Glucocorticoid Receptor Pathway Research Articles

Patient-derived organoids identify tailored therapeutic options and determinants of plasticity in sarcomatoid urothelial bladder cancer

Sarcomatoid Urothelial Bladder Cancer (SARC) is a rare and aggressive histological subtype of bladder cancer for which therapeutic options are limited and experimental models are lacking. Here, we report the establishment of a long-term 3D organoid-like model derived from a SARC patient (SarBC-01). SarBC-01 emulates aggressive morphological, phenotypical, and transcriptional features of SARC and harbors somatic mutations in genes frequently altered in sarcomatoid tumors such as TP53 (p53) and RB1 (pRB). High-throughput drug screening, using a library comprising 1567 compounds in SarBC-01 and conventional urothelial carcinoma (UroCa) organoids, identified drug candidates active against SARC cells exclusively, or UroCa cells exclusively, or both. Among those, standard-of-care chemotherapeutic drugs inhibited both SARC and UroCa cells, while a subset of targeted drugs was specifically effective in SARC cells, including agents targeting the Glucocorticoid Receptor (GR) pathway. In two independent patient cohorts and in organoid models, GR and its encoding gene NR3C1 were found to be significantly more expressed in SARC as compared to UroCa, suggesting that high GR expression is a hallmark of SARC tumors. Further, glucocorticoid treatment impaired the mesenchymal morphology, abrogated the invasive ability of SARC cells, and led to transcriptomic changes associated with reversion of epithelial-to-mesenchymal transition, at single-cell level. Altogether, our study highlights the power of organoids for precision oncology and for providing key insights into factors driving rare tumor entities.

Induction of SGK1 via glucocorticoid-influenced clinical outcome of triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive breast malignancy. Glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays a pivotal role in the cellular responses to various stresses including chemotherapy. Serum- and glucocorticoid-induced kinase-1 (SGK1) is known as an important downstream effector molecule in the GR signaling pathway, we attempted to explore its clinicopathological and functional significance in TNBC in which GR is expressed. We first immunolocalized GR and SGK1 and correlated the results with clinicopathological variables and clinical outcome in 131 TNBC patients. We also evaluated the effects of SGK1 on the cell proliferation and migration in TNBC cell lines with administration of dexamethasone (DEX) to further clarify the significance of SGK1. The status of SGK1 in carcinoma cells was significantly associated with adverse clinical outcome in TNBC patients examined and was significantly associated with lymph node metastasis, pathological stage, and lymphatic invasion of the patients. In particular, SGK1 immunoreactivity was significantly associated with an increased risk of recurrence in GR-positive TNBC patients. Subsequent in vitro studies also demonstrated that DEX promoted TNBC cell migration and the silencing of gene expression did inhibit the cell proliferation and migration of TNBC cells under DEX treatment. To the best of our knowledge, this is the first study to explore an association between SGK1 and clinicopathological variables and clinical outcome of TNBC patients. SGK1 status was significantly positively correlated with adverse clinical outcome of TNBC patients and promoted carcinoma cell proliferation and migration of carcinoma cells.

Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia

Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Abstract #1412847: Seasonal Variation in Growth Velocity in Asian Indian Children

Earlier studies on seasonality have shown greater height gain in children in spring and has been attributed to the effect of melatonin, glucocorticoid receptor pathway, ghrelin and a number of other factors on growth hormone. This study aimed to find seasonal variations in growth velocity in Asian Indian children between 3 and 18 years of age. This was a longitudinal prospective study conducted in 16 schools and preschools of NTR district of Andhrapradesh, India. Date of birth was documented and height was recorded using standard anthropometric technique, described by WHO, at three monthly intervals for one year. At the baseline visit, 4,186 children were recruited. Data pertaining to 859 students was eliminated because of the presence of chronic illnesses or height SDS +/-5 or being lost for follow-up. Of the 3,327 children analyzed, 1,627 were boys and 1,700 were girls. There was no significant seasonal variation in height velocity between any of the four seasons in either boys or girls. The amplitude of seasonal variation is described in proportion to the distance from the Equator. However, improving agricultural conditions and changing lifestyle habits might have contributed to the absence of seasonal variation in growth.

Abstract 3666: A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle

Abstract Skeletal muscle dysfunction due to the effects of cancer secretome is observed in multiple cancer types and extreme dysfunction is manifested as cachexia. Major preclinical studies on cancer-associated muscle defects utilized mouse models or mouse C2C12 mouse myoblast cell line for in vitro studies. Because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required to fully comprehend the effects of cancer secretome on skeletal muscle. Here, we report a simple method to establish skeletal muscle stem cell line (hMuSC), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with hMuSCs to myotube transition. Cancer cell line derived factors accelerated stem to myotube differentiation with accompanying changes including an increase in PAX7+/MyoD+ myogenic progenitor cells. Among the pathways activated by cancer-derived factors include inflammatory pathway involving CXCL8 (also called IL-8), glucocorticoid receptor (GR) pathway, and wound healing pathway. Furthermore, cancer-derived factors significantly altered splicing machinery in hMuSCs. Additionally, AKT and p53 pathways that function in metabolic/survival pathways of the skeletal muscle were adversely affected when hMuSCs were exposed to cancer cell-derived factors. Cancer-derived factors increased the expression levels of previously known cachexia-associated genes such as MT-2, ZIP14, and PDK4. Thus, the model system not only recapitulates results of previous mouse studies but also provides much needed human model system that can easily be adapted for large scale studies to explore the epigenomic changes during hMuSC differentiation and to screen for drugs that restore skeletal muscle function in various diseases. Citation Format: Ruizhong Wang, Brijesh Kumar, Poornima Bhat-Nakshatri, Aditi Sanjay Khatpe, Michael P. Murphy, Kristen E. Wanczyk, Emma H. Doud, Amber L. Mosley, Yunlong Liu, Duojiao Chen, Ed Simpson, Hongyu Gao, Harikrishna Nakshatri. A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3666.

A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle.

Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized invivo skeletal muscle model system to study cancer cachexia.

Cerebral iron deficiency may induce depression through downregulation of the hippocampal glucocorticoid-glucocorticoid receptor signaling pathway

BackgroundIron is a trace essential element to sustain the normal neurological function of human. Many researches had reported the involvement of iron deficiency (ID) in neural development and cognitive functions. However, the role of ID in pathogenesis of depression and its underlying mechanism are still unclear. MethodsIn this study, we first used chronic unpredicted mild stress (CUMS) and iron deprivation mouse models to clarify the pathogenesis role of cerebral ID in depression. Then the role of hippocampal glucocorticoid (GC)-glucocorticoid receptor (GR) pathway in cerebral ID induced depression were elucidated in iron deprivation mice and iron deficiency anemia patients. ResultsOur results revealed that both CUMS and iron deprivation could induce cerebral ID in mice, and combination of iron deprivation and CUMS could accelerate the onset and aggravate the symptoms of depression in mice. In hippocampus, ID led to neuronal injury and neurogenesis decrease, which might be related to downregulation of GC-GR signaling pathway caused GR dysfunction, thereby inhibiting the negative feedback regulation function of hippocampus on hypothalamic-pituitary-adrenal (HPA) axis. Moreover, the overactivity of HPA axis in iron deprivation mice and iron deficiency anemia patients also confirmed GR dysfunction. LimitationsIron deprivation led to food and water intake decrease of mice, which may affect the behavioral test. In addition, we mainly evaluated the role of hippocampal ID in depression, and the number of iron deficiency anemia patients was limited. ConclusionsOur results identified that cerebral iron homeostasis was a key factor for maintaining mental stability.

Effects of Simvastatin on Cartilage Homeostasis in Steroid-Induced Osteonecrosis of Femoral Head by Inhibiting Glucocorticoid Receptor.

Steroid-induced osteonecrosis of femoral head (SONFH) is one of the most common bone disorders in humans. Statin treatment is beneficial in preventing the development of SONFH through anti-inflammation effects and inhibition of the glucocorticoid receptor (GR). However, potential mechanisms of statin action remain to be determined. In this study, pulse methylprednisolone (MP) treatment was used to induce SONFH in broilers, and then MP-treated birds were administrated with simvastatin simultaneously to investigate the changes in cartilage homeostasis. Meanwhile, chondrocytes were isolated, cultured, and treated with MP, simvastatin, or GR inhibitor in vitro. The changes in serum homeostasis factors, cell viability, and expression of GR were analyzed. The results showed that the morbidity of SONFH in the MP-treated group increased significantly compared with the simvastatin-treated and control group. Furthermore, MP treatment induced apoptosis and high-level catabolism and low-level anabolism in vitro and vivo, while simvastatin significantly decreased catabolism and slightly recovered anabolism via inhibiting GR and the hypoxia-inducible factor (HIF) pathway. The GR inhibitor or its siRNA mainly affected the catabolism of cartilage homeostasis in vitro. In conclusion, the occurrence of SONFH in broilers was related to the activation of GR and HIF pathway, and imbalance of cartilage homeostasis. Simvastatin and GR inhibitor maintained cartilage homeostasis via GR and the HIF pathway.

The expression characteristics and clinical significance of ACP6, a potential target of nitidine chloride, in hepatocellular carcinoma

BackgroundAcid phosphatase type 6 (ACP6) is a mitochondrial lipid phosphate phosphatase that played a role in regulating lipid metabolism and there is still blank in the clinico-pathological significance and functional roles of ACP6 in human cancers. No investigations have been conducted on ACP6 in hepatocellular carcinoma (HCC) up to date.MethodsHerein, we appraised the clinico-pathological significance of ACP6 in HCC via organizing expression profiles from globally multi-center microarrays and RNA-seq datasets. The molecular basis of ACP6 in HCC was explored through multidimensional analysis. We also carried out in vitro and in vivo experiment on nude mice to investigate the effect of knocking down ACP6 expression on biological functions of HCC cells, and to evaluate the expression variance of ACP6 in xenograft of HCC tissues before and after the treatment of NC.ResultsACP6 displayed significant overexpression in HCC samples (standard mean difference (SMD) = 0.69, 95% confidence interval (CI) = 0.56–0.83) and up-regulated ACP6 performed well in screening HCC samples from non-cancer liver samples. ACP6 expression was also remarkably correlated with clinical progression and worse overall survival of HCC patients. There were close links between ACP6 expression and immune cells including B cells, CD8 + T cells and naive CD4 + T cells. Co-expressed genes of ACP6 mainly participated in pathways including cytokine-cytokine receptor interaction, glucocorticoid receptor pathway and NABA proteoglycans. The proliferation and migration rate of HCC cells transfected with ACP6 siRNA was significantly suppressed compared with those transfected with negative control siRNA. ACP6 expression was significantly inhibited by nitidine chloride (NC) in xenograft HCC tissues.ConclusionsACP6 expression may serve as novel clinical biomarker indicating the clinical development of HCC and ACP6 might be potential target of anti-cancer effect by NC in HCC.

Regulation of the kiss2 promoter in yellowtail clownfish (Amphiprion clarkii) by cortisol via GRE-dependent GR pathway.

Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of kiss gene. In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position −660 and −433 with GR1, and −912 and −775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position −573 in the presence of GR1 and by each GRE site located at position −883, −860, −851, and −843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.

Long lasting anxiety following early life stress is dependent on glucocorticoid signaling in zebrafish

Chronic adversity in early childhood is associated with increased anxiety and a propensity for substance abuse later in adulthood, yet the effects of early life stress (ELS) on brain development remain poorly understood. The zebrafish, Danio rerio, is a powerful model for studying neurodevelopment and stress. Here, we describe a zebrafish model of ELS and identify a role for glucocorticoid signaling during a critical window in development that leads to long-term changes in brain function. Larval fish subjected to chronic stress in early development exhibited increased anxiety-like behavior and elevated glucocorticoid levels later in life. Increased stress-like behavior was only observed when fish were subjected to ELS within a precise time window in early development, revealing a temporal critical window of sensitivity. Moreover, enhanced anxiety-like behavior only emerges after two months post-ELS, revealing a developmentally specified delay in the effects of ELS. ELS leads to increased levels of baseline cortisol, and resulted in a dysregulation of cortisol receptors’ mRNA expression, suggesting long-term effects on cortisol signaling. Together, these findings reveal a ‘critical window’ for ELS to affect developmental reprogramming of the glucocorticoid receptor pathway, resulting in chronic elevated stress.

Loss of glucocorticoid receptor phosphorylation contributes to cognitive and neurocentric damages of the amyloid-β pathway

Aberrant cortisol and activation of the glucocorticoid receptor (GR) play an essential role in age-related progression of Alzheimer's disease (AD). However, the GR pathways required for influencing the pathobiology of AD dementia remain unknown. To address this, we studied an early phase of AD-like progression in the well-established APP/PS1 mouse model combined with targeted mutations in the BDNF-dependent GR phosphorylation sites (serines 134/267) using molecular, behavioral and neuroimaging approaches. We found that disrupting GR phosphorylation (S134A/S267A) in mice exacerbated the deleterious effects of the APP/PS1 genotype on mortality, neuroplasticity and cognition, without affecting either amyloid-β deposition or vascular pathology. The dynamics, maturation and retention of task-induced new dendritic spines of cortical excitatory neurons required GR phosphorylation at the BDNF-dependent sites that amyloid-β compromised. Parallel studies in postmortem human prefrontal cortex revealed AD subjects had downregulated BDNF signaling and concomitant upregulated cortisol pathway activation, which correlated with cognitive decline. These results provide key evidence that the loss of neurotrophin-mediated GR phosphorylation pathway promotes the detrimental effects of the brain cortisol response that contributes to the onset and/or progression of AD dementia. These findings have important translational implications as they provide a novel approach to treating AD dementia by identifying drugs that increase GR phosphorylation selectively at the neurotrophic sites to improve memory and cognition.

Differential expression of genes regulated by the glucocorticoid receptor pathway in patients with pulmonary tuberculosis

AimsPrevious studies in TB patients showed an immuno-endocrine imbalance characterized by a disease-severity associated increase in plasma levels of proinflammatory cytokines and glucocorticoids (GCs).To analyze the potential immunomodulatory effect of circulating GCs over peripheral blood mononuclear cells (PBMC) from TB patients, we investigated the expression of positively (anti-inflammatory-related genes ANXA1; FKBP51; GILZ, NFKBIA, and NFKBIB) and negatively (inflammatory genes: IL-6, IL-1β, and IFN-γ) Glucocorticoids Receptors (GR)-regulated genes. Plasma concentrations of cytokines and hormones, together with specific lymphoproliferation were also assessed. Materials and methodsGene expression was quantified by RT-qPCR, specific lymphoproliferation by 3H-thymidine incorporation, whereas plasma cytokines and hormones levels by ELISA. Key findingsTranscripts of ANXA1, GILZ, NFKBIB, and NFKBIA appeared significantly increased in patients, whereas FKBP51, IL-6, IL-1β, and NF-κB remained unchanged. Upon analyzing according to disease severity, mRNA levels for ANXA1 and NFKBIB were even higher in moderate and severe patients. GILZ was increased in moderate cases, with NFKBIA and IL-1 β being higher in severe ones, who also displayed increased GRβ transcripts. TB patients had reduced plasma DHEA concentrations together with increased pro and anti-inflammatory cytokines (IFN-γ, IL-6, and IL-10) cortisol and cortisol/DHEA ratio, more evident in progressive cases, in whom their PBMC also showed a decreased mycobacterial-driven proliferation. The cortisol/DHEA ratio and GRα expression were positively correlated with GR-regulated genes mainly in moderate patients. SignificanceThe increased expression of cortisol-regulated anti-inflammatory genes in TB patients-PBMC, predominantly in progressive disease, seems compatible with a relatively insufficient attempt to downregulate the accompanying inflammation.

NUSAP1 Could be a Potential Target for Preventing NAFLD Progression to Liver Cancer.

Background: Non-alcoholic fatty liver disease (NAFLD) has gradually emerged as the most prevalent cause of chronic liver diseases. However, specific changes during the progression of NAFLD from non-fibrosis to advanced fibrosis and then hepatocellular carcinoma (HCC) are unresolved. Here, we firstly identify the key gene linking NAFLD fibrosis and HCC through analysis and experimental verification. Methods: Two GEO datasets (GSE89632, GSE49541) were performed for identifying differentially expressed genes (DEGs) associated with NAFLD progression from non-fibrosis to early fibrosis and eventually to advanced fibrosis. Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, protein-protein interaction (PPI) network were integrated to explore the potential function of the DEGs and hub genes. The expression of NUSAP1 was confirmed in vivo and in vitro NAFLD models at mRNA and protein level. Then, cell proliferation and migration under high fat conditions were verified by cell counting kit-8 (CCK-8) and wound-healing assays. The lipid content was measured with Oil Red O staining. Finally, the analysis of clinical survival curves was performed to reveal the prognostic value of the crucial genes among HCC patients via the online web-tool GEPIA2 and KM plotter. Results: 5510 DEGs associated with non-fibrosis NAFLD, 3913 DEGs about NAFLD fibrosis, and 739 DEGs related to NAFLD progression from mild fibrosis to advanced fibrosis were identified. Then, a total of 112 common DEGs were found. The result of enrichment analyses suggested that common DEGs were strongly associated with the glucocorticoid receptor pathway, regulation of transmembrane transporter activity, peroxisome, and proteoglycan biosynthetic process. Six genes, including KIAA0101, NUSAP1, UHRF1, RAD51AP1, KIF22, and ZWINT, were identified as crucial candidate genes via the PPI network. The expression of NUSAP1 was validated highly expressed in vitro and vivo NAFLD models at mRNA and protein level. NUSAP1 silence could inhibit the ability of cell proliferation, migration and lipid accumulation in vitro. Finally, we also found that NUSAP1 was significantly up-regulated at transcriptional and protein levels, and associated with poor survival and advanced tumor stage among HCC patients. Conclusion: NUSAP1 may be a potential therapeutic target for preventing NAFLD progression to liver cancer.

Antiproliferative Activity of Two Unusual Dimeric Flavonoids, Brachydin E and Brachydin F, Isolated from Fridericia platyphylla (Cham.) L.G.Lohmann: In Vitro and Molecular Docking Evaluation.

Despite the breakthrough in the development of anticancer therapies, plant-derived chemotherapeutics continue to be the basis of treatment for most types of cancers. Fridericia platyphylla is a shrub found in Brazilian cerrado biome which has cytotoxic, anti-inflammatory, and analgesic properties. The aim of this study was to investigate the antiproliferative potential of the crude hydroethanolic extract, subfraction (containing 59.3% of unusual dimeric flavonoids Brachydin E and 40.7% Brachydin F), as well as Brachydin E and Brachydin F isolated from F. platyphylla roots. The cytotoxic activity was evaluated in glioblastoma, lung, prostate, and colorectal human tumor cell lines. The crude hydroethanolic extract did not present cytotoxic activity, but its subfraction presented lower IC50 values for glioblastoma (U-251) and prostate adenocarcinoma (PC-3) cell lines. Brachydins E and F significantly reduced cell viability, proliferation, and clonogenic potential of PC-3, inducing them to the process of regulated cell death. In silico studies have indicated nuclear receptors as targets for Brachydins E and F, and molecular docking has pointed out their binding into glucocorticoid receptor (GR) ligand pocket. Targeting GR pathway has been described as a therapeutic strategy, especially for prostate cancer. These results suggest that Brachydin E and Brachydin F are promising compounds to be further explored for their antitumor effects.

Constructing a novel competing Endogenous RNAs network based on NR3C1 and X-linked inhibitor of apoptosis protein genes reveals potential prognostic biomarkers in colorectal cancer.

Long noncoding RNAs (lncRNAs) have been recognized as the main modulatory molecules in various cancers and perform as competing endogenous RNAs (ceRNAs). The nuclear hormone receptor superfamily of ligand-activated transcription factors (NR3C1) regulates numerous proliferative and metabolic processes such as tumorigenesis and metabolic diseases. Furthermore, X-linked inhibitor of apoptosis protein (XIAP) belongs to a family of the inhibitors of apoptosis proteins, is located downstream of the glucocorticoid receptor (GR or NR3C1) pathway, and cooperates with GR to suppress apoptosis. However, the underlying mechanisms of NR3C1 and XIAP in colorectal cancer (CRC) remain mainly unclear. This research aims to clarify the potential RNA biomarkers and to construct a novel ceRNA network in CRC. Multistep bioinformatics methods such as Lnc2cancer and miRDB databases were applied to identify candidate lncRNAs and miRNAs. The interaction energy between lncRNAs, NR3C1, and XIAP genes was analyzed by the LncRRIsearch database. Plus, microRNAs and lncRNA were evaluated via the Diana tools database to select microRNAs with the most binding scores. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was applied to verify RNA molecules' expression levels and their association with the clinicopathological factors in 30 CRC tissues compared to 30 adjacent tissues. QRT-PCR showed upregulation of KCNQ1OT1, NR3C1, and XIAP and downregulation of miR-421. The ceRNA network was constructed with 17 lncRNAs, 2 mRNAs, and 42 miRNAs. Thus, we explained the potential interactions between KCNQ1OT1 and miR-421 with NR3C1 and XIAP genes. Our study represents potential prognostic biomarkers and a new ceRNA network for further study in CRC.

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

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

Transcriptomic signaling pathways involved in a naturalistic model of inflammation-related depression and its remission

This study aimed at identifying molecular biomarkers of inflammation-related depression in order to improve diagnosis and treatment. For this, we performed whole-genome expression profiling from peripheral blood in a naturalistic model of inflammation-associated major depressive disorder (MDD) represented by comorbid depression in obese patients. We took advantage of the marked reduction of depressive symptoms and inflammation following bariatric surgery to test the robustness of the identified biomarkers. Depression was assessed during a clinical interview using Mini-International Neuropsychiatric Interview and the 10-item, clinician-administered, Montgomery–Asberg Depression Rating Scale. From a cohort of 100 massively obese patients, we selected 33 of them for transcriptomic analysis. Twenty-four of them were again analyzed 4–12 months after bariatric surgery. We conducted differential gene expression analyses before and after surgery in unmedicated MDD and non-depressed obese subjects. We found that TP53 (Tumor Protein 53), GR (Glucocorticoid Receptor), and NFκB (Nuclear Factor kappa B) pathways were the most discriminating pathways associated with inflammation-related MDD. These signaling pathways were processed in composite z-scores of gene expression that were used as biomarkers in regression analyses. Results showed that these transcriptomic biomarkers highly predicted depressive symptom intensity at baseline and their remission after bariatric surgery. While inflammation was present in all patients, GR signaling over-activation was found only in depressed ones where it may further increase inflammatory and apoptosis pathways. In conclusion, using an original model of inflammation-related depression and its remission without antidepressants, we provide molecular predictors of inflammation-related MDD and new insights in the molecular pathways involved.

Adolescent stress sensitizes the adult neuroimmune transcriptome and leads to sex-specific microglial and behavioral phenotypes.

Adolescent exposure to chronic stress, a risk factor for mood disorders in adulthood, sensitizes the neuroinflammatory response to a subsequent immune challenge. We previously showed that chronic adolescent stress (CAS) in rats led to distinct patterns of neuroimmune priming in adult male and female rats. However, sex differences in the neuroimmune consequences of CAS and their underlying mechanisms are not fully understood. Here we hypothesized that biological sex would dictate differential induction of inflammation-related transcriptomic pathways and immune cell involvement (microglia activation and leukocyte presence) in the hippocampus of male and female rats with a history of CAS. Adolescent rats underwent CAS (six restraint and six social defeat episodes during postnatal days 38-49), and behavioral assessments were conducted in adolescence and adulthood. Neuroimmune measures were obtained following vehicle or a systemic lipopolysaccharide (LPS) challenge in adulthood. CAS led to increased time in the corners of the open field in adolescence. In males, CAS also increased social avoidance. As adults, CAS rats displayed an exaggerated enrichment of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway and chemokine induction following LPS challenge, and increased number of perivascular CD45+ cells in the hippocampus. However, CAS females, but not males, showed exaggerated glucocorticoid receptor (GR) pathway enrichment and increased microglial complexity. These results provide further insight to the mechanisms by which peripheral immune events may influence neuroimmune responses differentially among males and females and further demonstrate the importance of adolescent stress in shaping adult responses.

Regulation of Gut Microbiota Disrupts the Glucocorticoid Receptor Pathway and Inflammation-related Pathways in the Mouse Hippocampus.

An increasing number of studies have recently indicated the important effects of gut microbes on various functions of the central nervous system. However, the underlying mechanisms by which gut microbiota regulate brain functions and behavioral phenotypes remain largely unknown. We therefore used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to obtain proteomic profiles of the hippocampus in germ-free (GF), colonized GF, and specific pathogen-free (SPF) mice. We then integrated the resulting proteomic data with previously reported mRNA microarray data, to further explore the effects of gut microbes on host brain functions. We identified that 61 proteins were upregulated and 242 proteins were downregulated in GF mice compared with SPF mice. Of these, 124 proteins were significantly restored following gut microbiota colonization. Bioinformatic analysis of these significant proteins indicated that the glucocorticoid receptor signaling pathway and inflammation-related pathways were the most enriched disrupted pathways. This study provides new insights into the pathological mechanisms of gut microbiota-regulated diseases.