Identification of glucocorticoid-related molecular signature by whole blood methylome analysis

Objective: Cushing’s syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing’s syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing’s syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. Design: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing’s syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. Methods: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850000 CpG sites). Both unsupervised (Principal Component Analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. Results: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing’s syndrome correction. In Cushing’s syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. Conclusions: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assess glucocorticoid action beyond hormone assays.

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in living cells. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work unveils likely pathophysiologically relevant quaternary assemblies of the nuclear receptor with important implications for glucocorticoid action and drug design.

DHEAS and Differential Blood Counts as Indirect Signs of Glucocorticoid Excess in Adrenal Non-Producing Adenomas

The majority of incidentally discovered adrenal tumors are later characterized as non-producing adrenocortical adenomas (NPA). We asked whether laboratory abnormalities in parameters that reflect glucocorticoid action can be found in patients with NPA despite their nature of being clinically unapparent. Since glucocorticoids are potent immunosuppressants we studied blood counts and differential blood counts along with corticotropin and dehydroepiandrostenedione sulfate (DHEAS) blood concentrations, as well as cortisol values before and after an overnight 1 mg dexamethasone suppression test. We compared the results of normal individuals, of patients with adrenal adenomas and normal hormone profiles and with subclinical autonomous glucocorticoid hypersecretion, as well as overt cortisol excess. We found that almost all indices of the blood counts were significantly different between the patients groups. In particular, patients with adrenal non-producing adenomas already showed signs of glucocorticoid excess, including relative lymphocytopenia, lowered DHEAS, and ACTH concentrations than control individuals. We also found that the extent of lymphocytopenia correlated with the concentrations of DHEAS and ACTH, and DHEAS correlated well with ACTH. We conclude that the basal ACTH and DHEAS values along with the differential blood counts give good information on the extent of glucocorticoid excess and that silent adrenal adenomas seem to oversecrete glucocorticoids at concentrations that already alter these parameters.

Glucocorticoid receptor triggers a reversible drug-tolerant dormancy state with acquired therapeutic vulnerabilities in lung cancer

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.

Glucocorticoids: Fuelling the Fire of Atherosclerosis or Therapeutic Extinguishers?

Glucocorticoids are steroid hormones with key roles in the regulation of many physiological systems including energy homeostasis and immunity. However, chronic glucocorticoid excess, highlighted in Cushing’s syndrome, is established as being associated with increased cardiovascular disease (CVD) risk. Atherosclerosis is the major cause of CVD, leading to complications including coronary artery disease, myocardial infarction and heart failure. While the associations between glucocorticoid excess and increased prevalence of these complications are well established, the mechanisms underlying the role of glucocorticoids in development of atheroma are unclear. This review aims to better understand the importance of glucocorticoids in atherosclerosis and to dissect their cell-specific effects on key processes (e.g., contractility, remodelling and lesion development). Clinical and pre-clinical studies have shown both athero-protective and pro-atherogenic responses to glucocorticoids, effects dependent upon their multifactorial actions. Evidence indicates regulation of glucocorticoid bioavailability at the vasculature is complex, with local delivery, pre-receptor metabolism, and receptor expression contributing to responses linked to vascular remodelling and inflammation. Further investigations are required to clarify the mechanisms through which endogenous, local glucocorticoid action and systemic glucocorticoid treatment promote/inhibit atherosclerosis. This will provide greater insights into the potential benefit of glucocorticoid targeted approaches in the treatment of cardiovascular disease.

MiR-122-5p: A Novel Biomarker of Glucocorticoid Action

Background: Glucocorticoids are among the most prescribed medications for various indications, and treatment with glucocorticoids is associated with increased morbidity and mortality. A biomarker allowing quantification of glucocorticoid action could improve treatment safety and efficacy. Objective: To identify and validate circulating biomarkers of glucocorticoid action using a clinical experimental study and multi-omic network analysis. Methods: In a randomized, controlled, crossover, single-blind trial, 10 subjects without endogenous glucocorticoid production (Addison’s disease) received intravenous hydrocortisone infusion in a circadian pattern (physiological glucocorticoid exposure) or isotonic saline (glucocorticoid withdrawal) over 22 hours. Food intake and sample collections were standardized during both treatment periods. The transcriptomes of peripheral blood mononuclear cells and adipose tissue, plasma miRNAome and serum metabolome were collected at 7 AM (end of infusion). These multi-omic data were compared between the two interventions, within and between subjects, using network analysis of higher order interactions along with statistical and machine learning approaches. Samples from 120 subjects with varying glucocorticoid exposure from independent studies were used for the replication of the miRNA findings. The study was pre-registered at ClinicalTrials.gov with identifier NCT02152553. Results: We identified a transcriptomic profile derived from both peripheral blood mononuclear cells and adipose tissue, and a multi-omic signature including genes, miRNAs and metabolites that were associated with glucocorticoid exposure. Within the multi-omic signature we identified a single microRNA (miR-122-5p, p=0.009) regulated by glucocorticoid exposure, which we then replicated as a novel biomarker of glucocorticoid action in 120 subjects from independent studies (0.01 ≤ p ≤ 0.05). Conclusions: The discovery of miR-122-5p as a novel circulating biomarker of glucocorticoid action may have a significant impact on clinical practice. Our data also improves the understanding of glucocorticoid action and may have impact on future studies on the mechanistic understanding for the role of glucocorticoids in the etiology of common diseases, such as cardiovascular disease and obesity.

HNF4A is required to specify glucocorticoid action in the liver

The glucocorticoid receptor (GR) is a nuclear hormone receptor critical to the regulation of energy metabolism and the inflammatory response. The actions of GR are highly dependent on cell type and environmental context. Here, we demonstrate the necessity for liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver-specificity of GR action. In normal mouse liver, the HNF4 motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites found within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodelled, with both loss and gain of GR recruitment evident. Lost sites are characterised by HNF4 motifs and weak GRE motifs. Gained sites are characterised by strong GRE motifs, and typically show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is further demonstrated by evidence of an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

Duality of glucocorticoid action in cancer: tumor-suppressor or oncogene?

Glucocorticoid receptor (GR) is a key homeostatic regulator involved in governing immune response, neuro-integration, metabolism and lung function. In conjunction with its pivotal role in human biology, GR action is critically linked to pathology of various disease types, including cancer. While pharmacological activation of GR has been used for treatment of various liquid cancers, its role in solid cancers is less clearly defined and seems to be cancer-type dependent. This review focuses on the molecular aspects of GR biology, spanning the structural and functional basis of response to glucocorticoids, as well as how this transcription factor operates in cancer, including the implications in disease development, progression and drug resistance.