wistar male rats were exposed to forced swimming for 15 min and sacrificed at different times. Behavioral response was analyzed, and it was compared with the gene and protein expression of GR, Nurr1, IL-1β and BDNF in the hippocampus for each time point. Behavioral phenotyping showed a group with high immobility (vulnerable) while another had low immobility (resilient). No significant differences were found in the Nurr1, IL-1β and BDNF mRNA levels between resilient and vulnerable rats at different recovery times except for Nr3c1 (gene for GR). However, exposure to stress caused significantly higher levels of GR, Nurr1 and IL-1β proteins of vulnerable compared to resilient rats. This variability of behavioral phenotypes is associated with a differential molecular response to stress that involves GR, Nurr1, and IL-1β as mediators in coping with stress. This contributes to identifying biomarkers of susceptibility to stress.

Sensitivity to behavioral stress impacts disease pathogenesis in dystrophin-deficient mice.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome.Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it.To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance.The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment.To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells.Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002).Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. DisclosuresBiondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Age-Specific Modulation of Prefrontal Cortex LTP by Glucocorticoid Receptors Following Brief Exposure to HFD

The corticolimbic circuits in general and the medial prefrontal cortex in particular, undergo maturation during juvenility. It is thus expected that environmental challenges in forms of obesogenic diet can exert different effects in juvenile animals compared to adults. Further, the relationship between glucocorticoids and obesity has also been demonstrated in several studies. As a result, glucocorticoid receptor (GR) antagonists are currently being tested as potential anti-obesity agents. In the present study, we examined the effects of short-term exposure to high-fat diet (HFD) on prefrontal long-term potentiation (LTP) in both juvenile and adult rats, and the role of glucocorticoid receptors (GRs) in modulating these effects. We found HFD impaired prefrontal LTP in both juveniles and adults, but the effects of GR modulation were age- and diet-dependent. Specifically, GR antagonist RU-486 reversed the impairment of LTP in juvenile animals following HFD, and had no effect on control-diet animals. In adult animals, RU-486 has no effect on HFD-impaired LTP, but abolished LTP in control-diet animals. Furthermore, impairments in the prefrontal LTP following HFD are involved with an increase in the mPFC GR levels only in the juveniles. Further, we found that in vivo application of GR agonists into adult mPFC rescued HFD-induced impairment in LTP, suggesting that these receptors might represent strategic therapeutic targets to potentially combat obesity and metabolic related disorder.

DMD – ANIMAL MODELS: EP.85 Oestrogen and HPA/renin-angiotensin-aldosterone pathways are associated with the behavioural sensitivity to stress in dystrophin-deficient mice

Mutation to the dystrophin gene can increase the sensitivity to behavioural stress in vertebrates, including the mdx mouse model of Duchenne muscular dystrophy. Hyper-sensitivity to behavioural stress in mdx mice can exacerbate some dystrophinopathy phenotypes and cause hypotension-induced death. However, using a large-scale screen of male and female mdx mice, we have discovered a sub-population that show wildtype-like responses to mild (forced downhill treadmill exercise) and moderate (scruff-restraint) behavioural stress. These “stress resistant” mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal (HPA) and renin-angiotensin-aldosterone (RAAS) pathways following behavioural stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to “stress-sensitive” mdx mice. At rest, “stress-resistant” mdx mice had a less severe striated muscle histopathology, including fewer central nucleated fibres, less fibrosis, greater vascular density, elevated expression levels of utrophin and greater exercise and skeletal muscle oxidative capacity. Most interestingly, female mdx mice were more “stress resistant”, as indicated by a greater level of physically activity following forced downhill treadmill exercise and scruff-restraint, relative to male mdx mice. Stress-resistance was completely abolished after ovariectomy. Replacement of oestradiol in ovariectomized female mdx mice completely rescued their “stress resistance”, again indicated by a greater physical activity response following forced downhill treadmill exercise, but not to scruff-restraint. Our data demonstrate that the differential sensitivity to behavioural stress in mdx mice is linked to oestrogen, activation of the HPA axis and the RAAS pathway and associated with striated muscle pathophysiology.

Lactobacillus paracasei Supplementation Prevents Early Life Stress-Induced Anxiety and Depressive-Like Behavior in Maternal Separation Model-Possible Involvement of Microbiota-Gut-Brain Axis in Differential Regulation of MicroRNA124a/132 and Glutamate Receptors.

This study was designed to investigate stressful social experience (SSE) in early life by examining how it can induce alterations in the microbiota-gut-brain axis. To test this, different experimental groups of pups experienced the presence of either a stranger (S) with mother (M+P+S) or without their mother (MS+S−M). Animals were assessed for anxiety-like behavior and high-throughput bacterial 16s rRNA sequencing was performed to analyze the structure of the gut microbiota. Our analysis revealed that early life SSE induced anxiety-like behavior and reduced the diversity and richness of gut microbiota. In the second experiment, all groups were supplemented with Lactobacillus paracasei HT6. The findings indicated that Lactobacillus supplementation had a significant beneficial effect on anxiety-like behavior in stressed rats (MS, M+P+S, and MS + S−M) accompanied by normalized levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), glucocorticoid receptor (GR), serotonin (5-HT), dopamine (DA), and noradrenaline (NA). Concomitantly, the expression of microRNA (miR)-124a was down-regulated and miR-132, caspase-3, glutamate receptors (GluR1, GluR 2; NR2A, and NR2B) were up-regulated in stressed groups but remained unchanged by Lactobacillus supplementation in stressed individuals. This indicates that stress-associated GluR1-GR altered interactions can be significantly prevented by Lactobacillus supplementation. Analysis of the fecal metabolite profile was undertaken to analyze the effect of Lactobacillus, revealing that five predicted neuroactive microbial metabolites were reduced by early life SSE. Our results showed a potential link between Lactobacillus supplementation and beneficial effects on anxiety-like behavior, the mechanism of which could be potentially mediated through stress hormones, neurotransmitters, and expression of miRNAs, glutamate receptors, and the microbiota-gut-brain axis.

Effect of progestin therapy on steroid receptor status and cellular proliferation in low-grade endometrial adenocarcinoma

<h3>Objectives:</h3> Low grade endometrioid endometrial cancer is a hormone sensitive disease however little is known about the role of steroid receptors in the development or treatment of disease. Our objective was to determine if steroid receptor status, or change in receptor status, predicts response to progesterone therapy in women with early stage, low grade endometrial cancer. <h3>Methods:</h3> A retrospective cohort of women was identified who underwent primary progesterone therapy for treatment of grade 1 endometrial adenocarcinoma. Steroid receptor (SR) status as well as Ki-67 expression was assessed with immunohistochemistry on pre- and post-treatment formalin fixed paraffin embedded biopsy specimens. Automated image analysis on digitized immunostained slides was performed. Estrogen Receptor (ER), Progesterone Receptor (PR), Androgen Receptor (AR) and Glucocorticoid Receptor (GR) were scored with an H-score and Ki-67 was reported as percent positive. Clinical and pathologic variables, including response to progesterone therapy were collected. SR levels and clinical characteristics were compared using paired t- and Fischer exact tests. <h3>Results:</h3> Fourteen women were included in this analysis, the majority of whom (13/14) had a progestin IUD for treatment of their carcinoma and the majority (9/14) were non-surgical candidates due to medical co-morbidities. Eight women had resolution of adenocarcinoma and six women did not have a response to therapy. Median treatment duration was 4.8 months (range 3.3 – 9.3). Responders were more likely to be younger and pre-menopausal. Two patients had mismatch repair deficient tumors, neither responded to progestin therapy. Both ER and PR declined significantly after progestin treatment (median H-score pre vs post for ER 183 vs 104, p=0.013; PR 110 vs 40, p<.001). GR levels increased (23 vs 47, p=0.003). AR demonstrated variable response. Neither pre-treatment, post-treatment, nor change in SR levels were associated with response to progestin therapy. Women with a response to therapy had lower Ki-67 levels prior to treatment compared to women who did not respond to therapy (26% vs 39%, p=0.033). <h3>Conclusions:</h3> Both ER and PR levels declined during progestin treatment in primary low grade endometrial adenocarcinoma suggesting a negative feedback loop from PR to ER signaling at the level of protein expression. Neither pre-treatment SR levels, nor change in SR receptor status predicted a response to progestin therapy which suggests more complex context-specific interactions of SRs and their ligands. Pre-treatment cellular proliferation as a biomarker for response to hormonal therapy warrants further study.

Effect of m-Trifluoromethyl-diphenyl diselenide on the Pain-Depression Dyad Induced by Reserpine: Insights on Oxidative Stress, Apoptotic, and Glucocorticoid Receptor Modulation.

Chronic pain and depression often coexist sharing common pathological mechanisms, and available analgesics and antidepressants have demonstrated limited clinical efficacy. Evidence has demonstrated that neuronal oxidative stress, apoptosis, and also glucocorticoid receptor dysregulation facilitate the occurrence and development of both chronic pain and depression. This study evaluated the effect of the organoselenium compound m-trifluoromethyl-diphenyl diselenide [(m-CF3-PhSe)2] in the pain-depression comorbidity induced by reserpine. Mice were treated with reserpine 0.5mg/kg for 3days (intraperitoneal, once a day), and in the next 2days, they were treated with (m-CF3-PhSe)2 10mg/kg (intragastric, once a day). Thirty minutes after the last administration of (m-CF3-PhSe)2, mice were subjected to the behavioral testing. (m-CF3-PhSe)2 treatment reverted the reserpine-increased thermal hyperalgesia and depressive-like behavior observed in the hot-plate test and forced swimming test, respectively. Reserpine provoked a decrease of crossings and rearings in the open-field test, while (m-CF3-PhSe)2 presented a tendency to normalize these parameters. Reserpine and/or (m-CF3-PhSe)2 treatments did not alter the locomotor activity of mice observed in the rota-rod test. These effects could be related to modulation of oxidative stress, apoptotic pathway, and glucocorticoid receptors, once (m-CF3-PhSe)2 normalized thiobarbituric acid reactive substances and 4-hydroxynonenal modified protein levels, markers of lipoperoxidation, poly(ADP-ribose) polymerase cleaved/total ratio, and glucocorticoid receptor levels increased by reserpine in the hippocampus. Considering that pain-depression dyad is a complex state of difficult treatment, this organoselenium compound could raise as an interesting alternative to treat pain-depression condition.

Abstract 1420: GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines

Abstract Androgen deprivation therapy (ADT) with or without antiandrogens such as enzalutamide is the mainstay of treatment in advanced prostate cancer (PC). However, new therapeutic strategies are needed for patients who relapse on antiandrogen therapy. The glucocorticoid receptor (GR) has been implicated as a potential antiandrogen bypass mechanism. We previously showed that ORIC-101, a potent and selective, orally bioavailable small molecule GR antagonist, reverses GR-mediated resistance to enzalutamide in preclinical PC models. In the clinic, we are evaluating ORIC-101 in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Activation of the PI3K/AKT pathway through PTEN loss is a poor prognostic factor and another potential resistance mechanism to anti-hormone directed therapies. Reciprocal crosstalk between androgen signaling and PI3K/AKT pathway activation forms the basis for the clinical development of AKT inhibitors (AKTi) in combination with ADT or antiandrogens (NCT03072238, NCT04087174). In this study, we tested preclinically whether activated GR confers resistance to the combination of AKT inhibitors with enzalutamide and whether co-treatment with ORIC-101 reverses GR-mediated resistance to an enzalutamide/AKTi doublet. We first evaluated the effects of enzalutamide plus AKTi treatment on GR levels, GR target genes and activity in three prostate cancer cell lines: PTEN-null line LNCaP and PTEN-wildtype lines VCaP and CWR22PC. We tested three AKT inhibitors: the ATP-competitive inhibitors ipatasertib (GDC-0068) and capivasertib (AZD5363), in addition to the allosteric inhibitor MK2206, in CSS media and 10% FBS, with or without supplementation of the synthetic glucocorticoid, dexamethasone. An increase in the protein and mRNA levels of GR and GR target genes was observed after 7 days of enzalutamide treatment. In the AKTi plus enzalutamide setting, AKT inhibition did not block the enzalutamide-mediated GR upregulation in either PTEN-null or PTEN-wildtype cells. We next evaluated whether increased GR activity mediates resistance to the enzalutamide/AKTi doublet with ipatasertib or capivasertib, and whether this resistance can be reversed with ORIC-101. GR activation with dexamethasone promoted tumor cell growth and androgen-regulated gene expression in the presence of enzalutamide and ipatasertib or capivasertib, using a 21-day proliferation assay in CWR22PC cells. ORIC-101 was able to reverse these dexamethasone-induced effects, confirmed by reduced secreted PSA levels and cell number measured at end of study. These findings indicate that GR upregulation and activation, an established resistance mechanism for antiandrogens, may drive resistance when combined with an AKT inhibitor, and the GR antagonist ORIC-101 is able to overcome this resistance and restore antitumor activity. Citation Format: Shravani Barkund, Haiying Zhou, Lori S. Friedman, Anneleen Daemen. GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1420.

Haloperidol and aripiprazole impact on the BDNF and glucocorticoid receptor levels in the rat hippocampus and prefrontal cortex: effect of the chronic mild stress

Objective. Changes in the brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) expression in the prefrontal cortex (PFC) and hippocampus (HIP) are associated with psychiatric diseases and stress response. Chronic mild stress (CMS) may alter BDNF as well as GR levels in both the PFC and the HIP. The aim of the present study was to find out whether chronic treatment with a typical antipsychotic haloperidol (HAL) and an atypical antipsychotic aripiprazole (ARI) may modify the CMS effect on the BDNF and GR expression in the above-mentioned structures. Methods. The rats were exposed to CMS for 3 weeks and from the 7th day of CMS injected with vehicle (VEH), HAL (1 mg/kg) or ARI (10 mg/kg) for 4 weeks. BDNF and GR mRNA levels were established in the PFC and the HIP by Real Time PCR, whereas, PFC and HIP samples were obtained by punching them from 500 µm thick frozen sections. C-Fos immunoreactivity was analyzed in the PFC and the HIP on 30 µm thick paraformaldehyde fixed sections. Weight gain and corticosterone (CORT) levels were also measured. Results. The CMS and HAL suppressed the BDNF and GR mRNA levels in the PFC. In the HIP, CMS elevated BDNF mRNA levels that were suppressed by HAL and ARI treatments. The CMS decreased the c-Fos immunoreactivity in the PFC in both HAL- and ARI-treated animals. In the HIP, HAL increased the c-Fos immunoreactivity that was again diminished in animals exposed to CMS. Stressed animals gained markedly less weight until the 7th day of CMS, however, later their weight gain did not differ from the unstressed ones or was even higher in CMS+HAL group. Un-stressed HAL and ARI animals gained less weight than the VEH ones. Neither CMS nor HAL/ARI affected the plasma CORT levels. Conclusion. The present data indicate that HAL and ARI in the doses 1 mg/kg or 10 mg/kg, respectively, does not modify the effect of the CMS preconditioning on the BDNF and GR mRNA levels in the PFC or the HIP. However, HAL seems to modify the CMS effect on the HIP activation.

Effects of dietary Astragalus Propinquus Schischkin polysaccharides on growth performance, immunological parameters, antioxidants responses and inflammation-related gene expression in Channa argus

The present study investigated the effects of dietary Astragalus Propinquus schischkin polysaccharides on growth, immune responses, antioxidants responses and inflammation-related genes expression in Channa argus. Channa argus were randomly divided into 5 groups and fed 5 levels diets of A. propinquus schischkin polysaccharides (0, 250, 500, 1000 and 2000 mg/kg) for 56 days. The results showed, dietary A. propinquus schischkin polysaccharides addition can increase the final body weight, weight gain and specific growth rate, decrease the feed conversion ratio of Channa argus. And dietary A. propinquus schischkin polysaccharides supplementation can increase the levels of serum superoxide dismutase, catalase, glutathione peroxidase, lysozyme, complement 3, complement 4, immunoglobulin M and alkaline phosphatase, decrease the levels of serum malondialdehyde, cortisol, aspartate aminotransferase and glutamic-pyruvic transaminase. Furthermore, dietary A. propinquus schischkin polysaccharides can decrease the gene expression levels of interleukin-1β, interleukin-, tumor necrosis factor-α and nuclear factor-κB, increase the gene expression levels of glucocorticoid receptor in liver, spleen, kidney, intestine. To sum up, dietary A. propinquus schischkin polysaccharides can accelerate growth, enhance immune responses and antioxidants responses, regulate inflammation-related genes expression in Channa argus and the optimum amount is 1000 mg/kg.

ALKBH5-mediated m6A-demethylation of USP1 regulated T-cell acute lymphoblastic leukemia cell glucocorticoid resistance by Aurora B.

Recent studies evidence that ubiquitin-specific proteases (USPs) are associated with the occurrence and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL). N6 -methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) exerts a carcinogenic effect in human cancers and improves the mRNA stability of USPs. Whether ubiquitin-specific protease 1 (USP1) controls chemoresistance of T-ALL is unknown. Our study demonstrated that USP1 expression was upregulated in glucocorticoid (GC)-resistant T-ALL patients and cells (CEM-C1). High expression of USP1 was correlated to the poor prognosis in T-ALL patients. Silencing USP1 increased CEM-C1 cell sensitivity to dexamethasone (Dex), reduced cell invasion, promoted cell apoptosis, and ameliorated glucocorticoid receptor (GR) expression. USP1 mediated T-ALL chemoresistance by interacting with and deubiquitination of Aurora B. Overexpression of USP1 reversed the amelioration effect of Aurora B inhibitor on CEM-C1 cell resistance to Dex. Mechanistically, ALKBH5 enhanced USP1 expression by reducing m6A level and mRNA stability in USP1 mRNA transcript. Downregulation of ALKBH5 reduced the levels of USP1 and Aurora B, facilitated CEM-C1 cell sensitivity to Dex, apoptosis, and GR expression, suppressed cell invasion. However, overexpression of USP1 reversed all the effects of ALKBH5 on CEM-C1 cells. In vivo results showed that tail vein injection of sh-USP1 resulted in a significant prolongation of mouse survival, suppressed tumor growth, maintained the normal weight of mice, reduced USP1 expression and facilitated GR expression. In conclusion, inhibition of ALKBH5-mediated m6A modification decreased USP1 expression and downregulation of USP1 ameliorated GC resistance of T-ALL through suppressing Aurora B expression and elevating GR level.

Effect of dexamethasone on gene expression of cannabinoid receptor type 1 and adenosine monophosphate-activated protein kinase in the hypothalamus of broilers (Gallus domesticus)

Hypothalamic neural circuits play a critical role in integrating peripheral signals and conveying information about energy and nutrient status. We detected cannabinoid receptor type 1 (CB1) distribution in the hypothalamus, liver, duodenum, jejunum, and ileum among 7- and 35-day-old broilers. The effects of dexamethasone (DEX) on CB1 gene expression were evaluated by in vitro and in vivo experiments on glucocorticoid receptor (GR) and adenosine monophosphate-activated protein kinase (AMPK) in the hypothalamus of broilers. In vitro, hypothalamic cells from 17-day-old broiler embryos were incubated with either 0.1% dimethyl sulfoxide or DEX (100 nmol/mL) for 1 h. In the in vivo study, 28-day-old broilers were injected with DEX for 24 h or 72 h. Results showed that CB1 was mainly expressed in the hypothalamus, and 72 h DEX treatment increased the expression. One-day treatment of broilers with DEX did not change the hypothalamic CB1 gene expression. Moreover, DEX treatment for 24 h and 72 h increased the mRNA level of hypothalamic AMPKα2 and GR. However, no differences were observed on the gene expression of CB1, GR, and AMPKα2 in hypothalamic cells with DEX-treated for 1 h. In conclusion, CB1 is mainly expressed in the hypothalamus of broilers; 72-h DEX exposure can regulate the CB1 system and AMPK signaling pathway of the broiler hypothalamus.

Anti-stress effects of combined block of glucocorticoid and mineralocorticoid receptors in the paraventricular nucleus of the hypothalamus.

Mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs) and corticotropin-releasing factor (CRF) in the paraventricular nucleus of hypothalamus (PVN) are involved in the response to stress. The present study investigated the role of GRs and MRs in the PVN in regulating depressive and anxiety-like behaviours. To model chronic stress, rats were exposed to corticosterone treatment via drinking water for 21 days, and GR antagonist RU486 and MR antagonist spironolactone, alone and combined, were directly injected in the PVN daily for the last 7 days of corticosterone treatment. Behavioural tests were run on days 22 and 23. Depressive- and anxiety-like behaviours were evaluated in forced swim test, sucrose preference test, novelty-suppressed feeding test and social interaction test. The expression of GRs, MRs and CRF were detected by western blot. Rats exposed to corticosterone exhibited depressive- and anxiety-like behaviours. The expression of GRs and MRs decreased, and CRF levels increased in the PVN. The intra-PVN administration of RU486 increased the levels of GRs and CRF without influencing depressive- or anxiety-like behaviours. The spironolactone-treated group exhibited an increase in MRs without influencing GRs and CRF in the PVN and improved anxiety-like behaviours. Interestingly, the intra-PVN administration of RU486 and spironolactone combined restored expression of GRs, MRs and CRF and improved depressive- and anxiety-like behaviours. In this rat model of stress, the simultaneous restoration of GRs, MRs and CRF in the PVN might play an important role in the treatment of depression and anxiety.

Small leucine zipper protein promotes the metastasis of castration-resistant prostate cancer through transcriptional regulation of matrix metalloproteinase-13.

Matrix metalloproteinases (MMPs) function as central modulators of tissue remodeling. Abnormal expression and altered activity of MMPs result in excessive extracellular matrix degradation and increased tumor metastasis in various cancers. Small leucine zipper protein (sLZIP), belonging to the leucine zipper transcription factor family, functions as a transcriptional regulator of genes involved in various cellular processes. However, its role in MMP expression and castration-resistant prostate cancer (CRPC) metastasis remains unclear. In this study, we investigated the role of sLZIP in MMP-13 expression and its involvement in CRPC metastasis. sLZIP increased MMP-13 transcription by directly binding to its promoter in CRPC cells. We found that the expression levels of glucocorticoid receptor (GR), which represses MMP transcription, were elevated in CRPC cells. However, sLZIP suppressed the inhibitory effect of GR and enhanced the secretion of MMP-13 in CRPC cells. sLZIP promoted cell migration and invasion; however, a specific MMP-13 inhibitor blocked sLZIP-induced cell motility. Depletion of sLZIP using the CRISPR/Cas9 system downregulated MMP-13 messenger RNA expression in PC3 cells. Mice injected with sLZIP-depleted PC3 cells showed significantly reduced metastatic tumor volume in the lung compared with mice injected with control PC3 cells. Our findings suggest that sLZIP plays an important role in MMP-13 induction and CRPC metastasis. Therefore, sLZIP inhibition could be a novel therapeutic strategy for metastatic GR-enriched CRPC.