Member Of Nuclear Receptor Family Research Articles

Molecular characterization and potential function of Rxrγ in gonadal differentiation of Chinese soft-shelled turtle (Pelodiscus sinensis)

Retinoid X receptor (RXR) is a member of the ligand-dependent nuclear receptor family. Previous studies revealed that RXRs are involved in reproduction in vertebrates. However, information on the function of RXRs in turtles is scarce. In this study, the Rxrγ cDNA sequence of Pelodiscus sinensis was cloned and analyzed, and a polyclonal antibody was constructed. RXRγ protein showed a positive signal in both mature and differentiated gonads of the turtle. Subsequently, the function of the Rxrγ gene in gonadal differentiation was confirmed using short interfering RNA (RNAi). The full-length cDNA sequence of the Rxrγ gene in P. sinensis was 2152 bp, encoding 407 amino acids and containing typical nuclear receptor family domains, including the DNA-binding domain (DBD), ligand-binding domain (LBD), and activation function 1 (AF1). Moreover, gonadal Ps-Rxrγ showed sexual dimorphism expression patterns in differentiated gonads. Real-time quantitative PCR results revealed that the Rxrγ gene was highly expressed in the turtle ovary. RNAi treatment increased the number of Sertoli cells in ZZ embryonic gonads. Furthermore, RNA interference upregulated Dmrt1 and Sox9 in ZZ and ZW embryonic gonads. However, Foxl2, Cyp19a1, Stra8, and Cyp26b1 were downregulated in embryonic gonads. The results indicated that Rxrγ participated in gonadal differentiation and development in P. sinensis.

The pro-inflammatory phenotype of human macrophages is restricted by the activity of the orphan nuclear receptor Nurr1

Abstract The nuclear receptor related 1 protein (Nurr1) is a member of the orphan nuclear hormone receptor family whose expression has been associated with several inflammatory diseases. Macrophages are key regulators of the inflammatory processes, yet information about the role of Nurr1 in human macrophages is limited. In the present study we examined the expression and activity of Nurr1 in steady state and activated human monocyte-derived macrophages. Pro- and anti-inflammatory macrophages were derived in vitro by culture of blood monocytes with the hematopoietic cytokines GM-CSF and M-CSF, respectively. Nurr1 expression was predominant in macrophages with a pro-inflammatory phenotype. Accordingly, its expression was induced by a variety of inflammatory stimuli such as TLR-2, -3 and -4 ligands, TNF, IFN-β, and IL-4. Pro-inflammatory macrophages exposed to the Nurr1 agonist C-DIM12 decreased their production of IL-1β, IL-6, and reactive oxygen species. Conversely, Nurr1 deficient macrophages produced enhanced levels of IL-6. Mechanistically, Nurr1 agonists partially abrogated the activation of the NF-κB signaling pathway in pro-inflammatory macrophages exposed to LPS, rendering cells with a lower rate of NF-κB p65 nuclear translocation. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages where it may constitute a brake to attenuate the synthesis of inflammatory mediators. Supported by grants from Secretaria de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)

Computational design and experimental confirmation of conformationally constrained peptides to compete with coactivators for pediatric PPAR[Formula: see text] by minimizing indirect readout effect.

The peroxisome proliferator-activated receptor-[Formula: see text] (PPAR[Formula: see text]) is a member of PPAR nuclear receptor family, and its antagonists have been widely used to treat pediatric metabolic disorders. Traditional type-1 and type-2 PPAR[Formula: see text] antagonists are all small-molecule compounds that have been developed to target the ligand-binding site (LBS) of PPAR[Formula: see text], which is not overlapped with the coactivator-interacting site (CIS) of PPAR[Formula: see text]. In this study, we described the rational design of type-3 peptidic antagonists that can directly disrupt PPAR[Formula: see text]-coactivator interaction by physically competing with coactivator proteins for the CIS site. In the procedure, seven reported PPAR[Formula: see text] coactivator proteins were collected and eight 11-mer helical peptide segments that contain the core PPAR[Formula: see text]-binding LXXLL motif were identified in these coactivators, which, however, possessed a large flexibility and intrinsic disorder when splitting from coactivator protein context, and thus would incur a considerable entropy penalty (i.e. indirect readout) upon binding to PPAR[Formula: see text] CIS site. By carefully examining the natively folded conformation of these helical peptides in their parent protein context and in their interaction mode with the CIS site, we rationally designed a hydrocarbon bridge across the solvent-exposed, ([Formula: see text], [Formula: see text]+ 4) residues to constrain their helical conformation, thus largely minimizing the unfavorable indirect readout effect but having only a moderate influence on favorable enthalpy contribution (i.e. direct readout) upon PPAR[Formula: see text]-peptide binding. The computational findings were further substantiated by fluorescence competition assays.

Impact of Phytochemicals on PPAR Receptors: Implications for Disease Treatments

Peroxisome proliferator-activated receptors (PPARs) are members of the ligand-dependent nuclear receptor family. PPARs have attracted wide attention as pharmacologic mediators to manage multiple diseases and their underlying signaling targets. They mediate a broad range of specific biological activities and multiple organ toxicity, including cellular differentiation, metabolic syndrome, cancer, atherosclerosis, neurodegeneration, cardiovascular diseases, and inflammation related to their up/downstream signaling pathways. Consequently, several types of selective PPAR ligands, such as fibrates and thiazolidinediones (TZDs), have been approved as their pharmacological agonists. Despite these advances, the use of PPAR agonists is known to cause adverse effects in various systems. Conversely, some naturally occurring PPAR agonists, including polyunsaturated fatty acids and natural endogenous PPAR agonists curcumin and resveratrol, have been introduced as safe agonists as a result of their clinical evidence or preclinical experiments. This review focuses on research on plant-derived active ingredients (natural phytochemicals) as potential safe and promising PPAR agonists. Moreover, it provides a comprehensive review and critique of the role of phytochemicals in PPARs-related diseases and provides an understanding of phytochemical-mediated PPAR-dependent and -independent cascades. The findings of this research will help to define the functions of phytochemicals as potent PPAR pharmacological agonists in underlying disease mechanisms and their related complications.

Analyzing the Androgen Receptor Interactome in Prostate Cancer: Implications for Therapeutic Intervention

The androgen receptor (AR) is a member of the ligand-activated nuclear receptor family of transcription factors. AR’s transactivation activity is turned on by the binding of androgens, the male sex steroid hormones. AR is critical for the development and maintenance of the male phenotype but has been recognized to also play an important role in human diseases. Most notably, AR is a major driver of prostate cancer (CaP) progression, which remains the second leading cause of cancer deaths in American men. Androgen deprivation therapies (ADTs) that interfere with interactions between AR and its activating androgen ligands have been the mainstay for treatment of metastatic CaP. Although ADTs are effective and induce remissions, eventually they fail, while the growth of the majority of ADT-resistant CaPs remains under AR’s control. Alternative approaches to inhibit AR activity and bypass resistance to ADT are being sought, such as preventing the interaction between AR and its cofactors and coregulators that is needed to execute AR-dependent transcription. For such strategies to be efficient, the 3D conformation of AR complexes needs to be well-understood and AR-regulator interaction sites resolved. Here, we review current insights into these 3D structures and the protein interaction sites in AR transcriptional complexes. We focus on methods and technological approaches used to identify AR interactors and discuss challenges and limitations that need to be overcome for efficient therapeutic AR complex disruption.

Signal Crosstalk and the Role of Estrogen Receptor beta (ERβ) in Prostate Cancer.

Prostate cancer remains the most prevalent cancer among men worldwide; however, as a sex hormone-dependent cancer, sex hormones and their receptor signaling play an important role in the development and progression of cancer. Most current treatment options for prostate cancer thus revolve around the inhibition of androgen signaling (eg, ADT), which, although effective in the early stages, eventually progresses to treatment-resistant prostate cancer with no effective follow-up options. Recent studies have shown that among the nuclear receptor family members, in addition to androgen receptors, estrogen receptor (ER) plays an important biological function as a transcription factor and regulatory protein in various cancers, acting either directly or indirectly by forming homodimers or heterodimers with ligands. In this paper, we review the application of ERβ in animal models and in vitro experiments in the last 5 years, as well as the presence and role of some of its splice variants. We summarize the overview and update of ERβ in prostate cancer, and provide a corresponding analysis of some current research disagreements. Its crosstalk action on some important cancer growth-related signaling pathways (eg, TGF-β and ERK), regulation of downstream target proteins (eg, nuclear translocation of EGFR and expression of oncogenic-related protein MMP-2), and interactions with related ERβ co-regulators (eg, ZFHX3), agonists, and antagonists in prostate cancer are highlighted, and the resulting effects on tumor progression are described. In addition, the paper describes its current potential clinical application as a novel therapeutic strategy and some of the challenges it faces.

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.

Aberrant mTOR/autophagy/Nurr1 signaling is critical for TSC-associated tumor development.

Tuberous sclerosis complex (TSC), an inherited neurocutaneous disease, is caused by mutations in either the TSC1 or TSC2 gene. This genetic disorder is characterized by the growth of benign tumors in the brain, kidneys, and other organs. As a member of the orphan nuclear receptor family, nuclear receptor related 1 (Nurr1) plays a vital role in some neuropathological diseases and several types of benign or malignant tumors. Here, we explored the potential regulatory role of TSC1/2 signaling in Nurr1 and the effect of Nurr1 in TSC-related tumors. We found that Nurr1 expression was drastically decreased by the disruption of the TSC1/2 complex in Tsc2-null cells, genetically modified mouse models of TSC, cortical tubers of TSC patients, and kidney tumor tissue obtained from a TSC patient. Deficient TSC1/2 complex downregulated Nurr1 expression in an mTOR-dependent manner. Moreover, hyperactivation of mTOR reduced Nurr1 expression via suppression of autophagy. In addition, Nurr1 overexpression inhibited cell proliferation and suppressed cell cycle progression. Therefore, TSC/mTOR/autophagy/Nurr1 signaling is partially responsible for the tumorigenesis of TSC. Taken together, Nurr1 may be a novel therapeutic target for TSC-associated tumors, and Nurr1 agonists or reagents that induce Nurr1 expression may be used for the treatment of TSC.

The estrogen-related receptor γ modulator, GSK5182, inhibits osteoclast differentiation and accelerates osteoclast apoptosis

Estrogen-related receptor γ (ERRγ), a member of the orphan nuclear receptor family, is a key mediator in cellular metabolic processes and energy homeostasis. Therefore, ERRγ has become an attractive target for treating diverse metabolic disorders. We recently reported that ERRγ acts as a negative regulator of osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In the present study, we explored the effects of an ERRγ-specific modulator, GSK5182, on ERRγ-regulated osteoclast differentiation and survival. Interestingly, GSK5182 increased ERRγ protein levels much as does GSK4716, which is an ERRγ agonist. GSK5182 inhibited osteoclast generation from bone-marrow-derived macrophages without affecting cytotoxicity. GSK5182 also attenuated RANKL-mediated expression of c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), pivotal transcription factors for osteoclastogenesis. Arrested osteoclast differentiation was associated with reduced RANK expression, but not with the M-CSF receptor, c-Fms. GSK5182 strongly blocked the phosphorylation of IκBα, c-Jun N-terminal kinase, and extracellular signal-regulated kinase in response to RANKL. GSK5182 also suppressed NF-κB promoter activity in a dose-dependent manner. In addition to osteoclastogenesis, GSK5182 accelerated osteoclast apoptosis by caspase-3 activation. Together, these results suggest that GSK5182, a synthetic ERRγ modulator, may have potential in treating disorders related to bone resorption.

A PROSS-designed extensively mutated estrogen receptor \u03b1 variant displays enhanced thermal stability while retaining native allosteric regulation and structure

Protein stability limitations often hamper the exploration of proteins as drug targets. Here, we show that the application of PROSS server algorithms to the ligand-binding domain of human estrogen receptor alpha (hERα) enabled the development of variant ERPRS* that comprises 24 amino acid substitutions and exhibits multiple improved characteristics. The protein displays enhanced production rates in E. coli, crystallizes readily and its thermal stability is increased significantly by 23 °C. hERα is a nuclear receptor (NR) family member. In NRs, protein function is allosterically regulated by its interplay with small molecule effectors and the interaction with coregulatory proteins. The in-depth characterization of ERPRS* shows that these cooperative effects are fully preserved despite that 10% of all residues were substituted. Crystal structures reveal several salient features, i.e. the introduction of a tyrosine corner in a helix-loop-helix segment and the formation of a novel surface salt bridge network possibly explaining the enhanced thermal stability. ERPRS* shows that prior successes in computational approaches for stabilizing proteins can be extended to proteins with complex allosteric regulatory behaviors as present in NRs. Since NRs including hERα are implicated in multiple diseases, our ERPRS* variant shows significant promise for facilitating the development of novel hERα modulators.

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Retinoid X receptor alpha (RXRα), a nuclear receptor of transcription factor, controls various physiological and pathological pathways including cellular growth, proliferation, differentiation, and apoptosis. Here, we report that RXRα is phosphorylated at its N-terminal A/B domain by cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis, triggering its translocation to the centrosome, where phosphorylated-RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) through its N-terminal A/B domain by a unique mechanism. The interaction promotes PLK1 activation, centrosome maturation, and mitotic progression. Levels of p-RXRα are abnormally elevated in cancer cell lines, during carcinogenesis in animals, and in clinical tumor tissues. An RXRα ligand XS060, which specifically inhibits p-RXRα/PLK1 interaction but not RXRα heterodimerization, promotes mitotic arrest and catastrophe in a tumor-specific manner. These findings unravel a transcription-independent action of RXRα at the centrosome during mitosis and identify p-RXRα as a tumor-specific vulnerability for developing mitotic drugs with improved therapeutic index.

Therapeutic Targeting of MDR1 Expression by RORγ Antagonists Resensitizes Cross-Resistant CRPC to Taxane via Coordinated Induction of Cell Death Programs.

Overexpression of ATP-binding cassette subfamily B member 1 (ABCB1)-encoded multidrug resistance protein 1 (MDR1) constitutes a major mechanism of cancer drug resistance including docetaxel (DTX) and cabazitaxel (CTX) resistance in castration-resistant prostate cancer (CRPC). However, no therapeutics that targets MDR1 is available at clinic for taxane sensitization. We report here that retinoic acid receptor-related orphan receptor γ (RORγ), a nuclear receptor family member, unexpectedly mediates MDR1/ABCB1 overexpression. RORγ plays an important role in controlling the functions of subsets of immune cells and has been an attractive target for autoimmune diseases. We found that its small-molecule antagonists are efficacious in resensitizing DTX and CTX cross-resistant CRPC cells and tumors to taxanes in both androgen receptor-positive and -negative models. Our mechanistic analyses revealed that combined treatment with RORγ antagonists and taxane elicited a robust synergy in killing the resistant cells, which involves a coordinated alteration of p53, Myc, and E2F-controlled programs critical for both intrinsic and extrinsic apoptosis, survival, and cell growth. Our results suggest that targeting RORγ with small-molecule inhibitors is a novel strategy for chemotherapy resensitization in tumors with MDR1 overexpression.

Change of Nurr1 expression in mouse hippocampal CA3 region following excitotoxic neuronal damage.

Objective(s):Nuclear receptor-related protein 1 (Nurr1), one of immediate-early genes, is a member of orphan nuclear receptor family. The aim of this study was to investigate the time-dependent change of Nurr1 protein expression in the mouse hippocampal CA3 region following kainic acid (KA)-induced excitotoxic neuronal damage.Materials and Methods:Male ICR mice were used as experimental animals, and 30 mg/kg KA was administered intraperitoneally. To confirm the KA-induced neuronal damage in the hippocampal CA3 region, Fluoro-Jade B histofluorescence staining was performed. In addition, the time-dependent change of Nurr1 protein expression was also examined using immunohistochemistry and western blot analysis. Results:Marked neuronal damage was observed in the hippocampal CA3 region at 24 hr after KA injection. In addition, both Nurr1 immunoreactivity and protein level were significantly increased at 6 hr and 12 hr after KA injection, and then decreased at 24 hr after KA injection.Conclusion:This result indicates that KA-induced alteration of Nurr1 protein expression may be associated with the neuronal degeneration in the hippocampal CA3 region after KA injection.

Estrogen receptor-α in female skeletal muscle is not required for regulation of muscle insulin sensitivity and mitochondrial regulation

ObjectiveEstrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. MethodsA novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women. ResultsTwelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. ConclusionsCollectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.

Abstract B037: Potential next-generation androgen receptor-targeted therapeutic for enzalutamide-resistant prostate cancer; In vivo characterization in immune-compromised SRG rats

Abstract Current treatment options for advanced castration-resistant prostate cancers (CRPC) are effective for a brief period before becoming refractory. It is important to use relevant in vivo models for candidate selection in the development of next-generation mechanistically-distinct drugs to treat castration- and drug- resistant prostate cancers. Here, we describe a series of AR pan-antagonists (selective AR degraders (SARDs)) that degrade the AR and AR splice variants. SARDs inhibit the wild-type and LBD mutant ARs comparably and inhibit the in vitro proliferation and in vivo growth of enzalutamide-sensitive and resistant prostate cancer xenografts. Xenograft studies conducted in immune-compromised SRG rats (Sprague Dawley Rag2-/- Il2rg -/-; Hera Biolabs) with three lead SARDs demonstrated regression of enzalutamide-sensitive and -resistant VCaP tumors both in castrated and in intact rats. SRG rats provide the benefit of abundant blood samples for weekly evaluation of rising PSA, which also indicated that the SARDs inhibit the rising PSA, while enzalutamide was inactive in enzalutamide-resistant model. Drug metabolism and pharmacokinetic (DMPK) studies, also conducted with SRG and wild-type rats and in combination with efficacy, indicate that the molecules possess all the necessary drug-like properties. The molecules exhibit a broad safety margin with no cross-reactivity with G-Protein Coupled Receptor, kinase, and nuclear receptor family members. Collectively, the SARDs exhibit the properties necessary for a next-generation prostate cancer drug and that use of SRG rats facilitated thorough characterization of their in vivo properties. Citation Format: Suriyan Udhayasuriyan Ponnusamy, Fallon K Noto, Bisoye Towobola Adedeji, Yali He, Dong-Jin Hwang, Sam Moody, Thirumagal Thiyagarajan, Tseten Yeshi Jamling, Duane D Miller, Ramesh Narayanan. Potential next-generation androgen receptor-targeted therapeutic for enzalutamide-resistant prostate cancer; In vivo characterization in immune-compromised SRG rats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B037. doi:10.1158/1535-7163.TARG-19-B037

Orally Bioavailable Androgen Receptor Degrader, Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer.

Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers. Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice. UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein-coupled receptor kinase and nuclear receptor family members. Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.

IPO5 promotes the proliferation and tumourigenicity of colorectal cancer cells by mediating RASAL2 nuclear transportation

BackgroundKaryopherin nuclear transport receptors play important roles in tumour development and drug resistance and have been reported as potential biomarkers and therapeutic targets for tumour treatment. However, IPO5, one of the karyopherin nuclear transport receptor family members, remains largely uncharacterized in tumour progression.MethodsThe TCGA data, quantitative reverse transcription-PCR (qRT-PCR), western blotting, and IHC analyses were used to detect IPO5 expression in CRC tissues. A series of in vivo and in vitro experiments was utilized to demonstrate the function of IPO5 in CRC tissues. Mass spectrometry (MS), CO-IP technology, subcellular fractionation, and immunofluorescence were utilized to investigate the possible mechanisms of CRC.ResultsIPO5 was highly expressed and positively correlated with the clinicopathological characteristics of colorectal cancer tissues. Functional experiments indicated that IPO5 could promote the development of CRC. Mechanistically, we screened RASAL2, one cargo of IPO5, and further confirmed that IPO5 bound to the NLS sequence of RASAL2, mediating RASAL2 nuclear translocation and inducing RAS signal activation, thereby promoting the progression of CRC.ConclusionsTogether, our results indicate that IPO5 is overexpressed in colorectal cancer cells. By transporting RASAL2, IPO5 may play a crucial role in CRC.

Abstract 5214: Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP

Abstract Luminal breast cancer represents approximately two thirds of all breast cancer cases and is characterized by the expression of hormone receptors, such as the estrogen receptor alpha (ER). ERα is a member of the steroid nuclear receptor family and is involved in a hormonal signaling pathway that drives tumor growth through upregulation of genes involved in cell cycle progression, such as MYC and CCND1. Antagonists of ER function are the standard of care treatment for ER+ luminal breast cancer. However, resistance to ER antagonists is common in advanced breast cancer cases. Therefore, there is an urgent need to investigate new therapeutics that can be utilized to treat tumors that are resistant to traditional therapies. p300/CBP are two paralogous acetyltransferases that catalyze histone 3, lysine 18 and 27 acetylation (H3K18ac and H3K27ac) at promoters and enhancers to promote gene expression. ER mediated transcription is critically reliant upon the recruitment of co-activators, including p300/CBP. However, the mechanistic role of p300/CBP catalytic activity in globally regulating ER mediated transcription remains poorly understood. Inhibition of p300/CBP, as a critical co-activator of ER, potentially represents an applicable strategy for inhibiting ER function in luminal breast tumors. Importantly, the catalytic histone acetyltransferase (HAT) domain of p300/CBP is under intense investigation as a target of small molecule therapeutics in cancer. We analyzed publicly available data and report that p300/CBP are upregulated at the mRNA level in breast cancer, including the luminal subtypes, and that their expression negatively correlates with patient survival. We also found that pharmacologic inhibition of p300/CBP HAT activity in ER+ cell lines potently suppresses ER mediated transcription, downregulates ER, c-Myc and Cyclin D1 protein levels and prevents estrogen induced growth in vitro. Studies are underway to understand how p300/CBP regulates ER signaling using genetic, pharmacologic, and biochemical methods to assess the therapeutic effects of pharmacologic p300/CBP inhibition on ER+ breast cancer. (Supported by Bankhead-Coley Research Program, and James and Esther King Biomedical Research Program, Florida Department of Health) Citation Format: Aaron Waddell, Iqbal Mahmud, Guimei Tian, Daiqing Liao. Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5214.

HEPATOPROTECTIVE ROLE OF LOVASTATIN - PREGNANE X RECEPTOR AGONIST IN LITHOCHOLIC ACID INDUCED LIVER CHOLESTASIS DISEASED RATS

Objective: Pregnane X receptor (PXR), member of nuclear receptor family, an integral component of the body defence mechanism against chemical insult are expressed in the liver, gastrointestinal system & lungs. Some studies have shown that the lovastatin is pregnane X receptor (PXR) activation effect.
 Methods: In the present study the hepatoprotective effect of lovastatin was investigated against lithocholic acid induced liver toxicity. Liver markers in serum and antioxidant enzymes in liver tissue were assessed by using standard procedures.
 Results: The level of liver marker (such as SGOT & SGPT) and bilirubin were increased significantly (p<0.05) and antioxidant enzyme (i.e. SOD, GSH and CAT) were significantly (p<0.05) decrease in lithocholic acid treated groups as compared to control group. Lovastatin at doses of 0.1, 0.2, 0.3 mg/kg showed significantly (p<0.05) decrease in the levels of liver marker (SGOT & SGPT) and bilirubin as compared to the positive control group in both pre & post treated models. The antioxidant enzymes such as Superoxide dismutase (SOD), Glutathione (GSH) and Catalase (CAT) content in liver tissue were significantly (p<0.05) increase after administration of lovastatin at dose dependent manner in both pre & post treated models.
 Conclusions: The results of the present study indicates that under the present experimental conditions, lovastatin showed hepatoprotective abilities against lithocholic acid induced hepatotoxicity in albino rat.
 Keywords: Hepatoprotection, lovastatin, Lithocholic Acid, Pregnane X Receptor

The FXR agonist obeticholic acid inhibits the cancerogenic potential of human cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive cancer with high resistance to chemotherapeutics. CCA is enriched in cancer stem cells, which correlate with aggressiveness and prognosis. FXR, a member of the metabolic nuclear receptor family, is markedly down-regulated in human CCA. Our aim was to evaluate, in primary cultures of human intrahepatic CCA (iCCA), the effects of the FXR agonist obeticholic acid (OCA), a semisynthetic bile acid derivative, on their cancerogenic potential. Primary human iCCA cell cultures were prepared from surgical specimens of mucinous or mixed iCCA subtypes. Increasing concentrations (0–2.5 μM) of OCA were added to culture media and, after 3–10 days, effects on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC/propidium iodide), cell migration and invasion (wound healing response and Matrigel invasion assay), and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR gene expression was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.05) and in mucinous iCCA vs mixed iCCA cells (p < 0.05) but was upregulated by addition of OCA. OCA significantly (p < 0.05) inhibited proliferation of both mucinous and mixed iCCA cells, starting at a concentration as low as 0.05 μM. Also, CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with its different affinity for FXR. OCA significantly induced apoptosis of both iCCA subtypes and decreased their in vitro cancerogenic potential, as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and Matrigel invasion. In general, these effects were more evident in mixed than mucinous iCCA cells. When tested together with Gemcitabine and Cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics, but mainly in mixed iCCA cells. OCA abolished the capacity of both mucinous and mixed iCCA cells to form colonies when administered together with Gemcitabine and Cisplatin. In subcutaneous xenografts of mixed iCCA cells, OCA alone or combined with Gemcitabine or Cisplatin markedly reduced the tumor size after 5 weeks of treatment by inducing necrosis of tumor mass and inhibiting cell proliferation. In conclusion, FXR is down-regulated in iCCA cells, and its activation by OCA results in anti-cancerogenic effects against mucinous and mixed iCCA cells, both in vitro and in vivo. The effects of OCA predominated in mixed iCCA cells, consistent with the lower aggressiveness and the higher FXR expression in this CCA subtype. These results, showing the FXR-mediated capacity of OCA to inhibit cholangiocarcinogenesis, represent the basis for testing OCA in clinical trials of CCA patients.