Glucocorticoid Receptor Status Research Articles

Abstract IA20: Hormonal action in breast cancer: Common features and unique properties that drive tumor development and outcome

Abstract Estrogen receptor (ER) occupies many thousands of regions throughout the human genome. Interestingly, the functional implications of most of these chromatin-bound sites remain completely elusive. In two interconnected stories, I will highlight distinct genomic features that differentiate ER genomic action between patient subgroups, but also between individual patients. Between patients subgroups, we identify clearlty distinct profiles of ER action, dictated by Glucocorticoid Receptor (GR) activity. We show that the activity status of GR determines luminal subtype identity and is associated with patient outcome. GR suppresses tumor growth by engaging with ER, leading to redistribution of ER on the chromatin and upregulation of the tumor suppressor gene ZBTB16. We importantly demonstrate that clinically available epigenetic inhibitors, which mimic GR-induced gene repression, can effectively suppress the growth of highly aggressive ER-positive breast cancer cells displaying absence of GR activity. In a second study, I will elaborate on the massive inter-patient heterogeneity of ER chromatin interactions, with direct biological and clinical implications. While the vast majorty of ER/DNA binding events are poorly conserved between patients, common ER sites are most-transcriptionally active and enriched for breast cancer risk SNPs. With that, we present a natural hierarchy of ER genomic action, identifying those regiosn with the most-direct impact on breast cancer development and progresion. Citation Format: Wilbert Zwart. Hormonal action in breast cancer: Common features and unique properties that drive tumor development and outcome [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA20.

From maternal glucocorticoid and thyroid hormones to epigenetic regulation of offspring gene expression: An experimental study in a wild bird species.

Offspring phenotype at birth is determined by its genotype and the prenatal environment including exposure to maternal hormones. Variation in both maternal glucocorticoids and thyroid hormones can affect offspring phenotype, but the underlying molecular mechanisms, especially those contributing to long-lasting effects, remain unclear. Epigenetic changes (such as DNA methylation) have been postulated as mediators of long-lasting effects of early-life environment. In this study, we determined the effects of elevated prenatal glucocorticoid and thyroid hormones on handling stress response (breath rate) as well as DNA methylation and gene expression of glucocorticoid receptor (GR) and thyroid hormone receptor (THR) in great tits (Parus major). Eggs were injected before incubation onset with corticosterone (the main avian glucocorticoid) and/or thyroid hormones (thyroxine and triiodothyronine) to simulate variation in maternal hormone deposition. Breath rate during handling and gene expression of GR and THR were evaluated 14 days after hatching. Methylation status of GR and THR genes was analyzed from the longitudinal blood cells sampled 7 and 14 days after hatching, as well as the following autumn. Elevated prenatal corticosterone level significantly increased the breath rate during handling, indicating an enhanced metabolic stress response. Prenatal corticosterone manipulation had CpG-site-specific effects on DNA methylation at the GR putative promoter region, while it did not significantly affect GR gene expression. GR expression was negatively associated with earlier hatching date and chick size. THR methylation or expression did not exhibit any significant relationship with the hormonal treatments or the examined covariates, suggesting that TH signaling may be more robust due to its crucial role in development. This study provides some support to the hypothesis suggesting that maternal corticosterone may influence offspring metabolic stress response via epigenetic alterations, yet their possible adaptive role in optimizing offspring phenotype to the prevailing conditions, context-dependency, and the underlying molecular interplay needs further research.

Expression and clinicopathological significance of glucocorticoid receptor, SGK1, and NDRG1 in hormone-naïve prostate carcinoma.

Glucocorticoid receptor (GR) has been implicated in prostate carcinoma growth and progression. Glucocorticoid receptor beta (GRβ) acts as an inhibitor of GR; however, its function is not well understood. Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a GR-responsive gene that phosphorylates N-myc downstream-regulated gene 1 (NDRG1) and is involved in cancer growth and invasion. However, the expression of GR, GRβ, SGK1, and NDRG1 in prostate cancer and their relationship with clinicopathological and functional significance remain unknown. The association between the status of GR, GRβ, SGK1, and NDRG1 immunoreactivity and clinicopathological variables was analyzed in patients with prostate carcinoma to explore their clinical significance. In prostate carcinoma cases, the relative abundance of GR and NDRG1 immunoreactivity was inversely and significantly associated with the primary tumor stage (pT), while GR immunoreactivity was inversely and significantly associated with the Ki-67 score. The relative expression status of NDRG1 was significantly associated with that of GR. However, no significant correlation was observed between any of the clinicopathological parameters and GRβ and SGK1 expression. Our findings indicate that GR and NDRG1 expression status is correlated with clinicopathological features in patients with prostate cancer.

189 GLUCOCORTICOID RECEPTOR AND SERUM- AND GLUCOCORTICOID-INDUCED KINASE-1 IN ESOPHAGEAL ADENOCARCINOMA AND ADJACENT BARRETT’S ESOPHAGUS

Abstract Barrett’s esophagus (BE) is a consequence of gastroesophageal reflux disease and is predisposed to esophageal adenocarcinoma (EAC). EAC is an exemplar model of inflammation-associated cancer. Glucocorticoids suppress inflammation through glucocorticoid receptor (GR) and serum- and glucocorticoid-induced kinase−1 (Sgk1) expressions. Methods We immunolocalized GR and Sgk1 in EAC and the adjacent BE tissues and studied their association with clinical disease course in 87 patients with EAC who underwent surgical resection (N = 58) or endoscopic submucosal dissection (N = 29). Results Low GR and Sgk1 expressions in adjacent BE tissues were associated with adverse clinical outcomes (P = 0.0008 and 0.034, respectively). Patients with low Sgk1 expression in EAC cells exhibited worse overall survival (P = 0.0018). In multivariate Cox regression analysis, low GR expression in the adjacent nonmalignant BE tissues was significantly associated with worse overall survival (P = 0.023). Conclusion The present study indicated that evaluation of GR and Sgk1 expressions in both the EAC cells and adjacent nonmalignant BE tissues could help to predict clinical outcomes following endoscopic and surgical treatments. In particular, the GR status in BE tissues adjacent to EAC was an independent prognostic factor.

The Impact of a Single Phosphorylation Site Mutation in the Glucocorticoid Receptor on the Molecular and Cellular Development of the Cerebral Cortex

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

38P Triple-negative breast cancers with expression of glucocorticoid receptor in immune cells show better prognosis

Glucocorticoid receptor (GR) is shown to have variable frequency of expression in invasive tumors of the breast. Investigation of additional nuclear receptors like GR in receptor negative tumors like triple negative breast cancer (TNBC) may have prognostic and therapeutic significance. Expression of GR was evaluated by immunohistochemistry in 175 tumors of invasive breast cancer with long term follow up. GR Expression was separately evaluated in invasive tumor cells, stromal cells and tumor infiltrating lymphocytes (TIL's). Staining pattern was categorised as positive when more than 1% of the cells stained in each subpopulation of cells. Disease free survival was analysed between GR positive and negative status by Kaplan Meier analysis. Of the 175 tumors, 121 (70%) were ER positive, 53 (30%) were ER negative and 29% (51) were triple negative. 74% (130/175) tumors showed expression of GR in invasive tumor cells while (84%) 147/175 had expression in TIL's. No significant difference in distribution of GR was noted between ER positive and ER negative tumors (78% vs 66%, p-0.1). Of the TNBC's 54% (28/51) and 70% (36/51) showed expression of GR in invasive tumor and TIL's respectively. Overall, GR positive tumors had significant better survival than GR negative tumors (mean survival time of 85 vs 59 months respectively, p-0.04) Contrary to the reports that GR expression in TIL's are associated with immunosuppressive activity in model systems, TNBC's with increased expression of GR in immune cells were associated with better survival (Mean survival time 74 vs 41 months, log rank test- p-0.03). TNBC tumors which were GR negative had higher lymph node metastases (p-0.04) and none of the other clinical features like age, menopausal state, tumor size and grade were different between GR positive and negative tumors within TNBC. Glucocorticoids (GC) are often used to alleviate the adverse symptoms during chemotherapy. Determining the GR status is of importance due to the pro cell survival effect of the glucocorticoids mediated through GR during chemotherapy. Though GC mediated effects on chemotherapy are controversial, our results indicate favourable effects in TNBC.

Differential regulation of the glucocorticoid receptor nucleocytoplasmic shuttling by TPR-domain proteins

A dimer of the heat-shock protein of 90-kDa (Hsp90) represents the critical core of the chaperone complex associated to the glucocorticoid receptor (GR) oligomer. The C-terminal end of the Hsp90 dimer shapes a functional acceptor site for co-chaperones carrying tetratricopeptide repeat (TPR) domains, where they bind in a mutually exclusive and competitive manner. They impact on the biological properties of the GR•Hsp90 complex and are major players of the GR transport machinery. Recently, we showed that the overexpression of a chimeric TPR peptide influences the subcellular distribution of GR. In this study, the functional role of endogenous proteins carrying TPR or TPR-like sequences on GR subcellular distribution was characterized. It is demonstrated that, contrarily to the positive influence of FKBP52 on GR nuclear accumulation, FKBP51 and 14-3-3 impaired this property. While SGT1α showed no significant effect, the overexpression of the Ser/Thr phosphatase PP5 resulted in a nearly equal nuclear-cytoplasmic redistribution of GR rather than its typical cytoplasmic localization in the absence of steroid. This observation led to analyse the influence of the phosphorylation status of GR, which resulted not linked to its nucleo-cytoplasmic shuttling mechanism. Nonetheless, it was evidenced that both PP5 and FKBP52 are related to the anchorage of the GR to nucleoskeleton structures. The influence of these TPR domain proteins on the steroid-dependent transcriptional activity of GR was also characterized. It is postulated that the pleiotropic actions of the GR in different cell types may be the consequence of the relative abundance of different TPR-domain interacting co-chaperones.

Glucocorticoid receptor and serum- and glucocorticoid-induced kinase-1 in esophageal adenocarcinoma and adjacent Barrett's esophagus.

Barrett's esophagus (BE) is a consequence of gastroesophageal reflux disease and is predisposed to esophageal adenocarcinoma (EAC). EAC is an exemplar model of inflammation-associated cancer. Glucocorticoids suppress inflammation through glucocorticoid receptor (GR) and serum- and glucocorticoid-induced kinase-1 (Sgk1) expressions. Therefore, we immunolocalized GR and Sgk1 in EAC and the adjacent BE tissues and studied their association with clinical disease course in 87 patients with EAC who underwent surgical resection (N = 58) or endoscopic submucosal dissection (N = 29). Low GR and Sgk1 expressions in adjacent BE tissues were associated with adverse clinical outcomes (P = 0.0008 and 0.034, respectively). Patients with low Sgk1 expression in EAC cells exhibited worse overall survival (P = 0.0018). In multivariate Cox regression analysis, low GR expression in the adjacent nonmalignant BE tissues was significantly associated with worse overall survival (P = 0.023). The present study indicated that evaluation of GR and Sgk1 expressions in both the EAC cells and adjacent nonmalignant BE tissues could help to predict clinical outcomes following endoscopic and surgical treatments. In particular, the GR status in BE tissues adjacent to EAC was an independent prognostic factor.

GR, Sgk1, and NDRG1 in esophageal squamous cell carcinoma: their correlation with therapeutic outcome of neoadjuvant chemotherapy

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly malignant neoplasm.The glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays pivotal roles in cellular response to various stresses of tumor cells, including chemotherapy. However, the status of the GC-GR pathway in ESCC, including its correlation with chemotherapeutic responses, is largely unknown.MethodsGR, serum-and glucocorticoid-regulated kinase 1 (Sgk1), and N-myc down regulation gene 1 (NDRG1) were immunolocalized in 98 patients with ESCC who had undergone esophagectomy following neoadjuvant chemotherapy (NAC) with 2 courses of 5-fluorouracil + cisplatin. We also examined biopsy specimens before NAC in 42 cases and compared the results between those before and after NAC.ResultsOverall survival (OS) of the patients treated with surgery following NAC was significantly shorter in the group with high GR than that with low GR status (P = 0.0473). Both OS and disease-free survival (DFS) were significantly shorter in both Sgk1- and NDRG1-high groups than in the low groups (OS: Sgk1, P = 0.0055; NDRG1, P = 0.0021; DFS: Sgk1, P = 0.0240; NDRG1, P = 0.0086). Biopsy specimens before NAC showed significantly shorter DFS in the high Sgk1 group (P = 0.0095), while both OS and DFS were shorter in the high NDRG1 group (OS, P = 0.0233; DFS, P = 0.0006) than in the respective low groups. In the high NDRG1 group of biopsy specimens before NAC, the tumor reduction rate by NAC was significantly attenuated (P = 0.021).ConclusionsHigh GR, Sgk1, and NDRG1 statuses in ESCC after NAC was significantly associated with an overall worse prognosis, with no significant changes in their expression levels before and after NAC. Therefore, increased activity of the GC-GR pathway with enhanced induction of Sgk1 and NDRG1 in carcinoma cells play pivotal roles in tumor progression and development of chemo-resistance in patients with ESCC undergoing NAC.

The Two Faces of Adjuvant Glucocorticoid Treatment in Ovarian Cancer.

Adjuvant glucocorticoid treatment is routinely used in the treatment of ovarian cancer to mitigate the undesirable side effects of chemotherapy, thereby enhancing tolerability to higher cytotoxic drug doses and frequency of treatment cycles. However, in vitro and preclinical in vivo and ex vivo studies indicate that glucocorticoids may spare tumor cells from undergoing cell death through enhanced cell adhesion, promotion of anti-inflammatory signaling, and/or inhibition of apoptotic pathways. The implications of laboratory studies showing potential negative impact on the efficacy of chemotherapy have been long overlooked since clinical investigations have found no apparent survival detriment attributable to adjuvant glucocorticoid use. Importantly, these clinical studies were not randomized and most did not consider glucocorticoid receptor status, a vital determinant of tumor response to glucocorticoid administration. Additionally, the clinically beneficial elements of increased chemotherapy treatment adherence and dosing afforded by adjuvant glucocorticoids may offset and therefore mask their anti-chemotherapy activities. This review summarizes the current evidence on the impact of glucocorticoids in ovarian cancer and discusses the need for further research and development of alternative strategies to ameliorate untoward side effects of chemotherapy.

High glucocorticoid receptor expression predicts short progression-free survival in ovarian cancer

ObjectiveTo investigate the association of tumor glucocorticoid receptor (GR) expression and patient outcome in ovarian cancer. MethodsGR expression was evaluated by immunohistochemistry using tissue microarrays of specimens from 481 patients with ovarian cancer and 4 patients with benign conditions. Low GR expression was defined as an intensity of 0 or 1+ and high GR as 2+ or 3+ in >1% of tumor cells. Analyses were performed to evaluate the relationship of GR expression with clinical characteristics, progression-free survival (PFS) and overall survival (OS). ResultsGR protein was highly expressed in 133 of 341 (39.0%) tumors from patients who underwent upfront cytoreduction surgery followed by adjuvant chemotherapy. High GR expression was more common in serous tumors (p<0.001), high grade tumors (p<0.001), and advanced stage tumors (p=0.037). Median PFS was significantly decreased in cases with high GR (20.4months) compared to those with low GR (36.0months, HR=1.66, 95% CI 1.29–2.14, p<0.001). GR remained an independent prognostic factor for PFS in multivariate analysis. OS was not associated with GR status. ConclusionsThese data suggest that high GR expression correlates with poor prognosis and support the hypothesis that modulating GR activity in combination with chemotherapy may improve outcomes.

Abstract P6-12-15: Efficacy results of a phase 1/2 study of glucocorticoid receptor (GR) antagonist mifepristone (MIFE) in combination with eribulin in GR-positive triple-negative breast cancer (TNBC)

Abstract Background: GR is variably expressed in TNBC and high expression is associated with poor prognosis in estrogen receptor-negative (ER-) early stage breast cancer. Treatment with mifepristone (MIFE) potentiates the effectiveness of chemotherapy in GR+ TNBC xenografts. Enrollment is complete in this study of patients with GR+ TNBC treated at the recommended Phase 2 dose (RP2D) of MIFE in combination with eribulin. Objectives: To determine the safety, tolerability, pharmacokinetics (PK) and clinical activity of the MIFE plus eribulin combination in pts with GR+ TNBC at the RP2D. Methods: Eligibility: In Part 1 (dose finding), pts with solid tumors; in Part 2 (expansion phase), pts with TNBC (GR result required at time of screening in Part 1, but could be pending at time of screening in Part 2). Up to 5 prior chemotherapy regimens for advanced disease; ECOG PS 0-1; adequate end-organ function. Design: 3 + 3 dose escalation scheme. After a 7-day lead-in of oral daily MIFE alone, MIFE was continued daily and eribulin was given on days 1 and 8 of a 21-day cycle. GR+ was defined as &amp;gt;10% of tumor cells with any intensity of GR staining. Results: 16 pts with metastatic breast cancer were treated in Part 1, and 21 pts with TNBC were treated in Part 2. Median age was 54 (range 30-81). MTD/RP2D was MIFE 300 mg/day + eribulin 1.1 mg/m2. Safety: DLT in Part 1 was neutropenia. Neutropenia occurred in 23/36 total patients (2 Grade [G] 1, 10 G3, 11 G4); 2 instances included neutropenic fever. Recovery of WBC was brisk with growth factor support. Neuropathy was observed in 8 pts (5 G1, 1 G2, 2 G3). Other most common AEs (fatigue, hypokalemia, nausea, alopecia) were mainly G1 or G2; among these, G3/G4 events were limited to fatigue (4 G3), hypokalemia (3 G3 and 1 G4) and nausea (1 G3). There were 2 instances of G1 vaginal bleeding. There was no impact of MIFE on eribulin PK. Efficacy: There were 23 evaluable pts with TNBC across Parts 1 and 2 treated at the RP2D: 21 GR+, 2 GR status unknown; median of 3 prior chemotherapy regimens; 1 patient had received prior eribulin. Responses were: 3 PR, 8 SD, 11 PD and one too early to assess. Median PFS was 9 weeks. Conclusions: MIFE plus eribulin was well tolerated and appears to be an active treatment regimen. Five TNBC patients had a PFS longer than the upper 95% CI for PFS (i.e., &amp;gt;15 wks) reported by Aogi et al. for TNBC treated with eribulin (Annals of Oncology 2012?23:144148). Clinical trial information: NCT02014337. Citation Format: Han HS, Wilks S, Paplomata E, Modiano MR, Becerra C, Braiteh FS, Spira AI, Pluard TJ, Richards DA, Conzen SD, Baker G, Fishman RS, Marcantonio A, O'Shaughnessy J, Nanda R. Efficacy results of a phase 1/2 study of glucocorticoid receptor (GR) antagonist mifepristone (MIFE) in combination with eribulin in GR-positive triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-15.

Dexamethasone Accumulation in Dexamethasone Sensitive and Resistant Acute Lymphoblastic Leukemia

Introduction:Glucocorticoids (GC) have been at the forefront of acute lymphoblastic leukemia (ALL) treatment for a number of decades. However, there is heterogeneity of response, both in terms of GC-related toxicity and leukemia cell sensitivity. Contributing factors include marked pharmacokinetic variability observed in children (Yang et al. JCO, 2008; Jackson et al.AACR annual meeting abstract CT115, 2016) and a several hundred fold range of GC cellular response. Cellular resistance can be caused by deletion of the glucocorticoid receptor (GR) but is more commonly downstream of the GR. One neglected upstream parameter relates to GC accumulation, which may be an important factor in ALL GC response, given the evidence for drug transporters in ALL cells. Therefore, this study aimed to determine whether variation in intracellular dexamethasone (dex) levels is a determinant of dex sensitivity in an ALL setting.Methods:A number of cell lines including PreB697, GC resistant PreB697 sub-lines and REH cells, along with primagraft material from 9 patients and 6 primary patient samples (5 presentation and 1 relapse) were studied. The relative sensitivity of cells to dex was assessed using Alamar Blue drug sensitivity assay. Two methods were developed to assess intracellular dex accumulation; a liquid-chromatography mass spectrometry (LC/MS) method and a flow cytometry method, using dex conjugated to the fluorochrome FITC analysed on a FACSCalibur flow cytometry machine. GR status of the cells was confirmed by western blotting.Results:Dex GI50 values (concentration giving 50% growth inhibition) ranged from 37nM in PreB697 cells to >1000nM in GC resistant sub-lines and REH cells. Dex GI50 values in patient ALL cells ranged from 2 to >1000nM. Dex resistant cells were defined as having a dex GI50 of >500nM. The mean GI50 of the dex sensitive cells was 3.8nM. Western blotting suggested wildtype GR status in all samples, with R3D11 and REH serving as hemizygous deleted and GR negative controls, respectively.The mean dex accumulation was measured in cells using an LC/MS assay developed from a fully validated assay measuring plasma dex concentrations (Jackson et al. NCRI annual meeting abstract BACR9, 2014). Dex was stable in RF10 media for at least 8 hours and there was no matrix effect of RF10 media on dex chromatograms compared to dex in plasma. An incubation concentration of 500nM was chosen as this is the observed median value of dex cell exposure clinically. Dex concentrations were quantifiable in cell numbers of 1 x 106after incubation with 500nM dex, allowing measurement of patient samples where limited numbers of cells are available.Dex accumulation in cell lines after incubation with 500nM dex for 4h was 2.1 and 1.8 pmol/million cells in PreB697 and dex resistant sub-lines, respectively (range for resistant subclones 1.2 - 2.1pmol/million cells). There was greater variability in patient cells with a 40-fold range seen, but dex accumulation was not significantly different between sensitive (mean, 1.0 pmol/million; range, 0.1-2.3) and resistant cells (1.4 pmol/million; range, 0.4-4.4) (unpaired students t-test, p=0.17).To assess intra-leukemia heterogeneity in terms of dex accumulation, a flow based assay was established using dex-FITC. Incubation conditions of 500nM dex-FITC at 37°C for 45 minutes were optimal. Dex-FITC accumulation did not differ significantly between sensitive and resistant cells; mean fluorescence intensity of 4.2 (range 1.5-5.9) versus 4.1 (range 2.0 - 9.1) in sensitive and resistant cells, respectively (p=0.97, unpaired students t test). Dex-FITC accumulation appeared uniform within the ALL samples examined.Conclusions:These data suggest that variations in dex accumulation are unlikely to play a role in dex resistance in ALL, at least in vitro. Advancement of the flow-based dex accumulation assay to include leukaemia-associated immunophenotype markers will allow measurement in dex-resistant MRD in vivo. Given that 35% of patients do not achieve plasma concentrations of 200nM dex (Jackson et al. AACR annual meeting abstract CT115, 2016), a combined approach incorporating pharmacokinetic assessments, drug accumulation and cellular response in ALL cells, may allow a comprehensive understanding of dex pharmacology in order to optimise its clinical utility. DisclosuresNo relevant conflicts of interest to declare.

Vitamin D interferes with glucocorticoid responsivenessin human peripheral blood mononuclear target cells.

Glucocorticoids (GCs) are widely used in the treatment of inflammatory and autoimmune diseases; however, patients are often resistant to GC effects. Current studies indicate that vitamin D reduces the risk or modifies the course of autoimmune diseases posing vitamin D supplementation as a prevention or therapeutic option. Herein, we investigated whether vitamin D can modify the response to GCs at the molecular level. To this end, peripheral blood mononuclear cells (PBMCs) were isolated from healthy vitamin D-deficient women and incubated with either the active metabolite 1,25(OH)2D3 (VitD) for 11days or dexamethasone (Dex) for the last 2days in the presence or absence of VitD. Ex vivo GC sensitivity was assessed by the expression of the glucocorticoid receptor (GR) responsive gene GILZ with RT-PCR. Long-term incubation of PBMCs with VitD significantly decreased the Dex-induced augmentation of GILZ expression. Since the intracellular concentration of GR and the GR nuclear translocation are critical determinants of GC sensitivity, we next evaluated the effect of VitD on these factors. RT-PCR and western-blot analysis revealed that VitD reduced the expression of GR. This effect was abolished by the HDAC-specific inhibitor trichostatin A, implying that HDAC was implicated in this effect. Moreover, NCoR1 mRNA was significantly decreased upon treatment with VitD either alone or as pre-treatment to Dex, suggesting that a possible increase in expression of this co-repressor was not involved. In addition, immunofluorescence analysis showed that VitD hindered the Dex-induced GRα nuclear translocation, an effect verified by subcellular fractionation and western-blot experiments. To further explore the underpinning mechanism, we examined the potential of VitD to: (1) strengthen the FK506-binding protein 5 (FKBP5) negative feedback loop and (2) modify the phosphorylation status of GR. Remarkably, VitD decreased FKBP5 expression and decreased phosphorylation at Ser211, while enhancing phosphorylation of GR at Ser203. Overall, VitD decreases the ex vivo GC sensitivity and this effect is, at least in part, attributed both to decrease of GR expression owing to a mechanism that engages HDAC and inhibition of GR translocation to nucleus via differential modulation of the phosphorylation state of GR. Our study provides, for the first time, evidence that long-term action of VitD induces GC resistance in PBMCs from healthy volunteers and offers a possible mechanistic basis for VitD-triggered attenuation of GC effects.

N-bromotaurine surrogates for loss of antiproliferative response and enhances cisplatin efficacy in cancer cells with impaired glucocorticoid receptor

Glucocorticoids (GCs) are frequently used in anticancer combination regimens; however, their continuous use adds selective pressure on cancer cells to develop GC-resistance via impairment of the glucocorticoid receptor (GR), therefore creating a need for GC-alternatives. Based on the drug repurposing approach and the commonalities between inflammation and neoplasia, drugs that are either in late-stage clinical trials and/or already marketed for GC-refractory inflammatory diseases could be evaluated as GC-substitutes in the context of cancer. Advantageously, unlike new molecular entities currently being de novo developed to restore GC-responsiveness of cancer cells, such drugs have documented safety and efficacy profile, which overall simplifies their introduction in clinical cancer trials. In this study, we estimated the potential of a well-established, multistage, cell line-based, mouse skin carcinogenesis model to be exploited as an initial screening tool for unveiling covert GC-substitutes. First, we categorized the cell lines of this model to GC-sensitive and GC-resistant, in correlation with their corresponding GR status, localization, and functionality. We found that GC-resistance starts in papilloma stages, due to a dysfunctional GR, which is overexpressed, DNA binding-competent, but transactivation-incompetent in papilloma, squamous, and spindle stages of the model. Then, aided by this tool, we evaluated the ability of N-bromotaurine, a naturally occurring, small-molecule, nonsteroid anti-inflammatory drug which is under consideration for use interchangeably/in replacement to GCs in skin inflammations, to restore antiproliferative response of GC-resistant cancer cells. Unlike GCs, N-bromotaurine inhibited cell-cycle progression in GC-resistant cancer cells and efficiently synergized with cisplatin, thus indicating a potential to be exploited instead of GCs against cancer.

Hypermethylation of glucocorticoid receptor gene promoter results in glucocorticoid receptor gene low expression in peripheral blood mononuclear cells of patients with systemic lupus erythematosus.

Our aim was to investigate the relationship between the DNA methylation status of glucocorticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Fifteen newly emerging SLE patients and fifteen healthy controls were enrolled in this study. DNA and total RNA were extracted from the PBMCs of the SLE patients and healthy controls. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfite-sequencing PCR. The mRNA expression of GRα, DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and growth arrest, and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. The mRNA expression of GRα was significantly declined in SLE patients, and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls. There were 38, 25, 30, and 49 CpG islands in amplified fragment of GR promoter 1D, 1E, 1F, and 1H, respectively. The overall mean methylation status of the 152 CpG islands of the four promoters was significantly elevated in SLE patients. There was a negative correlation between hypermethylation of GR promoter and GRα mRNA expression in SLE patients. This study demonstrated that hypermethylation of GRα promoter may result in GRα gene low expression in PBMCs of patients with SLE. This study also found that the global methylation status of PBMCs in SLE patients was obviously lower than healthy controls, and it was related to the elevated GADD45α mRNA expression in SLE patients. These conclusions have to be certified by larger-scale clinical studies.

Glucocorticoid Receptor Status Is a Principal Determinant of Variability in the Sensitivity of Non–Small-Cell Lung Cancer Cells to Pemetrexed

Pemetrexed is an S-phase targeted drug in front-line or maintenance therapy of advanced nonsquamous non-small-cell lung cancer (NSCLC) but methods are needed for predicting the drug response. Dexamethasone is typically administered the day before, the day of, and the day after pemetrexed. As dexamethasone strongly regulates many genes including p53 through the glucocorticoid receptor (GR), we hypothesized that dexamethasone influences tumor response to pemetrexed. Eight nonsquamous NSCLC cell line models with varied p53 and GRα/GRβ status were used for gene expression and cell-cycle analyses and for loss- or gain-of-function experiments. In three cell lines dexamethasone profoundly, but reversibly, suppressed the fraction of S-phase cells. Dexamethasone also reversibly repressed expression of thymidylate synthase and dihydrofolate reductase, which are primary targets of pemetrexed but are also quintessential S-phase enzymes as well as the S-phase-dependent expression of thymidine kinase 1. Dexamethasone also decreased expression of the major pemetrexed transporters, the reduced folate carrier and the proton coupled folate transporter. Only cells expressing relatively high GRα showed these dexamethasone effects, regardless of p53 status. In cells expressing low GRα, the dexamethasone response was rescued by ectopic GRα. Further, depletion of p53 did not attenuate the dexamethasone effects. The presence of dexamethasone during pemetrexed treatment protected against pemetrexed cytotoxicity in only the dexamethasone responsive cells. The results predict that in nonsquamous NSCLC tumors, reversible S-phase suppression by dexamethasone, possibly combined with a reduction in the drug transporters, attenuates responsiveness to pemetrexed and that GR status is a principal determinant of tumor variability of this response.

Importance of epigenetic mechanisms in visceral pain induced by chronic water avoidance stress

Epigenetic molecular mechanisms, which include DNA methylation and histone deacetylation, are implicated in the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Previously, we demonstrated that repeated water avoidance stress (WAS), a validated model of chronic psychological stress, induces heightened visceral pain behaviors in rodents that resemble irritable bowel syndrome (IBS) sequelae. However, the involvement of epigenetic molecular mechanisms in the pathophysiology of stress-induced visceral pain has not been explored. Our hypothesis is that epigenetic mechanisms within the central nervous system (CNS) are important to chronic stress-induced visceral hypersensitivity. Adult male F-344 rats with intracerebroventricular (i.c.v.) cannulae were exposed to 7 days of repeated WAS. Controls received a SHAM stress. Following the daily 1h stressor, trichostatin A (TSA; 100 ng/ml), a potent histone deacetylase inhibitor, or vehicle (VEH; 0.1% DMSO/saline,) as control was administered via the i.c.v. cannula. Visceral sensitivity was assessed 24h after the final WAS and quantified the visceromotor response (VMR) by recording the number of abdominal contractions in response to graded pressures (20-60 mmHg) of colorectal distensions (CRD). From a separate group of rats that were exposed to repeated WAS or SHAM stress, the amygdala was isolated to assess the methylation status of glucocorticoid receptor (GR) and corticotropin releasing-factor (CRF) genes via bisulfite sequencing and verified by pyrosequencing. GR and CRF gene expression was quantified via qRT-PCR. Stressed rats exhibited visceral hypersensitivity that was significantly attenuated by TSA. Compared to SHAM controls, methylation of the GR gene was increased following WAS while expression of the GR gene was decreased. Methylation of the CRF promoter was decreased with WAS with a concomitant increase in CRF expression. This study demonstrates the involvement of central epigenetic mechanisms in regulating stress-induced visceral hypersensitivity and provides a foundation for exploring the epigenetic mechanisms that may contribute to IBS-like symptomatology.

Folic acid supplementation prevents the changes in hepatic promoter methylation status and gene expression in intrauterine growth‐retarded piglets during early weaning period

During intrauterine life, genome interacts with maternal signals to influence the mRNA expression levels of specific genes persistently by regulating DNA methylation status. The objective of this study was to examine the responses of glucocorticoid receptor (GR), peroxisome proliferator-activated receptor alpha and gamma (PPARα and PPARγ) promoter methylation, mRNA expression of genes involved in energy metabolism and metabolite concentrations of intrauterine growth-retarded (IUGR) piglets to dietary folic acid supplementation. According to a 2 × 2 factorial arrangement, 16 IUGR and 16 normal birth weight (NBW) piglets were fed a basal diet or a basal diet supplemented with 5 mg/kg of folic acid from weaning (day 14) to day 35 of age. Triglycerides in hepatic tissue and plasma were significantly elevated in control diets-fed IUGR piglets compared with NBW piglets but were decreased by dietary folic acid supplementation. Hepatic mRNA expression levels of GR, PPARα, PPARγ, fatty acid synthase and phosphoenolpyruvate carboxykinase (PEPCK) in IUGR piglets fed a control diet were significantly higher than that in NBW piglets, and promoter methylation status of GR, PPARα and PPARγ in IUGR piglets was reduced significantly compared with NBW piglets. However, the changes in gene expression and DNA methylation status of IUGR piglets were reversed by dietary folic acid supplementation. Hepatic DNA methyltransferase activity was greater with dietary folic acid supplementation regardless of birth weight. Taken together, these results demonstrated that folic acid supplementation during early period of life could prevent the changes of promoter methylation status and gene expressions in the liver of IUGR piglets.

Social, biological, and tumor characteristics of 139 African American (AA) women with newly diagnosed breast cancer in Chicago.

6064 Background: Previous work by our group has shown that social isolation, social stress, and increased vigilance are associated with larger and more aggressive mammary tumors in animal models. In this study, we seek to better characterize these relationships among AA women with newly diagnosed breast cancer. Methods: AA women newly diagnosed with breast cancer were recruited from 3 hospitals in the Chicago area from 2005-2009. Women underwent in-home interviews which focused on psychosocial functioning and social networks. In addition, 4 daily salivary cortisol samples were self-collected over a 3 day period. Additional neighborhood and community level data were collected using direct neighborhood mapping and geocoded information. Tumor samples were analyzed by IHC for ER, PR, HER2 and glucocorticoid receptor (GR) status. Results: 139 women were included in the sample. Median age 52. Stage: 5%DCIS, 29% I, 31% II, 30% III, 5% IV. 34% were triple negative (TN) and of 71 evaluable cases, 64% were GR+. GR status was associated with invasive cancer (69% invasive cancers GR+ vs. 29% of DCIS cases). 32% of pts were clinically depressed, 68% had moved in the previous 10 years, 31% had suffered a sexual assault, and loneliness scores were higher than a same-race Cook County control sample. In multivariate modeling, number of sexual assaults in the neighborhood was associated with estrogen-receptor (ER) negative status (b = -0.189, p < 0.05) and poorer histological grade of tumor (b = 0.143, p < 0.05). Felt loneliness was associated with HER2neu receptor negative status (b = -0.096, p < 0.05). 67% of patients had no diurnal cortisol variation (flat response) and cortisol group (flat versus circadian) was significantly associated with the individual factors mentioned above. Conclusions: AA women have high rates of TN tumors and GR+ tumors which is being recognized as an additional poor prognostic factor. They also suffer from numerous social stressors which appear to be linked to tumor biology and a flattened stress response. Continued work on understanding the interaction of these social variables and stress response on tumor biology and adherence to therapy is needed. No significant financial relationships to disclose.