AR Activity Research Articles

Antiandrogenic and Estrogenic Activity Evaluation of Oxygenated and Nitrated Polycyclic Aromatic Hydrocarbons Using Chemically Activated Luciferase Expression Assays.

To establish the risk of the endocrine disrupting activity of polycyclic aromatic compounds, especially oxygenated and nitrated polycyclic aromatic hydrocarbons (oxy-PAHs and nitro-PAHs, respectively), antiandrogenic and estrogenic activities were determined using chemically activated luciferase expression (CALUX) assays with human osteoblast sarcoma cells. A total of 27 compounds including 9 oxy-PAHs (polycyclic aromatic ketones and quinones) and 8 nitro-PAHs was studied. The oxy-PAHs of 7H-benz[de]anthracen-7-one (BAO), 11H-benzo[a]fluoren-11-one (B[a]FO), 11H-benzo[b]fluoren-11-one (B[b]FO), and phenanthrenequinone (PhQ) exhibited significantly the potent inhibition of AR activation. All nitro-PAHs exhibited high antiandrogenic activities (especially high for 3-nitrofluoranthene (3-NFA) and 3-nitro-7H-benz[de]anthracen-7-one (3-NBAO)), and the AR inhibition was confirmed as noncompetitive for 3-NFA, 3-NBAO, and 1,3-dinitropyrene (1,3-DNPy). Antiandrogenic activity of 3-NFA demonstrated characteristically a U-shaped dose-response curve; however, the absence of fluorescence effect on the activity was confirmed. The prominent estrogenic activity dependent on dose-response curve was confirmed for 2 oxy-PAHs (i.e., B[a]FO and B[b]FO). Elucidating the role of AR and ER on the effects of polycyclic aromatic compounds (e.g., oxy- and nitro-PAHs) to endocrine dysfunctions in mammals and aquatic organisms remains a challenge.

Activation of androgen receptors alters hippocampal synaptic plasticity and memory retention through modulation of L-type calcium channels

AimsIt has been revealed that membrane androgen receptor activation modulates avoidance memory and synaptic plasticity. In a previous study, we showed that Calcineurin, a calcium dependent phosphatase, could be a potential mediator of these AR effects. Also, it is reported that AR activation leads to L-type calcium channel activation. The aim of the current study is to test whether L-type calcium channels are downstream of AR and whether this signal pathway mediates the impairment effect of androgenic steroids on passive avoidance memory and synaptic plasticity. Materials and methodsWe measured the effect of Nandrolone Decanoate (AR agonist), AR antagonist (Nilutamide) plus ND or L-type calcium channel inhibitor (Nifedipine) plus ND on passive avoidance performance of adolescent male rats. For extracellular field potential recordings hippocampal slices were perfused with ND, Nilutamide-ND or Nifedipine-ND. Key findingsOur results clarified that AR activation by ND could impair avoidance behavior as step through latency decreased in ND-treated group while application of both Nilutamide and Nifedipine reestablished normal avoidance behavior. Also, LTP induction in the CA1 area of hippocampus was diminished by ND perfusion and both AR antagonist and L-type calcium channel inhibitor application lead to normal LTP. These findings support our hypothesis that activation of L-type calcium channels are involved in ARs mechanism effects on both avoidance behavior and hippocampal synaptic plasticity. SignificanceUnderstanding the biological effects of AR agonists on cognitive processes and its cellular mechanism may be a new/supplementary way to treating fear-related disorders.

In vitro bioanalytical assessment of the occurrence and removal of bioactive chemicals in municipal wastewater treatment plants in Korea

Effluents of wastewater treatment plants (WWTPs) contain various organic micropollutants, some of which can exert negative effects on the quality of receiving waters or drinking water sources. This study monitored two full-scale WWTPs in Korea for the occurrence and removal of bioactive chemicals for a one-year period using a battery of in vitro bioassays as a complementary approach to chemical analysis. Bioassays covering different endpoints were employed, such as hormone receptor activation (AR and ERα), xenobiotic metabolism (PAH and PXR), oxidative stress response (Nrf2), and cytotoxicity. The WWTP influents showed AR, ERα, and PAH activities at ng/L - μg/L and PXR and Nrf2 activities at μg/L - mg/L as bioanalytical equivalent concentrations of a reference compound for each bioassay. These bioactivities decreased along with the WWTP treatment train, with significant removals achieved by the secondary biological treatment processes. Cytotoxicity was observed only for some municipal wastewater (M-WWTP) influents but was below the limit of quantification for most cases. The influent and effluent bioactivities observed in this study were mostly comparable to those reported in other WWTPs in the literature. Comparison of the bioactivities with the effect-based trigger (EBT) values indicates that the impact of WWTP effluents on receiving water quality was low for most endpoints. For Nrf2, however, further investigation is required to evaluate the observed high bioactivities compared with the current EBT. The observed ERα activity could partly be explained by the presence of some steroid estrogens. Overall, our results contribute to an important database for the concentrations and removal efficiencies of bioactive chemicals in WWTPs and demonstrate bioassays as a useful tool for urban water quality monitoring.

Estrogenic, androgenic, and glucocorticoid activities and major causative compounds in river waters from three Asian countries.

Estrogen, androgen, and glucocorticoid receptors (ER, AR, and GR) agonist activities in river water samples from Chennai and Bangalore (India), Jakarta (Indonesia), and Hanoi (Vietnam) were evaluated using a panel of chemical-activated luciferase gene expression (CALUX) assays and were detected mainly in the dissolved phase. The ER agonist activity levels were 0.011-55ng estradiol (E2)-equivalent/l, higher than the proposed effect-based trigger (EBT) value of 0.5ng/l in most of the samples. The AR agonist activity levels were < 2.1-110ng dihydrotestosterone (DHT)-equivalent/l, and all levels above the limit of quantification exceeded the EBT value of 3.4ng/l. GR agonist activities were detected in only Bangalore and Hanoi samples at dexamethasone (Dex)-equivalent levels of < 16-150ng/l and exceeded the EBT value of 100ng/l in only two Bangalore samples. Major compounds contributing to the ER, AR, and GR agonist activities were identified for water samples from Bangalore and Hanoi, which had substantially higher activities in all assays, by using a combination of fractionation, CALUX measurement, and non-target and target chemical analysis. The results for pooled samples showed that the major ER agonists were the endogenous estrogens E2 and estriol, and the major GR agonists were the synthetic glucocorticoids Dex and clobetasol propionate. The only AR agonist identified in major androgenic water extract fractions was DHT, but several unidentified compounds with the same molecular formulae as endogenous androgens were also found.

Progression of prostate cancer reprograms MYC-mediated lipid metabolism via lysine methyltransferase 2A

BackgroundThe activities of MYC, the androgen receptor, and its associated pioneer factors demonstrate substantial reprogramming between early and advanced prostate cancer. Although previous studies have shown a shift in cellular metabolic requirements associated with prostate cancer progression, the epigenetic regulation of these processes is incompletely described. Here, we have integrated chromatin immunoprecipitation sequencing (ChIP-seq) and whole-transcriptome sequencing to identify novel regulators of metabolism in advanced prostate tumors characterized by elevated MYC activity.ResultsUsing ChIP-seq against MYC, HOXB13, and AR in LNCaP cells, we observed redistribution of co-bound sites suggestive of differential KMT2A activity as a function of MYC expression. In a cohort of 177 laser-capture microdissected foci of prostate tumors, KMT2A expression was positively correlated with MYC activity, AR activity, and HOXB13 expression, but decreased with tumor grade severity. However, KMT2A expression was negatively correlated with these factors in 25 LuCaP patient-derived xenograft models of advanced prostate cancer and 99 laser-capture microdissected foci of metastatic castration-resistant prostate cancer. Stratified by KMT2A expression, ChIP-seq against AR and HOXB13 in 15 LuCaP patient-derived xenografts showed an inverse association with sites involving genes implicated in lipid metabolism, including the arachidonic acid metabolic enzyme PLA2G4F. LuCaP patient-derived xenograft models grown as organoids recapitulated the inverse association between KMT2A expression and fluorine-18 labeled arachidonic acid uptake in vitro.ConclusionsOur study demonstrates that the epigenetic activity of transcription factor oncogenes exhibits a shift during prostate cancer progression with distinctive phenotypic effects on metabolism. These epigenetically driven changes in lipid metabolism may serve as novel targets for the development of novel imaging agents and therapeutics.

Reciprocal regulation of CIP2A and AR expression in prostate cancer cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples (p = 0.023). Furthermore, the protein level of CIP2A was positively correlated with patients’ short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A level, associated with high protein and phosphorylation levels of AR. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR protein levels and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced protein levels of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR protein levels, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa.

FOXA1 regulates alternative splicing in prostate cancer.

SummaryDysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an “exon definition” mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

1381P A transcriptomic signature of AR activity prognosticates development of castration-resistance following metastasis-directed therapy in oligometastatic castration-sensitive prostate cancer

Prospective reports suggest metastasis-directed therapy (MDT) in oligometastatic castration-sensitive prostate cancer (omCSPC) is associated with improved treatment outcomes. When the addition of androgen-deprivation therapy (ADT) to MDT for omCSPC is needed is unclear. Here we assess the ability of an androgen receptor activity (AR-A) signature to provide prognostic information for omCSPC patients treated with MDT without and with ADT.

BluePrint breast cancer molecular subtyping recognizes single and dual subtype tumors with implications for therapeutic guidance

PurposeBluePrint (BP) is an 80-gene molecular subtyping test that classifies early-stage breast cancer (EBC) into Basal, Luminal, and HER2 subtypes. In most cases, breast tumors have one dominant subtype, representative of a single activated pathway. However, some tumors show a statistically equal representation of more than one subtype, referred to as dual subtype. This study aims to identify and examine dual subtype tumors by BP to understand their biology and possible implications for treatment guidance.MethodsThe BP scores of over 15,000 tumor samples from EBC patients were analyzed, and the differences between the highest and the lowest scoring subtypes were calculated. Based upon the distribution of the differences between BP scores, a threshold was determined for each subtype to identify dual versus single subtypes.ResultsApproximately 97% of samples had one single activated BluePrint molecular subtype, whereas ~ 3% of samples were classified as BP dual subtype. The most frequently occurring dual subtypes were the Luminal-Basal-type and Luminal-HER2-type. Luminal-Basal-type displays a distinct biology from the Luminal single type and Basal single type. Burstein’s classification of the single and dual Basal samples showed that the Luminal-Basal-type is mostly classified as ‘luminal androgen receptor’ and ‘mesenchymal’ subtypes, supporting molecular evidence of AR activation in the Luminal-Basal-type tumors. Tumors classified as Luminal-HER2-type resemble features of both Luminal-single-type and HER2-single-type. However, patients with dual Luminal-HER2-type have a lower pathological complete response after receiving HER2-targeted therapies in addition to chemotherapy in comparison with patients with a HER2-single-type.ConclusionThis study demonstrates that BP identifies tumors with two active functional pathways (dual subtype) with specific transcriptional characteristics and highlights the added value of distinguishing BP dual from single subtypes as evidenced by distinct treatment response rates.

Analysis of Capital Market Reactions To The Merger of Indonesian Sharia Banks: BRIS Stocks Evidence

This study aims to obtain evidence of the stock market’s reaction to the merger of Indonesian Islamic banks and its prediction power to JCI. The variables used in this study are abnormal returns and trading volume activity with an observation period of 60 days before the event and 60 days after the event. We use 2 types of time series data, which are daily data to analyze AR and trading volume activity, and weekly data to analyze the causality relationship. The results showed that there were no differences in abnormal returns before and after the merger, but there were differences in trading volume activity before and after the merger. In addition, this study also aims to determine the reciprocal relationship using Granger causality between the JCI and BRIS stocks prices before and after the merger of Indonesian Islamic banks. The results of the study show that there is no reciprocal relationship between the Composite Stocks Price Index and the stock’s price of BRIS and vice versa.

Abstract 989: Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases

Abstract Introduction: Androgen receptor splice variant 7 (AR-V7) is expressed in metastases from patients with castration resistant prostate cancer (CRPC) and shows a high level of inter- and intra-tumoral variability. However, the downstream activity generated by AR-V7 in tissue has not been shown. Whether AR-V7 is active in tissue or whether AR-V7 is a non-functioning biomarker with full-length AR is not known. To address this question, we constructed a tissue microarray (TMA) of 56 metastases from 27 patients with CRPC to analyze spatial gene expression using the GeoMx Digital Spatial Profiler. Immunostaining was performed to define epithelial, vascular, and stromal compartments. The stained tissues were then hybridized with barcoded tagged oligonucleotides targeting 2093 unique genes, which included those representing AR, AR-V cryptic exons, AR and neuroendocrine activity, and immune cell markers. One 500 um region of interest (ROI) was assessed per tissue core (approximately 1200 cells). A sequential section from the same TMA was then stained with AR-V7 and AR-C-terminal specific antibodies. ROIs for RNA and protein were selected to be similar between slides. In addition to DSP, each metastasis was assessed by RNA-seq on the bulk tissue. Results: The most differentially expressed genes (FDR&amp;lt;0.05) based on association with AR-V7 staining were known downstream AR regulated genes including KLK2 and 3, FKBP5, NKX3.1, TMPRSS2, FASN, and TARP. Additionally, genes associated with proliferation and stemness, e.g., POLB, KRT1, SOX2, were significantly expressed. Since 93% of patients were on ADT at time of tissue collection and over 80% also had been treated with either abiraterone or enzalutamide, the increase in AR downstream genes would not be expected to occur from ligand activation of AR-FL. We also have previously shown that knock-down of AR-V7 in LNCaP95 cells results in loss of AR binding to AREs. In these metastases, then, activation of AR downstream genes would be a result of AR-V7 nuclear transport of AR-FL through AR-V7/AR-FL heterodimers or transcriptional activation by AR-V7 homodimers. Of further note, AR cryptic exons 1, 2, and 5 were also significantly expressed in AR-V7 positive ROIs (p&amp;lt; 0.0001). RNA-seq intron/exon junction reads were used to demonstrate that additional AR-Vs, most commonly AR-V9, were also expressed in tissues positive for AR-V7, suggesting that AR splicing is a common event in CRPC. Finally, expression of neuroendocrine (NE) genes INSM1 and TUBB2 were not expressed in AR-V7 positive ROIs (negatively correlated, p&amp;lt;0.001), indicating that AR-V7 and NE phenotypes cannot co-exist in the same cell. Conclusion: AR-V7 continues to drive prostate cancer through activation of the AR-cistrome. Its expression is heterogenous in metastases along with NE cells, suggesting that in the presence of AR-V7 and NE markers, therapy needs to be directed at the N-terminus of AR and NE components. Citation Format: Cynthia C. Sprenger, Ilsa Coleman, Michelle Kriner, Lauren Brady, Margaret Hoang, Martine Roudier, Mamatha Damodarasamy, Radhika Patel, Lawrence True, Peter Nelson, Michael Haffner, Stephen Plymate. Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 989.

Abstract 1530: Derivatives of betulinic acid act as modulators of the androgen receptor and report cytotoxicity towards cancer cell lines

Abstract INTRODUCTION Triterpenoids are naturally occurring substances with a broad spectrum of beneficial effects for human health. Betulinic acid belongs to the group of pentacyclic triterpenoids and is well known for its antitumor activities. The library of about 1000 betulinic acid-derived triterpenes was collected and analyzed for cytotoxic activity on cancer and non-cancer cell lines (A549, CCRF-CEM, CEM-DNR, HCT116, K562, K562-TAX, U2OS, BJ and MRC-5) in our Institute over the last 20 years. Steroidal triterpenes are precursors of cholesterol, the precursor of testosterone, the fundamental ligand of the androgen receptor that is the key regulator in several physiological and pathological processes. Finding new modulators of androgen receptor is critical for the treatment of various androgen-dependent diseases, including prostate cancer or anabolic deficiencies. Betulinic acid and its derivatives showed structural similarity with steroids and therefore were selected to be tested for androgen receptor activity modulation. METHODS The stably transfected cell line HEK 293T GFP-AR was used to determine the translocation activity of the androgen receptor from the cytoplasm to the nucleus after the treatment with the derivatives. The high throughput fluorescent microscopy device and image analysis comprised the data, and compounds with the proved activity were selected for subsequent analysis. The luciferase reporter gene assay was further used to evaluate the effect of compounds on the transcriptional activity of the androgen receptor. The MTS assay was performed to assess the cytotoxicity. RESULTS Betulinic acid and its eight derivatives were identified to have 5-6 - fold higher translocation activity of AR (ratio cytoplasm GFP intensity/nuclear GFP intensity) than the negative control. Validation on luminescence luciferase reporter assay confirmed 4-5 - fold induction for five derivatives, whereas three derivatives were not active. Interestingly only two of them showed substantial cytotoxicities (IC50&amp;lt; 50 µM) as they possess significant inhibitory activity against all tested cancer cell lines with favorable therapeutic index against the non-cancer cell lines. CONCLUSION Derivatives of betulinic acid were proven to have modulatory activity on the androgen receptor pathway. The two derivatives of betulinic acid also displayed cytotoxicity towards almost all of the tested cancer cell lines. There could be not only AR modulation responsible for anticancer activity as the derivatives of betulinic acid are well know disruptors of mitochondrion functions as well. The synergy of these two mechanisms of action could potentiate the antitumor activity of the selected compounds. This work was supported by ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, CZ-OPENSCREEN - LM2018130, EATRIS-CZ - LM2018133. Citation Format: Alzbeta Srovnalova, Sona Gurska, Milan Urban, Jan Sarek, Jiri Rehulka, Petr Dzubak, Marian Hajduch. Derivatives of betulinic acid act as modulators of the androgen receptor and report cytotoxicity towards cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1530.

Antioxidation effect of alginate beads containing thyme, rosemary or geranium essential oils in lard and coconut oil

SummaryThe alginate beads encapsulated thyme (AT), rosemary (AR) and geranium (AG) essential oils (EOs) were prepared to inhibit lipid oxidation in lard and coconut oil. The result showed that beads dried in hot air oven (45 °C) encapsulated higher amount of EOs than drying by desiccation method. Sachets containing AT, AR and AG were applied in PET bottles containing lard and coconut oils and stored at 45 °C. The DPPH scavenging activities of AT, AR and AG were 85.39%, 78.26%, and 78.12%, respectively. During storage, released EOs reduced PV and TBARS of lard and coconut oil and did not affect their colour acceptability scores. From PV, FFA and TBARS, AT at 0.50 g exhibited the best results in terms of lipid oxidation inhibition, although encapsulation percentage and release were significantly lower than AG and AR. Porous in alginate beads could entrap lipid oxidation products, including volatile compounds and water vapour. Besides, the hydrophilic property of polymer matrix led to water absorption, which lower aw value. In week3, the odour acceptability scores of control lard failed, while those packed with AT, AR and AG were acceptable. Alginate beads containing EOs can be used to extend the storage life of lard and coconut oil.

Non-drug efflux function of ABCC5 promotes enzalutamide resistance in castration-resistant prostate cancer via upregulation of P65/AR-V7

Drug resistance is responsible for castration-resistant prostate cancer (CRPC)-associated mortality. While ATP binding cassette subfamily C member 5 (ABCC5) has been reported to regulate multiple drug resistance, its drug-efflux function may not be the main reason underlying resistance to enzalutamide, an androgen receptor inhibitor. Here, we aimed to determine whether the non-drug efflux function of ABCC5 affects enzalutamide resistance. The ABCC5 expression data in patients with prostate cancer (PCa) were retrieved from The Cancer Genome Atlas and Gene Expression Omnibus, and their correlation with disease prognosis was analyzed. Immunohistochemical staining was performed on a cohort of 80 patient samples. Proliferation of enzalutamide-resistant 22RV1 and C4-2B cells was investigated using CCK-8, EdU, and colony formation assays. The effect of ABCC5 silencing on enzalutamide resensitization was evaluated in vitro and in vivo. Functional assays indicated that ABCC5 depletion resensitized enzalutamide-resistant cells to inhibit cell growth and impeded xenograft tumor proliferation. Mechanistically, luciferase and ChIP assays confirmed that P65 regulated AR expression and activity by binding to its promoter, while ABCC5-mediated resistance effected by AR-V7 (one of the widely studied AR splicing variants that meditate AR antagonist resistance) upregulation could be reversed by P65 knockdown. Furthermore, activation of the NF-κB pathway reversed the effects of ABCC5 knockdown by extra AR-V7 expression. Thus, ABCC5 might be a novel target for enzalutamide-resistant CRPC treatment.

Interactions between β3‐adrenergic receptors and bFGF‐induced effects in rat aortic smooth muscle cells

BackgroundBasic fibroblast growth factor (bFGF)‐mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury‐induced neointima formation and subsequent vascular restenosis, a major event that hinders the long‐term success of angioplasty. The function of β3‐adrenergic receptors (β3‐ARs) in vascular injury‐induced neointima formation has not yet been defined.ObjectivesOur current study explored the possible role of β3‐ARs in vascular injury‐induced neointima formation by testing its effects on bFGF‐induced VSMC migration and proliferation.Methodsβ3‐AR expression in rat carotid arteries was examined at 14 days following a balloon catheter‐induced injury. The effects of β3‐AR activation on bFGF‐induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular‐signal‐regulated kinase/ mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested.ResultsWe found that vascular injury induced upregulation of β3‐ARs in neointima. Pretreatment of VSMCs with a selective β3‐AR agonist, CL316,243 significantly potentiated bFGF‐induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF‐induced cell migration and that inhibiting AKT phosphorylation reduced bFGF‐mediated cell proliferation.ConclusionOur results suggest that activation of β3‐ARs enhances bFGF‐mediated effects on VSMCs by interacting with bFGF‐mediated ERK and AKT phosphorylation and β3‐ARs may play a role in vascular injury‐induced neointima formation.

In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function

Objective &amp; HypothesisActivation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.MethodsWe employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.ResultsDatabase screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.ConclusionThese findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.

Cosmogenic production of Ar37 in the context of the LUX-ZEPLIN experiment

We estimate the amount of $^{37}$Ar produced in natural xenon via cosmic ray-induced spallation, an inevitable consequence of the transportation and storage of xenon on the Earth's surface. We then calculate the resulting $^{37}$Ar concentration in a 10-tonne payload~(similar to that of the LUX-ZEPLIN experiment) assuming a representative schedule of xenon purification, storage and delivery to the underground facility. Using the spallation model by Silberberg and Tsao, the sea level production rate of $^{37}$Ar in natural xenon is estimated to be 0.024~atoms/kg/day. Assuming the xenon is successively purified to remove radioactive contaminants in 1-tonne batches at a rate of 1~tonne/month, the average $^{37}$Ar activity after 10~tonnes are purified and transported underground is 0.058--0.090~$\mu$Bq/kg, depending on the degree of argon removal during above-ground purification. Such cosmogenic $^{37}$Ar will appear as a noticeable background in the early science data, while decaying with a 35~day half-life. This newly-noticed production mechanism of $^{37}$Ar should be considered when planning for future liquid xenon-based experiments.

Abstract BS1-2: A new perspective on androgen receptor action in estrogen receptor-α positive breast cancer

Abstract Resistance to estrogen receptor alpha (ER) target therapies is the major cause of breast cancer death. Therapeutic engagement of steroid receptors that impinge on, but do not ablate, ER signalling is an emerging new treatment strategy. The AR is expressed in the majority of breast tumors and represents an exceptional therapeutic target, especially as a range of drugs including AR agonists, antagonists and selective AR modulators (SARMs) are available. Interest in targeting AR for treatment of breast cancer has escalated over the past decade. Despite clinical correlations and preclinical studies supporting a protective role for AR in breast cancer, especially in ER-positive disease, enthusiasm was largely directed toward antagonizing AR with anti-androgenic drugs used to treat men with prostate cancer, a disease definitively driven by oncogenic AR activity. While some pre-clinical studies supported use of an AR antagonist for ER-positive breast cancer, this strategy has had minimal success in clinical trials. Our most recent preclinical studies using a diverse range of in vivo breast cancer models (Hickey et al, Nature Medicine 2021) provides compelling evidence for (i) AR being a tumor suppressor in ER+ breast cancer, and (ii) an AR agonist, not an antagonist, treatment strategy being the optimal therapeutic approach. Citation Format: W Tilley. A new perspective on androgen receptor action in estrogen receptor-α positive breast cancer. [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr BS1-2.

Additional activation of the AR gene may be involved in the development of the castration resistance phenotype in prostate cancer

IntroductionSeveral studies have already shown that changes in the AR gene may be associated with a more aggressive disease phenotype and even castration-resistant prostate cancer. Thus, we investigated cytogenetic and molecular alterations linked to AR. Materials and methodsTo evaluate AR methylation, we performed a cytogenetic-molecular analysis using fluorescence in situ hybridization that uses specific probes for the AR gene (Xq11.12) and the X chromosome centromere. For AR activity, we performed a qualitative analysis of human androgen receptor activity. To analyze the expression of AR in PC3 and LNCaP cell lines, we used qPCR assays. ResultsIn the qPCR assay, we found downregulation of AR in the PC3 cell line compared with the LNCaP. We found the presence of X chromosome polysomy in PC-3 and LNCaP cell lines by FISH assay. In the HUMARA-Q assay, we found two X chromosomes/cell and the activity of both AR in the PC-3 cell line. In LNCaP cells, we found two X chromosomes/cell and methylation of only one AR. ConclusionCastration-resistant prostate cancer phenotype represents a significant challenge in the setting of urological management. The X chromosomes and AR-linked alterations may contribute to a better understanding of the disease. However, further studies should be performed in an attempt to elucidate as much as possible the role of AR in the castration-resistant prostate cancer phenotype.

Lignans with α-glucosidase, protein tyrosine phosphatase 1B, and aldose reductase inhibitory activities from the fruits of Viburnum cylindricum

Viburnum cylindricum Buch.-Ham. ex D. Don is widely cultivated in China due to its considerable economic value. In this study, the EtOAc fraction (VCFE) of V. cylindricum fruits (VCF) was first reported to obviously reduce postprandial blood glucose levels in normal and diabetic ICR mice at 100 mg/kg, and significantly inhibit α-glucosidase, PTP1B, and AR activities in vitro. In order to clarify its antidiabetic components, nine undescribed lignans, including seven 9,9′-epoxylignans, viburindrins A−G (1−7), one rare variable 9,9′-epoxylignan, viburindrin H (8), and one furofuran lignan, viburindrin I (9), were isolated from VCFE by bioassay-guided fractionation, and their structures including absolute configurations were identified by comprehensive spectroscopic analyses and the comparison of the experimental and calculated ECD spectra. Compounds 1−8 exhibited significant inhibitory activities against these enzymes. Among them, compounds 1, 4, and 8 possessed the most potent inhibitory effects on α-glucosidase, PTP1B, and AR with the IC50 values of 14.21, 6.14, and 1.08 μM, respectively. In addition, their inhibitory kinetics and molecular modeling analyses were also investigated. These results uncovered that VCF with the active lignans could be the potential resource for the improvement and treatment of diabetes and related complications.