Investigation of selective glucocorticoid receptor modulation in high-grade serous ovarian cancer PDX models.

OBJECTIVE: In ovarian cancer (OvCa), tumor cell high glucocorticoid receptor (GR) has been associated with poor patient prognosis. In vitro, GR activation inhibits chemotherapy-induced OvCa cell death in association with transcriptional upregulation of genes encoding anti-apoptotic proteins. A recent randomized phase II study demonstrated improvement in progression-free survival (PFS) for heavily pre-treated OvCa patients randomized to receive therapy with a selective GR modulator (SGRM) plus chemotherapy compared to chemotherapy alone. We hypothesized that SGRM therapy would improve carboplatin response in OvCa patient-derived xenograft (PDX). METHODS: Six high-grade serous (HGS) OvCa PDX models expressing GR mRNA (NR3C1) and protein were treated with chemotherapy +/- SGRM. Tumor size was measured longitudinally by peritoneal transcutaneous ultrasonography. RESULTS: One of the 6 GR-positive PDX models showed a significant improvement in PFS with the addition of a SGRM. Interestingly, the single model with an improved PFS was least carboplatin sensitive. Possible explanations for the modest SGRM activity include the high carboplatin sensitivity of 5 of the PDX tumors and the potential that SGRMs activate the tumor invasive immune cells in patients (absent from immunocompromised mice). The level of tumor GR protein expression alone appears insufficient for predicting SGRM response. CONCLUSION: The significant improvement in PFS shown in 1 of the 6 models after treatment with a SGRM plus chemotherapy underscores the need to determine predictive biomarkers for SGRM therapy in HGS OvCa and to better identify patient subgroups that are most likely to benefit from adding GR modulation to chemotherapy.

FXR Agonism with Bile Acid Mimetic Reduces Pre-Clinical Triple-Negative Breast Cancer Burden.

Bariatric surgery is associated with improved outcomes for several cancers, including breast cancer (BC), although the mechanisms mediating this protection are unknown. We hypothesized that elevated bile acid pools detected after bariatric surgery may be factors that contribute to improved BC outcomes. Patients with greater expression of the bile acid receptor FXR displayed improved survival in specific aggressive BC subtypes. FXR is a nuclear hormone receptor activated by primary bile acids. Therefore, we posited that activating FXR using an established FDA-approved agonist would induce anticancer effects. Using in vivo and in vitro approaches, we determined the anti-tumor potential of bile acid receptor agonism. Indeed, FXR agonism by the bile acid mimetic known commercially as Ocaliva ("OCA"), or Obeticholic acid (INT-747), significantly reduced BC progression and overall tumor burden in a pre-clinical model. The transcriptomic analysis of tumors in mice subjected to OCA treatment revealed differential gene expression patterns compared to vehicle controls. Notably, there was a significant down-regulation of the oncogenic transcription factor MAX (MYC-associated factor X), which interacts with the oncogene MYC. Gene set enrichment analysis (GSEA) further demonstrated a statistically significant downregulation of the Hallmark MYC-related gene set (MYC Target V1) following OCA treatment. In human and murine BC analyses in vitro, agonism of FXR significantly and dose-dependently inhibited proliferation, migration, and viability. In contrast, the synthetic agonism of another common bile acid receptor, the G protein-coupled bile acid receptor TGR5 (GPBAR1) which is mainly activated by secondary bile acids, failed to significantly alter cancer cell dynamics. In conclusion, agonism of FXR by primary bile acid memetic OCA yields potent anti-tumor effects potentially through inhibition of proliferation and migration and reduced cell viability. These findings suggest that FXR is a tumor suppressor gene with a high potential for use in personalized therapeutic strategies for individuals with BC.

Vitamin D Receptor Polymorphisms in a Spanish Cohort of Parkinson's Disease Patients.

Background: Vitamin D receptor (VDR) is a nuclear hormone receptor widely expressed in the substantia nigra. Its association with an increased risk of Parkinson's disease (PD) is based on vitamin D deficiency and/or different polymorphisms in its gene receptor. This fact has been demonstrated by several case-control studies. Materials and Methods: Consequently, we investigated the association between VDR ApaI, BsmI, FokI, and TaqI gene polymorphisms and PD in a Spanish cohort that included 54 cases and 17 healthy controls. The detection of single nucleotide polymorphisms (SNPs) was performed using a polymerase chain reaction-restriction fragment length polymorphism. Results: Our data indicate that the SNPs were not associated with the age of onset of PD, nor with the occurrence of motor symptoms. However, only BsmI polymorphism was significantly associated with PD in this Spanish cohort. In fact, BsmI genotype was five times higher among PD patients than among controls, and the A allele was considered as a genetic risk for PD. Additionally, the combination of FokI and BsmI polymorphisms was significantly associated with PD and could represent a risk factor. Conclusion: We conclude that ApaI, TaqI, and FokI polymorphisms were not associated with PD, but BsmI could be a risk factor for PD in this randomized population.

The steroid hormone ADIOL promotes learning by reducing neural kynurenic acid levels.

Reductions in brain kynurenic acid levels, a neuroinhibitory metabolite, improve cognitive function in diverse organisms. Thus, modulation of kynurenic acid levels is thought to have therapeutic potential in a range of brain disorders. Here we report that the steroid 5-androstene 3ß, 17ß-diol (ADIOL) reduces kynurenic acid levels and promotes associative learning in Caenorhabditis elegans We identify the molecular mechanisms through which ADIOL links peripheral metabolic pathways to neural mechanisms of learning capacity. Moreover, we show that in aged animals, which normally experience rapid cognitive decline, ADIOL improves learning capacity. The molecular mechanisms that underlie the biosynthesis of ADIOL as well as those through which it promotes kynurenic acid reduction are conserved in mammals. Thus, rather than a minor intermediate in the production of sex steroids, ADIOL is an endogenous hormone that potently regulates learning capacity by causing reductions in neural kynurenic acid levels.

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.

Mitotic genome-bookmarking by nuclear hormone receptors: A novel dimension in epigenetic reprogramming and disease assessment.

Arrival of multi-colored fluorescent proteins and advances in live cell imaging has immensely contributed to our understanding of intracellular trafficking of nuclear receptors and their roles in gene regulatory functions. These regulatory events need to be faithfully propagated from progenitor to progeny cells. This is corroborated by multiple converging mechanisms that include histone modifications and lately, the phenomenon of 'mitotic genome-bookmarking' by specific transcription factors. This phenomenon refers to the retention and feed-forward transmission of progenitor's architectural blueprint of active transcription status which is silenced and preserved during mitosis. Upon mitotic exit, this phenomenon ensures accurate reactivation of transcriptome, proteome, cellular traits and phenotypes in the progeny cells. In addition to diverse modes of genome-bookmarking by nuclear receptors, a correlation between disease-associated receptor polymorphism and disruption of this phenomenon is apparent. However, breakthrough technologies shall reveal finer details of this phenomenon to help achieve normalcy in receptor-specific diseases.

Loss of functional peroxisomes leads to increased mitochondrial biogenesis and reduced autophagy that preserve mitochondrial function.

Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in mitochondria are a function of preserving cellular function or a response to cellular damage caused by the absence of peroxisomes. To address this, we developed conditional hepatocyte-specific Pex16 deficient (Pex16 KO) mice that develop peroxisome loss and subjected them to a low-protein diet to induce metabolic stress. Loss of PEX16 in hepatocytes led to increased biogenesis of small mitochondria and reduced autophagy flux but with preserved capacity for respiration and ATP capacity. Metabolic stress induced by low protein feeding led to mitochondrial dysfunction in Pex16 KO mice and impaired biogenesis. Activation of PPARα partially corrected these mitochondrial disturbances, despite the absence of peroxisomes. The findings of this study demonstrate that the absence of peroxisomes in hepatocytes results in a concerted effort to preserve mitochondrial function, including increased mitochondrial biogenesis, altered morphology, and modified autophagy activity. Our study underscores the relationship between peroxisomes and mitochondria in regulating the hepatic metabolic responses to nutritional stressors.

NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans.

Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.

Constitutively Active Androgen Receptor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the predominant type of liver cancer and a leading cause of cancer-related death globally. It is also a sexually dimorphic disease with a male predominance both in HCC and in its precursors, non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of the androgen receptor (AR) in HCC has been well documented; however, AR-targeted therapies have failed to demonstrate efficacy in HCC. Building upon understandings of AR in prostate cancer (PCa), this review examines the role of AR in HCC, non-androgen-mediated mechanisms of induced AR expression, the existence of AR splice variants (AR-SV) in HCC and concludes by surveying current AR-targeted therapeutic approaches in PCa that show potential for efficacy in HCC in light of AR-SV expression.

Loss of heat shock factor initiates intracellular lipid surveillance by actin destabilization.

Cells sense stress and initiate response pathways to maintain lipid and protein homeostasis. However, the interplay between these adaptive mechanisms is unclear. Herein, we demonstrate how imbalances in cytosolic protein homeostasis affect intracellular lipid surveillance. Independent of its ancient thermo-protective properties, the heat shock factor, HSF-1, modulates lipid metabolism and age regulation through the metazoan-specific nuclear hormone receptor, NHR-49. Reduced hsf-1 expression destabilizes the Caenorhabditis elegans enteric actin network, subsequently disrupting Rab GTPase-mediated trafficking and cell-surface residency of nutrient transporters. The ensuing malabsorption limits lipid availability, thereby activating the intracellular lipid surveillance response through vesicular release and nuclear translocation of NHR-49 to both increase nutrient absorption and restore lipid homeostasis. Overall, cooperation between these regulators of cytosolic protein homeostasis and lipid surveillance ensures metabolic health and age progression through actin integrity, endocytic recycling, and lipid sensing.

NHR-23 and SPE-44 regulate distinct sets of genes during Caenorhabditis elegans spermatogenesis.

Spermatogenesis is the process through which mature male gametes are formed and is necessary for the transmission of genetic information. While much work has established how sperm fate is promoted and maintained, less is known about how the sperm morphogenesis program is executed. We previously identified a novel role for the nuclear hormone receptor transcription factor, NHR-23, in promoting Caenorhabditis elegans spermatogenesis. The depletion of NHR-23 along with SPE-44, another transcription factor that promotes spermatogenesis, caused additive phenotypes. Through RNA-seq, we determined that NHR-23 and SPE-44 regulate distinct sets of genes. The depletion of both NHR-23 and SPE-44 produced yet another set of differentially regulated genes. NHR-23-regulated genes are enriched in phosphatases, consistent with the switch from genome quiescence to post-translational regulation in spermatids. In the parasitic nematode Ascaris suum, MFP1 and MFP2 control the polymerization of Major Sperm Protein, the molecule that drives sperm motility and serves as a signal to promote ovulation. NHR-23 and SPE-44 regulate several MFP2 paralogs, and NHR-23 depletion from the male germline caused defective localization of MSD/MFP1 and NSPH-2/MFP2. Although NHR-23 and SPE-44 do not transcriptionally regulate the casein kinase gene spe-6, a key regulator of sperm development, SPE-6 protein is lost following NHR-23+SPE-44 depletion. Together, these experiments provide the first mechanistic insight into how NHR-23 promotes spermatogenesis and an entry point to understanding the synthetic genetic interaction between nhr-23 and spe-44.

Lactic acid bacteria-derived γ-linolenic acid metabolites are PPARδ ligands that reduce lipid accumulation in human intestinal organoids.

Gut microbiota regulate physiological functions in various hosts, such as energy metabolism and immunity. Lactic acid bacteria, including Lactobacillus plantarum, have a specific polyunsaturated fatty acid saturation metabolism that generates multiple fatty acid species, such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and trans-fatty acids. How these bacterial metabolites impact host physiology is not fully understood. Here, we investigated the ligand activity of lactic acid bacteria-produced fatty acids in relation to nuclear hormone receptors expressed in the small intestine. Our reporter assays revealed two bacterial metabolites of γ-linolenic acid (GLA), 13-hydroxy-cis-6,cis-9-octadecadienoic acid (γHYD), and 13-oxo-cis-6,cis-9-octadecadienoic acid (γKetoD) activated peroxisome proliferator-activated receptor delta (PPARδ) more potently than GLA. We demonstrate that both γHYD and γKetoD bound directly to the ligand-binding domain of human PPARδ. A docking simulation indicated that four polar residues (T289, H323, H449, and Y473) of PPARδ donate hydrogen bonds to these fatty acids. Interestingly, T289 does not donate a hydrogen bond to GLA, suggesting that bacterial modification of GLA introducing hydroxy and oxo group determines ligand selectivity. In human intestinal organoids, we determined γHYD and γKetoD increased the expression of PPARδ target genes, enhanced fatty acid ß-oxidation, and reduced intracellular triglyceride accumulation. These findings suggest that γHYD and γKetoD, which gut lactic acid bacteria could generate, are naturally occurring PPARδ ligands in the intestinal tract and may improve lipid metabolism in the human intestine.

COUP-TFII in Kidneys, from Embryos to Sick Adults.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear hormone receptor of unknown ligands. This molecule has two interesting features: (1) it is a developmental gene, and (2) it is a potential hormone receptor. Here, we describe the possible roles of COUP-TFII in the organogenesis of the kidneys and protection from adult renal diseases, primarily in mouse models. COUP-TFII is highly expressed in embryos, including primordial kidneys, and is essential for the formation of metanephric mesenchyme and the survival of renal precursor cells. Although the expression levels of COUP-TFII are low and its functions are unknown in healthy adults, it serves as a reno-protectant molecule against acute kidney injury. These are good examples of how developmental genes exhibit novel functions in the etiology of adult diseases. We also discuss the ongoing research on the roles of COUP-TFII in podocyte development and diabetic kidney disease. In addition, the identification of potential ligands suggests that COUP-TFII might be a novel therapeutic target for renal diseases in the future.

Remembering your A, B, C's: Alzheimer's disease and ABCA1.

The function of ATP binding cassette protein A1 (ABCA1) is central to cholesterol mobilization. Reduced ABCA1 expression or activity is implicated in Alzheimer's disease (AD) and other disorders. Therapeutic approaches to boost ABCA1 activity have yet to be translated successfully to the clinic. The risk factors for AD development and progression, including comorbid disorders such as type 2 diabetes and cardiovascular disease, highlight the intersection of cholesterol transport and inflammation. Upregulation of ABCA1 can positively impact APOE lipidation, insulin sensitivity, peripheral vascular and blood-brain barrier integrity, and anti-inflammatory signaling. Various strategies towards ABCA1-boosting compounds have been described, with a bias toward nuclear hormone receptor (NHR) agonists. These agonists display beneficial preclinical effects; however, important side effects have limited development. In particular, ligands that bind liver X receptor (LXR), the primary NHR that controls ABCA1 expression, have shown positive effects in AD mouse models; however, lipogenesis and unwanted increases in triglyceride production are often observed. The longstanding approach, focusing on LXRß vs. LXRα selectivity, is over-simplistic and has failed. Novel approaches such as phenotypic screening may lead to small molecule NHR modulators that elevate ABCA1 function without inducing lipogenesis and are clinically translatable.

Mechanisms of Feedback Regulation of Vitamin A Metabolism.

Vitamin A is an essential nutrient required throughout life. Through its various metabolites, vitamin A sustains fetal development, immunity, vision, and the maintenance, regulation, and repair of adult tissues. Abnormal tissue levels of the vitamin A metabolite, retinoic acid, can result in detrimental effects which can include congenital defects, immune deficiencies, proliferative defects, and toxicity. For this reason, intricate feedback mechanisms have evolved to allow tissues to generate appropriate levels of active retinoid metabolites despite variations in the level and format, or in the absorption and conversion efficiency of dietary vitamin A precursors. Here, we review basic mechanisms that govern vitamin A signaling and metabolism, and we focus on retinoic acid-controlled feedback mechanisms that contribute to vitamin A homeostasis. Several approaches to investigate mechanistic details of the vitamin A homeostatic regulation using genomic, gene editing, and chromatin capture technologies are also discussed.

NHR-80 senses the mitochondrial UPR to rewire citrate metabolism for lipid accumulation in Caenorhabditis elegans.

Mitochondria are known as the powerhouse of the cell. Dysfunction of mitochondria homeostasis induces the mitochondrial unfolded protein response (UPRmt), altering cellular metabolism. How cells sense the UPRmt to rewire metabolism is largely unknown. Here, we show that inactivation of either the citric/tricarboxylic acid (TCA) cycle enzymes aco-2 or idha-1, which encode aconitase and isocitrate dehydrogenase respectively, leads to citrate accumulation. In Caenorhabditis elegans, both in vitro and in vivo, citrate accumulation consequently triggers the UPRmt and also promotes lipid accumulation. The transcription factor DVE-1 binds to the promoter of the nuclear hormone receptor nhr-80 to transactivate its expression. NHR-80 then upregulates lipogenesis and lipid accumulation, shifting excess citrate for use in lipogenesis and for storage as triacylglycerol in lipid droplets. Inactivation of DVE-1 or NHR-80 fully abolishes the citrate-induced lipid accumulation. Therefore, our work uncovers a DVE-1-NHR-80-lipogenesis axis linking the transmission of the mitochondrial stress signal to lipid metabolism.

Differential expression of nuclear hormone receptors by dendritic cell subsets in human vaginal mucosa and skin.

Nuclear hormone receptors (NHRs) expressed by dendritic cells (DCs), the major immune inducers and regulators, could play important roles in host immunity. Assessment of NHRs expressed by DCs in the vaginal mucosa (VM), in comparison with those expressed by DCs in other tissues, will thus help us understand the immunology of human vagina. This study identified 16 NHR transcripts that are differentially expressed among 8 different antigen-presenting cell (APC) subsets isolated from human VM, skin, and blood. The expression profiles of NHRs were largely tissue specific. VM APCs expressed increased levels of LXRA, RXRA, ESRRA, ESRRAP2, and PPARG, whereas skin and blood APCs expressed increased levels of NURR1, NOR1 and RARA. Of interest, female sex hormone receptors, ESR1 and PGR, were found to be mainly expressed by non-APC cell types in the VM; ESR1 by HLA-DR+CD34+ and PGR by HLA-DR- cells. ERα and PR were expressed by vimentin+ cells in the VM, but not in human skin. ERα, but not PR, was also expressed in CD10+ cells in the lamina propria of VM. In conclusion, NHR expression by APC subsets is tissue- and cell type-specific. Future studies on the roles of individual NHRs expressed by different cell types, including DC subsets, in the human VM are warranted.

MicroRNAs based regulation of cytokine regulating immune expressed genes and their transcription factors in COVID-19.

BACKGROUND: Coronavirus disease 2019 is characterized by the elevation of a broad spectrum of inflammatory mediators associated with poor disease outcomes. We aimed at an in-silico analysis of regulatory microRNA and their transcription factors (TF) for these inflammatory genes that may help to devise potential therapeutic strategies in the future. METHODS: The cytokine regulating immune-expressed genes (CRIEG) were sorted from literature and the GEO microarray dataset. Their co-differentially expressed miRNA and transcription factors were predicted from publicly available databases. Enrichment analysis was done through mienturnet, MiEAA, Gene Ontology, and pathways predicted by KEGG and Reactome pathways. Finally, the functional and regulatory features were analyzed and visualized through Cytoscape. RESULTS: Sixteen CRIEG were observed to have a significant protein-protein interaction network. The ontological analysis revealed significantly enriched pathways for biological processes, molecular functions, and cellular components. The search performed in the miRNA database yielded ten miRNAs that are significantly involved in regulating these genes and their transcription factors. CONCLUSION: An in-silico representation of a network involving miRNAs, CRIEGs, and TF, which take part in the inflammatory response in COVID-19, has been elucidated. Thus, these regulatory factors may have potentially critical roles in the inflammatory response in COVID-19 and may be explored further to develop targeted therapeutic strategies and mechanistic validation.

Protocadherin 19 Clustering Epilepsy and Neurosteroids: Opportunities for Intervention.

Steroids yield great influence on neurological development through nuclear hormone receptor (NHR)-mediated gene regulation. We recently reported that cell adhesion molecule protocadherin 19 (encoded by the PCDH19 gene) is involved in the coregulation of steroid receptor activity on gene expression. PCDH19 variants cause early-onset developmental epileptic encephalopathy clustering epilepsy (CE), with altered steroidogenesis and NHR-related gene expression being identified in these individuals. The implication of hormonal pathways in CE pathogenesis has led to the investigation of various steroid-based antiepileptic drugs in the treatment of this disorder, with mixed results so far. Therefore, there are many unmet challenges in assessing the antiseizure targets and efficiency of steroid-based therapeutics for CE. We review and assess the evidence for and against the implication of neurosteroids in the pathogenesis of CE and in view of their possible clinical benefit.