Endogenous Glucocorticoid Receptor Research Articles

8644 Dynamic Insights into Glucocorticoid Receptor Dysfunction: Novel patient-derived T437I Mutation and Its Impact on Glucocorticoid Receptor Function

Abstract Disclosure: T. Tettey: None. S. Kim: None. K. Wagh: None. Q. Lao: None. L.R. Schiltz: None. D.A. Stavreva: None. D.M. Presman: None. D.P. Merke: None. G.L. Hager: None. The glucocorticoid receptor (GR) is a hormone regulated transcription factor which binds chromatin to regulate many genes, including stress-response and inflammatory genes. Dexamethasone, a GR agonist, is widely used in the treatment of severe inflammation, including COVID-19. GR is vital for life and several GR mutations have been linked to severe pathophysiological conditions. Here we report a novel mutation in the DNA binding domain (DBD) of GR (T437I) in a patient with partial glucocorticoid resistance, a rare genetic disorder characterized by generalized insensitivity to glucocorticoids and a consequent hyperactivation of the hypothalamic pituitary adrenal axis. While the clinical manifestations of this hT437I GR mutation are apparent, its molecular mechanisms remain unexplored. Employing a comprehensive approach that integrates biophysical methods, molecular biology, and genomics, we investigate the molecular underpinnings of this genetic anomaly. Using CRISPR/Cas9 gene editing, we introduce GFP or Halo-tagged hT437I GR mutant into GR null mouse cells. Our findings indicate that the T437I GR variant exhibits normal hormone binding and nuclear translocation. Live-cell imaging using number and brightness analysis reveals that the mutant receptor maintains a wildtype-like oligomeric state but adopts a heterodimeric conformation in the presence of endogenous GR. Global transcriptomics analyses of the mouse hT437I mutant show that most dexamethasone-regulated genes are unresponsive following hormone treatment. We hypothesize that this mutation within the DBD compromises the binding of GR to regulatory sites of target genes. Leveraging fluorescence high-resolution microscopy, we employ single molecule tracking to study the dynamic behavior of Halo-tagged hT437I GR in mouse cells. Our results indicate that the GR mutant has a higher dwell-time on chromatin compared to the wild-type receptor. This suggests that the hT437I mutation impairs the receptor's binding to genomic sites, potentially leading to a failure in regulating GR target genes. This ongoing exploration of the molecular intricacies aims to deepen our understanding of the clinical phenotype observed in the patient. Presentation: 6/2/2024

OR33-02 The Effects Of Timing And Patterns Of Hormone Stimulation On Single Molecule Dynamics And Gene Regulation In Vivo

Abstract Disclosure: D.A. Stavreva: None. L. Hoang: None. K. Wagh: None. R. Chari: None. A. Upadhyaya: None. G.L. Hager: None. Even though most studies on hormonal signaling are performed under continuous stimulation, secretion of many hormones follows a circadian pattern, comprised of periodic (ultradian) pulses with short duration. Using glucocorticoid receptor (GR) signaling as a model system we have previously demonstrated that receptor occupancy of regulatory elements precisely tracks the hormonal pulses resulting in cyclic GR-mediated transcription (gene pulsing) (Stavreva et al., NCB 2009). Using various genomic approaches, we have also uncovered the impact of ultradian and constant stimulation on the lifetimes of chromatin accessibility and long-range promoter/enhancer interactions (Stavreva et al., Genome Res. 2015). By visualizing RNA synthesis from single-copy GR-regulated transcription sites (TSs) we demonstrated treatment-specific RNA bursting patterns. Moreover, our single molecule tracking (SMT) experiments showed that transcription factor (TF) dynamics determine burst duration and frequency (Stavreva et al., 2019). To further study the in vivo dynamics of various TFs we tagged the endogenous GR as well as the estrogen, progesterone receptor (ER and PR, respectively), and the large Pol II subunit (Rpb1) with Halo tag in MCF7 cells. We use SMT in combination with other microscopy and biochemical approaches to i) map the dynamics of TFs in response to constant and ultradian hormone stimulations, and ii) determine their effects on transcription regulation. These studies will help us better understand the importance of timing and pattern of hormone treatment for proper gene regulation in different physiological contexts. Presentation: Sunday, June 18, 2023

The glucocorticoid receptor modulator relacorilant reverses the immunosuppressive effects of cortisol

Cortisol, an endogenous glucocorticoid receptor (GR) agonist, controls a broad transcriptional program that affects T-cell activation, pro-inflammatory cytokine secretion, apoptosis, and immune-cell trafficking. The degree to which endogenous cortisol blunts the anti-tumor immune response checkpoint inhibitors stimulate had not been assessed. We addressed this question using relacorilant, a selective GR modulator (SGRM) that competitively antagonizes the effects of cortisol activity. GR expression in human tumor and immune cells positively correlated with PD-L1 expression and tumor infiltration of Th2 and Treg cells, and negatively correlated with Th1-cell infiltration. In vitro, cortisol inhibited, and relacorilant restored, T-cell activation and pro-inflammatory cytokine secretion in human peripheral blood mononuclear cells. In the ovalbumin-expressing EG7 and MC38 immune-competent tumor models, relacorilant significantly improved anti-PD-1 antibody efficacy and showed favorable effects on antigen-specific T-cells and systemic TNFα and IL-10. These data characterize the broad immunosuppressive effects of endogenous cortisol and highlight the potential of combining an SGRM with an immune checkpoint inhibitor.

A Novel Model Using AAV9-Cre to Knockout Adult Leydig Cell Gene Expression Reveals a Physiological Role of Glucocorticoid Receptor Signalling in Leydig Cell Function

Glucocorticoids are steroids involved in key physiological processes such as development, metabolism, inflammatory and stress responses and are mostly used exogenously as medications to treat various inflammation-based conditions. They act via the glucocorticoid receptor (GR) expressed in most cells. Exogenous glucocorticoids can negatively impact the function of the Leydig cells in the testis, leading to decreased androgen production. However, endogenous glucocorticoids are produced by the adrenal and within the testis, but whether their action on GR in Leydig cells regulates steroidogenesis is unknown. This study aimed to define the role of endogenous GR signalling in adult Leydig cells. We developed and compared two models; an inducible Cre transgene driven by expression of the Cyp17a1 steroidogenic gene (Cyp17-iCre) that depletes GR during development and a viral vector-driven Cre (AAV9-Cre) to deplete GR in adulthood. The delivery of AAV9-Cre ablated GR in adult mouse Leydig cells depleted Leydig cell GR more efficiently than the Cyp17-iCre model. Importantly, adult depletion of GR in Leydig cells caused reduced expression of luteinising hormone receptor (Lhcgr) and of steroidogenic enzymes required for normal androgen production. These findings reveal that Leydig cell GR signalling plays a physiological role in the testis and highlight that a normal balance of glucocorticoid activity in the testis is important for steroidogenesis.

Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets.

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

Genetic deletion of the glucocorticoid receptor in Cx3cr1+ myeloid cells is neuroprotective and improves motor recovery after spinal cord injury

Glucocorticoid receptors (GRs), part of the nuclear receptor superfamily of transcription factors (TFs), are ubiquitously expressed in all cell types and regulate cellular responses to glucocorticoids (e.g., cortisol in humans; corticosterone in rodents). In myeloid cells, glucocorticoids binding to GRs can enhance or repress gene transcription, thereby imparting distinct and context-dependent functions in macrophages at sites of inflammation. In experimental models and in humans, glucocorticoids are widely used as anti-inflammatory treatments to promote recovery of function after SCI. Thus, we predicted that deleting GR in mouse myeloid lineage cells (i.e., microglia and monocyte-derived macrophages) would enhance inflammation at the site of injury and worsen functional recovery after traumatic spinal cord injury (SCI). Contrary to our prediction, the intraspinal macrophage response to a moderate (75 kdyne) spinal contusion SCI was reduced in Cx3cr1-Cre;GRf/f conditional knockout mice (with GR specifically deleted in myeloid cells). This phenotype was associated with improvements in hindlimb motor recovery, myelin sparing, axon sparing/regeneration, and microvascular protection/plasticity relative to SCI mice with normal myeloid cell GR expression. Further analysis revealed that macrophage GR deletion impaired lipid and myelin phagocytosis and foamy macrophage formation. Together, these data reveal endogenous GR signaling as a key pathway that normally inhibits mechanisms of macrophage-mediated repair after SCI.

Class 3 phosphoinositide 3-kinase promotes hepatic glucocorticoid receptor stability and transcriptional activity.

AimLipid kinase class 3 phosphoinositide 3‐kinase (PI3K) and nuclear receptor transcription factor glucocorticoid receptor (GR) play essential physiological roles in metabolic adaptation to fasting by activating lysosomal degradation by autophagy and metabolic gene expression, yet their functional interaction is unknown. The requirement of class 3 PI3K for GR function was investigated in liver tissue.MethodsInactivation of class 3 PI3K was achieved through deletion of its essential regulatory subunit Vps15, by expressing Cre‐recombinase in the livers of Vps15f/f mice. The response to both 24‐h fasting and synthetic GR ligand, dexamethasone (DEX) was evaluated in control and mutant mice. Liver tissue was analysed by immunoblot, RT‐qPCR, and LC‐MS.ResultsVps15 mutant mice show decreased transcript levels of GR targets, coupled with lower nuclear levels of total and phosphorylated on Ser211, GR protein. Acute DEX treatment and 24‐h fasting both failed to re‐activate expression of GR targets in the livers of Vps15 mutant mice to the levels observed in controls. Decreased levels of endogenous GR ligand corticosterone and lower expression of 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1), a metabolic enzyme that controls corticosterone availability, were found in the livers of Vps15 mutants. Hepatic Vps15 depletion resulted in the activation of nuclear Akt1 signalling, which was paralleled by increased polyubiquitination of GR.ConclusionIn the liver, class 3 PI3K is required for corticosterone metabolism and GR transcriptional activity.

Glucocorticoid Mediated Transcriptional Activity in Human Corneal Epithelial Cells Lacking the Glucocorticoid Receptor

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

Impact of relacorilant, a selective glucocorticoid receptor antagonist, on the immunosuppressive effects of endogenous cortisol.

3091 Background: Cortisol, an endogenous glucocorticoid receptor (GR) agonist, controls a broad transcriptional program that affects T-cell activation, pro-inflammatory cytokine secretion, and immune cell trafficking. By selectively antagonizing GR, relacorilant may reverse the immunosuppressive effects of cortisol in solid tumor cancers. Methods: Immune cells and GR expression were assessed by IHC and calculated based on The Cancer Genome Atlas (TCGA) data. Human PBMCs were stimulated with αCD3+IL-12 +/- cortisol or cortisol + relacorilant. EG7 tumor-bearing mice were treated with anti-PD1 (RMP1-14) ip Q5D +/- relacorilant QD. Whole blood mRNA was measured via Nanostring, hematology was performed using standard complete blood count assays, and cytokines were assessed by immunoassays in study NCT02762981. Results: GR expression was observed in human tumor and immune cells. Its abundance was positively correlated with tumor infiltration of TH2, Treg, and PDL1+ cells ( P< .001) and negatively correlated with TH1 cells ( P< .001). In PBMCs, cortisol inhibited, and relacorilant restored, CD8+ T-cell activation ( P< .001) and pro-inflammatory cytokine secretion (TNFα P= .006, IFNγ P< .05). In the EG7 syngeneic model, relacorilant increased αPD1 efficacy ( P= .007) and decreased circulating IL-10 ( P< .002). In patients with advanced solid tumors, relacorilant + nab-paclitaxel systemically suppressed the expression of canonical GR-controlled genes ( ptgs2 P< .001) and genes encoding candidate-immunomodulatory drug targets ( cxcl8, ptger4, ido1; P< .001). In a small subset of patients (n = 11), sustained clinical response was associated with increased T-cell count ( P= .06) and IFNγ ( P= .03), as well as decreased Tregs ( P= .06) and IL-10 ( P= .03). Conclusions: Evidence of T-cell activation by relacorilant was observed in PBMCs, syngeneic mouse tumors, and patients with sustained response in a Phase 1 study. This supports the hypothesis that relacorilant can reverse immune suppression by endogenous cortisol in solid tumor cancers. Clinical studies with immune checkpoint inhibitors and relacorilant are planned.

Using Chromatin-Nuclear Receptor Interactions to Quantitate Endocrine, Paracrine, and Autocrine Signaling.

Hormone-activated nuclear receptors (NRs) control myriad cellular processes. The classical paradigm for hormone delivery is secretion from endocrine organs and blood-borne distribution to responding cells. However, many hormones can also be synthesized in the same tissues in which responding cells are found (paracrine signaling). In both endocrine and paracrine signaling, numerous factors affect hormone availability to target cell NRs, including hormone access to and sequestration by carrier proteins, transport across cell membranes, metabolism, and receptor availability. These factors can differ dramatically during development, between anatomical locations, and across cell types, and may cause highly variable responses to the same hormone signal. This has been difficult to study because current approaches are unable to quantify cell-intrinsic exposure to NR hormone ligands, precluding assessment of cell-specific hormone access and signaling. We have used the ligand-dependent interaction of the endogenous glucocorticoid (GC) receptor with chromatin as a biosensor that quantifies systemic access of GCs to cells within tissues at the single cell level, showing that tissues are buffered against circulating GCs. This approach also showed highly targeted paracrine GC signaling within the thymus, where GCs promote the positive selection of thymocytes with moderate affinity for self-antigens and the development of a safe and effective T-cell repertoire. We believe that this and complementary biosensor approaches will be useful to identify endocrine and paracrine target cells in situ and quantify their exposure to hormones regardless of the mode of delivery.

PlexinB1 Promotes Nuclear Translocation of the Glucocorticoid Receptor.

Androgen receptor (AR) and glucocorticoid receptor (GR) are nuclear receptors whose function depends on their entry into the nucleus where they activate transcription of an overlapping set of genes. Both AR and GR have a role in resistance to androgen deprivation therapy (ADT), the mainstay of treatment for late stage prostate cancer. PlexinB1, a receptor for semaphorins, has been implicated in various cancers including prostate cancer and has a role in resistance to ADT. We show here that activation of PlexinB1 by Sema4D and Sema3C results in translocation of endogenous GR to the nucleus in prostate cancer cells, and that this effect is dependent on PlexinB1 expression. Sema4D/Sema3C promotes the translocation of GR-GFP to the nucleus and mutation of the nuclear localization sequence (NLS1) of GR abrogates this response. These findings implicate the importin α/β system in the Sema4D/Sema3C-mediated nuclear import of GR. Knockdown of PlexinB1 in prostate cancer cells decreases the levels of glucocorticoid-responsive gene products and antagonizes the decrease in cell motility and cell area of prostate cancer cells upon dexamethasone treatment, demonstrating the functional significance of these findings. These results show that PlexinB1 activation has a role in the trafficking and activation of the nuclear receptor GR and thus may have a role in resistance to androgen deprivation therapy in late stage prostate cancer.

The Selective Progesterone Receptor Modulator Ulipristal Acetate Inhibits the Activity of the Glucocorticoid Receptor.

The selective progesterone modulator ulipristal acetate (ulipristal) offers a much-needed therapeutic option for the clinical management of uterine fibroids. Although ulipristal initially passed safety evaluations in Europe, postmarketing analysis identified cases of hepatic injury and failure, leading to restrictions on the long-term use of ulipristal. One of the factors potentially contributing to significant side effects with the selective progesterone modulators is cross-reactivity with other steroid receptors. To determine whether ulipristal can alter the activity of the endogenous glucocorticoid receptor (GR) in relevant cell types. Immortalized human uterine fibroid cells (UtLM) and hepatocytes (HepG2) were treated with the synthetic glucocorticoid dexamethasone and/or ulipristal. Primary uterine fibroid tissue was isolated from patients undergoing elective gynecological surgery and treated ex vivo with dexamethasone and/or ulipristal. In vivo ulipristal exposure was performed in C57Bl/6 mice to measure the effect on basal gene expression in target tissues throughout the body. Dexamethasone induced the expression of established glucocorticoid-target genes period 1 (PER1), FK506 binding protein 51 (FKBP5), and glucocorticoid-induced leucine zipper (GILZ) in UtLM and HepG2 cells, whereas cotreatment with ulipristal blocked the transcriptional response to glucocorticoids in a dose-dependent manner. Ulipristal inhibited glucocorticoid-mediated phosphorylation, nuclear translocation, and DNA interactions of GR. Glucocorticoid stimulation of PER1, FKBP5, and GILZ was abolished by cotreatment with ulipristal in primary uterine fibroid tissue. The expression of glucocorticoid-responsive genes was decreased in the lung, liver, and uterus of mice exposed to 2 mg/kg ulipristal. Interestingly, transcript levels of Fkbp5 and Gilz were increased in the hippocampus and pituitary. These studies demonstrate that ulipristal inhibits endogenous glucocorticoid signaling in human fibroid and liver cells, which is an important consideration for its use as a long-term therapeutic agent.

Repression of the Glucocorticoid Receptor Increases Hypoxic-Ischemic Brain Injury in the Male Neonatal Rat

Hypoxic-ischemic encephalopathy (HIE) resulting from asphyxia is the most common cause of neonatal brain damage and results in significant neurological sequelae, including cerebral palsy. The current therapeutic interventions are extremely limited in improving neonatal outcomes. The present study tests the hypothesis that the suppression of endogenous glucocorticoid receptors (GRs) in the brain increases hypoxic-ischemic (HI) induced neonatal brain injury and worsens neurobehavioral outcomes through the promotion of increased inflammation. A mild HI treatment of P9 rat pups with ligation of the right common carotid artery followed by the treatment of 8% O2 for 60 min produced more significant brain injury with larger infarct size in female than male pups. Intracerebroventricular injection of GR siRNAs significantly reduced GR protein and mRNA abundance in the neonatal brain. Knockdown of endogenous brain GRs significantly increased brain infarct size after HI injury in male, but not female, rat pups. Moreover, GR repression resulted in a significant increase in inflammatory cytokines TNF-α and IL-10 at 6 h after HI injury in male pups. Male pups treated with GR siRNAs showed a significantly worsened reflex response and exhibited significant gait disturbances. The present study demonstrates that endogenous brain GRs play an important role in protecting the neonatal brain from HI induced injury in male pups, and suggests a potential role of glucocorticoids in sex differential treatment of HIE in the neonate.

Abstract LB-260: Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in U.S. men, disproportionately affecting African American men and certain Hispanic/Latino populations. Patients with advanced PCa are treated with androgen deprivation therapy (ADT) and docetaxel (DTX)-based chemotherapy with effective but transient results due to therapy resistance. While the role of glucocorticoid receptor (GR) in promoting ADT resistance has been well characterized, comparatively little is known about its contribution to PCa stemness and chemoresistance. Our group demonstrated recently that DTX-resistance is associated with upregulation of a stemness transcriptomic program and enhanced cancer stem cell (CSC) properties in PCa cells. We also demonstrated that GR signaling upregulates the DTX-resistance associated proteins Clusterin and LEDGF/p75 in a panel of PCa cells. The proto-oncogene β-catenin promotes CSC survival in many cancers, including PCa, and several studies have demonstrated a crosstalk between AR and β-catenin signaling pathways contributing to ADT resistance. Given the structural similarities between AR and GR and the importance of GR in therapy resistance, this study was designed to investigate the interaction between GR and β-catenin and its contribution to CSC survival and DTX resistance. Using a panel of PCa cell lines including PC3, DU145, 22rv1, and their corresponding DTX-resistant cell lines, we showed by quantitative immunoblotting that GR is upregulated and β-catenin is activated in PC3 and 22rv1 DTX-resistant cells compared to sensitive cells. Co-immunoprecipitation studies revealed interaction between endogenous GR and β-catenin in these cells. We next assessed the effect of the potent GR ligand dexamethasone (Dex) on CSC maintenance, and observed that Dex treatment of PC3 cells increased tumorsphere number and expression of established CSC markers measured by multicolor flow cytometry. To identify potential GR target genes that could mediate the acquisition of CSC properties and DTX resistance, we performed RNA-sequencing analysis in four different Dex-treated and -untreated PCa cell lines. This analysis revealed several candidate GR target genes differentially regulated in a cell-type dependent manner. To determine the effects of the GR/β-catenin interaction on downstream GR target genes and β-catenin target genes, we performed immunoblotting of specific candidate target genes on lysates from cells treated with Dex alone or in combination with COR-10829 (selective GR inhibitor, and MSAB (β-catenin inhibitor). Interestingly, we also observed that the dual blockade of GR and β-catenin markedly reduced tumorsphere formation in DTX-resistant cells, a phenomenon not observed with either GR or β-catenin inhibition alone. In summary, our findings revealed a novel interaction between GR and β-catenin in PCa cells, and suggest that co-targeting the GR/β-catenin signaling axis in advanced PCa patients could be a potential strategy to overcome therapy resistance. Citation Format: Shannalee R. Martinez, Carlos J. Diaz Osterman, Xin S. Chen, Charles Wang, Lubo Zhang, Carlos A. Casiano. Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-260.

Abstract 1016: The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade

Abstract Expression of the constitutively active androgen receptor splice variant AR-V7 is associated with resistance to anti-androgen therapies and worse clinical outcomes in prostate cancer. Dimerization is a requirement for AR-V7 transcriptional activity, but whether that occurs through homodimerization or heterodimerization remains poorly understood. Because NR3C-family nuclear hormone receptors rely on a highly conserved D-box motif within the DNA-binding domain to mediate dimerization, we hypothesized that AR-V7 can heterodimerize with other NR3C-family nuclear hormone receptors, with a particular interest in the glucocorticoid receptor (GR). Like AR-V7, GR is upregulated in prostate cancer following androgen blockade, regulates canonical AR target genes, and is associated with resistance to anti-androgen therapies. Using NanoBRET technology in HEK293 cells, we found that AR-V7 interacts with liganded NR3C-family nuclear hormone receptors, including the full-length androgen receptor, the progesterone receptor, the mineralocorticoid receptor, and, most notably, GR. Using co-immunoprecipitation and proximity ligation assays, we demonstrate that AR-V7 associates with GR following androgen blockade in both the VCaP cell line and the CWR-22RV1 cell line, two models of prostate cancer that express endogenous AR-V7 and GR. Preliminary ChIP-Seq experiments in these cell lines suggest AR-V7 and GR chromatin occupancy at the same genomic sites in a ligand-dependent manner. Taken altogether, this data suggests AR-V7 can utilize GR as an alternative binding partner to initiate heterodimerization and transcriptional activation. Citation Format: Eric Bueter, David Hosfield, Erin McAuley, Phillip Selman, Geoffrey Greene, Suzanne D. Conzen, Russell Z. Szmulewitz. The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1016.

SUN-019 The Selective Progesterone Receptor Modulator Ulipristal Acetate Blocks Glucocorticoid Receptor Transactivation

Progesterone is a crucial regulator of the female reproductive system, but also contributes to the pathogenesis of certain gynecological diseases. As such, selective progesterone receptor modulators (SPRMs) have shown potential for the treatment of such diseases. The SPRM Ulipristal acetate (UPA) offers a much-needed therapeutic option for the clinical management of uterine fibroids. Although UPA initially passed safety evaluations in Europe, post-marketing analysis identified cases of liver injury and hepatic failure, leading to temporary restrictions on UPA use. The structural analysis predicted low glucocorticoid receptor (GR) antagonist properties of UPA, however the anti-glucocorticoid activity of Ulipristal has been demonstrated on reporter constructs in HeLa cells. To determine whether UPA can alter the activity of endogenous GR in various relevant cell types, immortalized human uterine fibroid cells (UtLM) and hepatocytes (HepG2) were treated with the synthetic glucocorticoid dexamethasone and/or UPA. Co-treatment of 100 nM UPA with dexamethasone reduced glucocorticoid-induced expression of PER1, FKBP5, and GILZ in HepG2 and UtLM cells by 30-55%. At 1μM, UPA completely blocked glucocorticoid regulation of these genes in both cell lines. As a comparison, the extent of glucocorticoid inhibition by UPA was comparable to the known PR and GR antagonist RU 486. To discover whether the anti-glucocorticoid activity of UPA was mediated by PR, we knocked down PR expression by siRNA and found that the inhibitory activity of UPA does not require PR, suggesting a direct effect on GR. To determine the mechanism by which UPA inhibits GR activity, we evaluated hallmarks of GR transactivation. As hormone-dependent phosphorylation at S211 is associated with transcriptionally active GR, we evaluated p211 status in response to dexamethasone treatment with and without UPA. In the presence of UPA, dexamethasone-mediated phosphorylation at 211 was significantly diminished. Furthermore, dexamethasone-dependent nuclear translocation of GR was reduced by approximately 70% in both cell lines. Reduced activation of GR resulted in diminished GR recruitment to known regulatory regions in the GILZ promoter in the presence of UPA. We next evaluated the effect of UPA on glucocorticoid-mediated transcription in primary cells. Co-treatment of dexamethasone with UPA blunted glucocorticoid responsiveness in primary human uterine fibroid tissue cultured ex vivo. To determine whether UPA could inhibit endogenous glucocorticoid signaling, mice were treated with vehicle or 2 mg/kg UPA and various tissues were isolated for gene expression analysis. UPA inhibited basal expression of Per1, Fkbp5, and Gilz in the uterus and liver, and lung but not the spleen. These studies suggest that UPA selectively regulates GR activity, which is an important consideration for its use as a long-term therapeutic agent.

Infralimbic dopamine D2 receptors mediate glucocorticoid-induced facilitation of auditory fear memory extinction in rats

The infralimbic (IL) cortex of the medial prefrontal cortex plays an important role in the extinction of fear memory. Also, it has been showed that both brain glucocorticoid and dopamine receptors are involved in many processes such as fear extinction that drive learning and memory; however, the interaction of these receptors in the IL cortex remains unclear. We examined a putative interaction between the effects of glucocorticoid and dopamine receptors stimulation in the IL cortex on fear memory extinction in an auditory fear conditioning paradigm in male rats. Corticosterone (the endogenous glucocorticoid receptor ligand), or RU38486 (the synthetic glucocorticoid receptor antagonist) microinfusion into the IL cortex 10 min before test 1 attenuated auditory fear expression at tests 1–3, suggesting as an enhancement of fear extinction. The effect of corticosterone, but not RU38486 was counteracted by the dopamine D2 receptor antagonist sulpiride pre-treatment administered into the IL (at a dose that failed to alter freezing behavior on its own). In contrast, intra-IL infusion of the dopamine D1 receptor antagonist SCH23390 pre-treatment failed to alter freezing behavior. These findings provide evidence for the involvement of the IL cortex D2 receptors in CORT-induced facilitation of fear memory extinction.

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies.

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1α, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples.

Abstract 4571: Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors

Abstract Androgen receptor (AR) signalling is critical to prostate cancer progression and androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer. Acquired resistance to ADT results from androgen-independent reactivation of the AR pathway primarily. Prostate tumours also bypass androgen blockade by the upregulation of the glucocorticoid receptor (GR). Both AR and GR are nuclear receptors that bind their respective ligands in the cytoplasm and then translocate to the nucleus where they act as transcription factors. Plexins are cell surface receptors for semaphorins. PlexinB1 is overexpressed and mutated in several cancers including prostate cancer and deletion of the plexinB1 gene reduces metastasis in mouse models, indicating an oncogenic role for plexinB1 in prostate cancer. We have found that activation of plexinB1 by semaphorin4D (Sema4D) results in AR phosphorylation at serine 81, activation of AR-transcriptional activity and increased expression of androgen-responsive genes, effects reversed by plexinB1 knockdown. PlexinB1 activation increases the translocation of both AR-GFP constructs and endogenous AR to the nucleus in prostate cancer cells. Furthermore knockdown of AR in the prostate cancer cell line LNCaP reduces Sema4D-induced anchorage-independent growth of these cells. To determine if the Sema4D/plexinB1 signalling pathway also affects translocation of other nuclear receptors to the nucleus, we have also studied the effect of Sema4D treatment on nuclear trafficking of the glucocorticoid receptor (GR). Prostate cancer cells were transfected with a GR-GFP construct and the subcellular localisation of GR-GFP was monitored upon Sema4D treatment by immunocytochemistry and confocal microscopy. The nuclear translocation of endogenous GR was also assessed following Sema4D stimulation by immunocytochemistry and subcellular fractionation studies. We found that activation of plexinB1 by Sema4D results in a significant increase in the percentage of cells with nuclear GR-GFP and in the nuclear localisation of endogenous GR in prostate cancer cells. These findings show that plexinB1 activation has a role in the trafficking and activation of nuclear receptors and so may have a role in resistance to ADT in late stage prostate cancer. Citation Format: Magali Williamson, John R. Masters. Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4571.

Helix 8 of the ligand binding domain of the glucocorticoid receptor (GR) is essential for ligand binding

Membrane association of estrogen receptors (ER) depends on cysteine palmitoylation and two leucines in the ligand binding domain (LBD), conserved in most steroid receptors. The role of this region, corresponding to helix 8 of the glucocorticoid receptor (GR) LBD, on membrane association of GR was studied in 4B cells, expressing endogenous GR, and Cos-7 cells transfected EGFP-GR constructs. 4B cells preloaded with radiolabeled palmitic acid showed no radioactivity incorporation into immunoprecipitated GR. Moreover, mutation C683A (corresponding to ER palmitoylation site) did not affect corticosterone-induced membrane association of GR. Mutations L687–690A, L682A, E680G and K685G prevented membrane and also nuclear localization through reduced ligand binding. L687–690A mutation decreased association of GR with heat shock protein 90 and transcriptional activity, without overt effects on receptor protein stability. The data demonstrate that palmitoylation does not mediate membrane association of GR, but that the region 680–690 (helix 8) is critical for ligand binding and receptor function.