Beta-adrenergic Receptor Signaling Research Articles

Potential Role of Pig UCP3 in Modulating Adipocyte Browning via the Beta-Adrenergic Receptor Signaling Pathway.

Adipose tissue plays an important role in regulating body temperature and metabolism, with white adipocytes serving as storage units for energy. Recent research focused on the browning of white adipocytes (beige adipocytes), causing thermogenesis and lipolysis. The process of browning is linked to the activation of uncoupling protein (UCP) expression, which can be mediated by the β3 adrenergic receptor pathway. Transcriptional factors, such as peroxisome proliferator activated receptor γ (PPARγ) and PPARγ coactivator 1 alpha, play vital roles in cell fate determination for fat cells. Beige adipocytes have metabolic therapeutic potential to combat diseases such as obesity, diabetes mellitus, and dyslipidemia, owing to their significant impact on metabolic functions. However, the molecular mechanisms that cause the induction of browning are unclear. Therefore, research using animal models and primary culture is essential to provide an understanding of browning for further application in human metabolic studies. Pigs have physiological similarities to humans; hence, they are valuable models for research on adipose tissue. This study demonstrates the browning potential of pig white adipocytes through primary culture experiments. The results show that upregulation of UCP3 gene expression and fragmentation of lipid droplets into smaller particles occur due to isoproterenol stimulation, which activates beta-adrenergic receptor signaling. Furthermore, PPARγ and PGC-1α were found to activate the UCP3 promoter region, similar to that of UCP1. These findings suggest that pigs undergo metabolic changes that induce browning in white adipocytes, providing a promising approach for metabolic research with potential implications for human health. This study offers valuable insights into the mechanism of adipocyte browning using pig primary culture that can enhance our understanding of human metabolism, leading to cures for commonly occurring diseases.

Abstract 3162: Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin

Abstract Introduction: Epidemiological studies showed that beta-adrenergic antagonists caused a low incidence of several cancer types. Glioblastoma highly expresses beta-2-adrenergic receptors. GBM accounts for 50% of central nervous system cancers. After standard-of-care therapeutic intervention, there is a survival time of 15 months. Our goal is to develop a translational therapy that will trigger the death of GBM cells while preserving healthy cells Methodology: Salmeterol xinafoate is a biased agonist that activates the G-protein pathway. Biased agonists activate signaling through the G-protein or the beta-arrestin pathway of G-protein coupled receptors. Dose-response experiments were performed using beta1 and beta2-adrenergic receptor antagonists and biased beta2-adrenergic receptor agonists to determine loss of glioma cell viability in GBM5, GBM12, and LN229 glioma cell lines. Western immunoblotting gauged survivin expression levels in all three lines. Epoxomicin was used to inhibit the proteasome complex. Annexin V and propidium iodide were employed to measure apoptotic or necrotic cell death. Results: Disrupting beta2-adrenergic receptor signaling by salmeterol xinafoate led to cell death of glioma cells but spared healthy neuron-glia cells using similar doses. Salmeterol xinafoate revealed greater efficacy for killing glioma cells than beta1 and beta2-adrenergic receptor antagonists. To determine the mechanism of glioma cell death, LN229 cells were treated with escalating doses of Salmeterol xinafoate, and at 24 hours, annexin V staining showed early apoptosis, and Annexin V/propidium iodide co-staining also showed late apoptosis. Propidium iodide revealed minor necrosis in LN229 glioma cells. Western immunoblotting showed salmeterol xinafoate downregulated survivin in a dose-dependent manner. However, the epoxomicin's inhibition of the proteasome complex prevented dose-escalated downregulation of survivin for all three glioma cell lines. Conclusion: The bitopic structure of salmeterol enables this drug to bind to the beta2-adrenergic receptors ligand binding site and an exosite site by its aryloxyalkyl tail that may contribute to the increased efficacy. Salmeterol xinafoate triggers glioma cell death by apoptosis at least in one glioma cell line. Salmeterol Xinafoate down-regulates survivin through protease degradation. Promising results show that the FDA-approved drug salmeterol xinafoate enables a favorable transition to the clinic. Repurposing this drug for GBMs may be combined with the current standard-of-care chemotherapies to enhance more significant loss of tumor volume and increase patient survival. Citation Format: Orli Algranatti, Ji Yae Lee, James M. Angelastro. Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3162.

Repurposing beta-blockers for combinatory cancer treatment: effects on conventional and immune therapies.

Beta-adrenergic receptor signaling regulates cellular processes associated with facilitating tumor cell proliferation and dampening anti-tumor immune response. These cellular processes may lead to compromised tumor control and cancer progression. Based on this ramification, Beta-blockers (BBs) have emerged as a potential treatment by inhibiting beta-adrenergic receptor signaling. This review aimed to investigate the relationship between the use of BBs and tumor progression and treatment response. Therefore, the authors explored several aspects: the potential synergistic relationship of BBs with chemotherapy and immunotherapy in enhancing the effectiveness of chemotherapeutic and immunotherapeutic treatments and their role in boosting endogenous immunity. Further, this review explores the distinctions between the major types of BBs: Non-selective Beta Blockers (NSBBs) and Selective Beta Blockers (SBBs), and their contributions to combinatory cancer treatment. In this review, we presented a perspective interpretation of research findings and future directions. Overall, this review discusses the potential and challenge that BBs present in improving the effectiveness and outcome of cancer treatment.

117P Beta-blockers in lung cancer patients receiving immunotherapy

The treatment standard of cancer has dramatically diverted with immune checkpoint inhibitors (ICI). Despite the proven clinical advantage, some tumors do not respond to ICI. The expression of PD-L1, the tumor microenvironment (TME), and the tumor mutational burden are essential to the success of ICI and are currently considered biomarkers predictive of response. Increased Beta-adrenergic receptor signaling has been shown to promote the creation of an immunosuppressive TME. The annulment of this pathway provides a more responsive TME and may enhance the activity of ICI, and the use of beta-blockade for this purpose has shown conflicting results.

Abstract 106: In Utero Morphine Exposure Dysregulates The Endogenous Opioid System (EOS) And Increases Risk Factors For Cardiovascular Disease In The Adulthood Offspring

Chronic opioid exposure and opioid use disorder are associated with nearly a 2-fold increased risk of CVD and 16% increase in coronary artery disease. Opioid abuse during pregnancy increases 5 fold the incidence of neonatal opioid withdrawal syndrome (NOWS) and is associated with low birth weight; however, the effect of OUD among pregnant women on their offspring health is understudied. Previously, we have shown that both male and female offspring from dams exposed to mu-opioid receptor agonist morphine (MOR) during the whole pregnancy are smaller and display high blood pressure. This study aimed to investigate the potential underlying mechanisms associated with increased blood pressure in MOR- exposed offspring. Sprague Dawley dams were treated with escalating doses of MOR (5-20 mg/kg/day, s.c) or saline (VEH) from gestational day 1 to 18, and pups were weaned on standard diet. Lipid profile was assessed as a risk factor for coronary artery disease using FPLC. MOR exposed offspring showed increased LDL (F-MOR vs. VEH ~2 fold; M-MOR vs. VEH ~1.5 fold mg/dL; n=6-8, p<0.05). Leptin levels, normalized to body fat, were not different between groups (F-MOR 219.8±35 vs F-VEH 181.4±15.8 pg/g fat; M-MOR 207.2±26.7 vs M-VEH 200.3±23.3 pg/g fat, n=7-8). Although proteinuria was not different in MOR exposed rats vs. VEH (F-MOR 4.6±0.8 vs F-VEH 5.5±1mg/ml; M-MOR 29.1±1.4 vs M-VEH 37.5±2.95 mg/mL; n=7-8), creatinine clearance was reduced in MOR-exposed males but not females (M-MOR 0.51±0.1 vs M-VEH 0.87±0.3 mL/min, n=7-8). MOR-exposed offspring showed a greater reduction in the pressor response to mecamylamine (F-MOR -46.25±6 vs F-VEH -18.3±1.3, M-MOR -50.5±0.28 vs M-VEH -25±5.1 delta mmHg, p<0.05). Proenkephalin, an endogenous opioid peptide that has been shown to inhibit the beta adrenergic receptor signaling, showed a ~3-fold reduction in heart and a ~7.5-fold reduction in the kidney cortex and outer medulla mRNA expression vs. VEH (-2ddCt, n=6-8, p<0.05). Maternal MOR exposure increases the sympathetic tone, which could be associated with increased sympathetic outflow to the kidney and the heart. Reduced proenkephalin could be involved in potential crosstalk between adrenergic and opioid receptor signaling exacerbating the actions of catecholamines.

Involvement of isoproterenol-induced intracellular Zn2+ dynamics in the basolateral amygdala in conditioned fear memory.

Beta-adrenergic receptors in the basolateral amygdala play an essential role in fear memory, while the physiological role of intracellular Zn2+ remains to be clarified. Intracellular Zn2+ level was decreased 5 min after local injection of 500μM isoproterenol (2μl), a nonselective beta-adrenergic receptor agonist into the basolateral amygdala, suggesting that intracellular Zn2+ dynamic is linked with beta-adrenergic receptor signaling in the basolateral amygdala. When isoproterenol was injected into the basolateral amygdala 20min prior to long-term potentiation (LTP) induction, LTP at perforant pathway-basolateral amygdala was enhanced and conditioned fear memory was also augmented, suggesting that isoproterenol leads to utilization of Zn2+ to consolidate fear memory followed by lowering intracellular Zn2+. We postulated that synaptic Zn2+ dynamics under conditioned fear experience regulates conditioned fear memory in cooperation with beta-adrenergic receptor signaling. When either intracellular Zn2+ chelator (ZnAF-2DA) or extracellular Zn2+ chelator (CaEDTA) was locally injected into the basolateral amygdala in the same manner, LTP was also enhanced. The local injection of ZnAF-2DA augmented fear memory. It is likely that the decrease in availability of intracellular Zn2+ by Zn2+ chelators under fear experience affects the function of Zn2+-required proteins followed by augmentation of fear memory and its related LTP. The present study suggests that beta-adrenergic receptor signaling is linked with intracellular Zn2+ signaling in the basolateral amygdala to consolidate conditioned fear memory. Because intracellular Zn2+ signaling is required for fear memory, the decrease in availability of intracellular Zn2+ may augment fear memory and its related LTP under non-physiological condition.

Beta 2-Adrenergic Receptor in Circulating Cancer-Associated Cells Predicts for Increases in Stromal Macrophages in Circulation and Patient Survival in Metastatic Breast Cancer.

The usage of beta blockers in breast cancer (BC) patients is implicated in the reduction in distant metastases, cancer recurrence, and cancer mortality. Studies suggest that the adrenergic pathway is directly involved in sympathetic-driven hematopoietic activation of pro-tumor microenvironmental proliferation and tumor cell trafficking into the circulation. Cancer-associated macrophage-like cells (CAMLs) are pro-tumor polynucleated monocytic cells of hematopoietic origin emanating from tumors which may aid in circulating tumor cell (CTC) dissemination into the circulation. We examined the linkage between Beta-2 adrenergic receptor (B2AR) signaling in CAMLs and CTCs by establishing expression profiles in a model BC cell line (MDA-MB-231). We compared the model to CAMLs and CTCs found in patents. Although internalization events were observed in patients, differences were found in the expression of B2AR between the tumor cell lines and the CAMLs found in patients. High B2AR expression on patients’ CAMLs was correlated with significantly more CAMLs in the circulation (p = 0.0093), but CTCs had no numerical relationship (p = 0.1565). High B2AR CAML expression was also significantly associated with a larger size of CAMLs (p = 0.0073), as well as being significantly associated with shorter progression-free survival (p = 0.0097) and overall survival (p = 0.0265). These data suggest that B2AR expression on CAMLs is closely related to the activation, intravasation, and growth of CAMLs in the circulation.

Adopting a Phosphoproteomics Approach to Investigate a Role for Protein Phosphatase 2A (PP2A) Regulatory Subunit B55α in Cardiac β-Adrenergic Receptor Signalling

Protein kinases are recognised as integral regulators of phosphorylation within beta-adrenergic receptor (β-AR) signalling pathways in the heart. In contrast, the role of protein phosphatases and dephosphorylation in β-AR pathways is less well defined. In vitro evidence suggests that protein phosphatase 2A (PP2A) regulatory subunit B55α may modulate gene transcription as a key downstream effector of β-AR signalling in cardiomyocytes. The goal of this study was to determine how loss of PP2A-B55α impacts β-AR signalling in cardiomyocytes.

Chronic fatigue and physical distress influence the outcomes of immunotherapy treatment in patients with recurrent ovarian cancer

<h3>Objectives:</h3> <i>In vivo</i> preclinical studies have shown that chronic stress augments tumor proliferation and promotes an immunosuppressive tumor microenvironment through activation of beta-adrenergic receptor signaling. The deleterious effects of chronic stress and resulting fatigue on immune responses have also been well characterized in several disease entities, however the impact of these on response to immune check point blockade is still not known. Our objective was to investigate the relationship between the different dimensions of patient reported fatigue and distress on response to immune check point inhibitor (ICI) treatment in a cohort of women undergoing immunotherapy treatment for recurrent ovarian cancer. <h3>Methods:</h3> A single-arm phase 2 clinical trial (NCT02853318) was recently completed at our center on 40 patients with recurrent platinum-sensitive or platinum-resistant ovarian cancer using anti-PD1 pembrolizumab in combination with bevacizumab and oral cyclophosphamide, showing an overall response rate of 47.5% with a median progression free survival (PFS) of 10.0 months. A validated 30-item Multidimensional Fatigue Symptom Inventory-Short Form (MFSI-SF) was administered to all patients at baseline, after 3 months and 6 months of treatment, and at the time of disease progression to assess the multidimensional nature of fatigue and chronic stress (general, physical, emotional and mental fatigue, and vigor). Each subscale has a maximum score of 24 points to generate a total overall MFSI-SF score up to 96, whereby a higher score indicates greater exhaustion. Scores for each dimension as well as the composite scores were correlated with PFS using Pearson correlation. <h3>Results:</h3> The overall MFSI-SF fatigue score at baseline prior to starting ICI was 31.3 (n=40, SD ±10.8) and remained stable throughout the treatment until disease progression, indicating that the ICI combination treatment did not lead to increased fatigue in any dimensions. Patients who had shorter PFS (< 10 months) scored significantly higher on the physical (p=0.005) and overall fatigue measures (p=0.02) throughout the clinical trial compared to patients with durable clinical responses (PFS > 10 months), despite similar score on vigor subscales (p=0.9). There was no statistical difference in general, emotional and mental fatigue scores between patients with absent or abbreviated response to ICI, compared to patients with a durable clinical response. <h3>Conclusions:</h3> Patients experiencing greater chronic physical distress and chronic overall fatigue had poor response to an immune checkpoint inhibition combination with shorter PFS compared to the median among a cohort of women undergoing treatment for recurrent ovarian cancer. These findings further support the emerging evidence on the adverse effects of chronic fatigue and stress on beta-adrenergic signaling pathways, which may well modulate immunotherapy treatment responses.

Crosstalk between beta-adrenergic and insulin signaling mediates mechanistic target of rapamycin hyperactivation in liver of high-fat diet-fed male mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. While increased nutrient intake and sympathetic activity have been associated with the disease, the pathogenesis of NAFLD remains incompletely understood. We investigated the impact of the interaction of high dietary fat and sugar intake with increased beta‐adrenergic receptor (β‐AR) signaling on the activity of nutrient‐sensing pathways and fuel storage in the liver. C57BL/6J mice were fed a standard rodent diet (STD), a high‐fat diet (HFD), a high‐fat/high‐sugar Western diet (WD), a high‐sugar diet with mixed carbohydrates (HCD), or a high‐sucrose diet (HSD). After 6 week on diets, mice were treated with isoproterenol (ISO) and the activity of liver mTOR complex 1 (mTORC1)‐related signaling analyzed by immunoblotting and correlated with tissue triglyceride and glycogen contents. ISO‐stimulated AKT‐ and ERK‐mediated activation of mTORC1 in STD‐fed mice. Consumption of all four high‐calorie diets exacerbated downstream activation of ribosomal protein S6 kinase beta‐1 (S6K1) in response to ISO. S6K1 activity was greater with the fat‐enriched HFD and WD and correlated with the presence of metabolic syndrome and a stronger activation of AKT and ERK1/2 pathways. Fat‐enriched diets also increased triglyceride accumulation and inhibited glycogen mobilization under β‐AR stimulation. In conclusion, crosstalk between β‐AR and insulin signaling may contribute to HFD‐induced hepatic steatosis through ERK1/2‐ and AKT‐mediated hyperactivation of the mTORC1/S6K1 axis. The findings provide further rationale for the development of therapies aimed at targeting augmented β‐AR signaling in the pathogenesis of NAFLD.

Characterization and modelling of the abnormal electrophysiological response under beta-adrenergic stimulation in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Introduction Hypertrophic Cardiomyopathy (HCM) is the most common inheritable heart pathology and the main cause of sudden cardiac death in young adults. HCM patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have shown an abnormal response of HCM myocytes to β-adrenergic stimulation (β-ARS), with prolongation of their action potential duration (APD). The mechanisms underlying this aberrant response to sympathetic stimulation remain unknown. Purpose To investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS using human-based experimental and computational methodologies. Methods Experimental ionic currents, action potential and calcium transient were recorded in human adult cardiomyocytes from control and HCM patients. Isoproterenol (10-7 mol/L) was used to elicit β-ARS. Whole-cell ruptured patch voltage clamp experiments were conducted to characterise L-type calcium and potassium currents, with recordings performed before and after 3 min of drug exposure. The latest models of human ventricular electrophysiology and beta-adrenergic receptor signalling were integrated and calibrated using the human measured data. Simulations under isoproterenol were performed to quantify the effects of β-ARS on the action potential and calcium transient. The role of the main ion currents affected by β-ARS and by HCM remodelling was evaluated. Results In vitro, isoproterenol shortened APD (-16 ± 3%) in control, while prolonging APD in HCM myocytes (+23 ± 8%). Analysis of the measured data indicated two possible mechanisms contributing to APD prolongation in HCM myocytes. Firstly, a protracted L-type calcium current, presenting slower inactivation kinetics in HCM compared to control. The relative increase of potassium currents under β-ARS was also lower in HCM myocytes. The developed in silico models of β-ARS replicated the behaviour observed in the experimental data, based on slower L-type calcium current inactivation kinetics and a smaller increase of potassium currents in HCM. In absence of β-ARS, simulated HCM cardiomyocytes exhibited prolonged APD compared to control (525 ± 88 vs 281 ± 56 ms, p &amp;lt; 0.001). Under β-ARS, APD in control was reduced (-16.46%), whereas APD was prolonged in HCM (+11.63%). Further analysis showed that the reduction of the potassium currents increment under β-ARS was the main cause of the APD prolongation in HCM myocytes, with L-type calcium inactivation minimally contributing to APD prolongation. Conclusions In this study we assessed the effects of β-ARS on ion currents and APD in control and HCM myocytes. Our modelling results suggest that the increase of potassium repolarising currents under β-ARS is greatly reduced in HCM cardiomyocytes, being the main mechanism underlying their APD prolongation. This APD prolongation may have severe consequences in HCM patients, increasing the risk of exercise-induced arrhythmias. Abstract Figure.

β-Caryophyllene, a natural bicyclic sesquiterpene attenuates β-adrenergic agonist-induced myocardial injury in a cannabinoid receptor-2 dependent and independent manner

The downregulation of cannabinoid type-2 receptors (CB2R) have been reported in numerous diseases including cardiovascular diseases (CVDs). The activation of CB2R has recently emerged as an important therapeutic target to mitigate myocardial injury. We examined whether CB2R activation can protect against isoproterenol (ISO)-induced myocardial injury (MI) in rats. In the present study, we investigated the cardioprotective effect of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid in rat model of MI. Rats were pre- and co-treated with BCP (50 mg/kg, orally) twice daily for 10 days along with subcutaneous injection of ISO (85 mg/kg) at an interval of 24 h for two days (9th and 10th days). AM630 (1 mg/kg), a CB2 receptor antagonist, was injected intraperitoneal as a pharmacological challenge prior to BCP treatment to reveal CB2R-mediated cardioprotective mechanisms of BCP. Desensitization of beta-adrenergic receptor (β-AR) signaling, receptor phosphorylation and recruitment of adapter β-arrestins were observed in ISO-induced MI in rats. ISO injections caused impaired cardiac function, elevated the levels of serum cardiac marker enzymes, and enhanced oxidative stress markers along with altered PI3K/Akt and NrF2/Keap1/HO-1 signaling pathways. ISO also promoted lysosomal dysfunction along with activation of NLRP3 inflammasomes and TLR4-NFκB/MAPK signaling and triggered rise in proinflammatory cytokines. There was a concomitant mitochondrial dysfunction followed by the activation of endoplasmic reticulum (ER) stress-mediated Hippo signaling and intrinsic pathway of apoptosis as well as altered autophagic flux/mTOR signaling in ISO-induced MI. Furthermore, ISO also triggered dyslipidemia evidenced by altered lipids, lipoproteins and lipid marker enzymes along with ionic homeostasis malfunction. However, treatment with BCP resulted in significant protective effects on all biochemical and molecular parameters analyzed. The cardioprotective effects were further strengthened by preservation of cardiomyocytes and cell organelles as observed in histopathological and ultrastructural studies. Interestingly, treatment with AM630, a CB2R antagonist was observed to abrogate the protective effects of BCP on the biochemical and molecular parameters except hyperlipidemia and ionic homeostasis in ISO-induced MI in rats. The present study findings demonstrate that BCP possess the potential to protect myocardium against ISO-induced MI in a CB2-dependent and independent manner.

Downregulation of USP33 inhibits Slit/Robo signaling pathway and is associated with poor patient survival of glioma.

Gliomas are the most common malignant tumors in the central nervous system originating from brain glial cells. Although characterized as highly invasive and highly malignant, few molecular targeting therapies have been developed. Ubiquitin Specific Protease 33 (USP33), a gene encoding a deubiquinating enzyme important in a variety of processes, including Slit-dependent cell migration and beta-2 adrenergic receptor signaling, participates in the development of several malignant tumors, however, its role in the development of glioma has not been evaluated. Real-time quantitative PCR was performed to examine the expression of USP33 in glioma tissues and cell lines. Immunohistochemistry was performed to determine USP33 expressions in glioma tissue microarray. Transwell assay was performed to analyze the effect of USP33 on glioma cell line migration. The Kaplan-Meier method and log rank test were applied to evaluate the prognostic value of USP33 expression. Univariate and multivariate Cox proportional hazard regression models were used to identify the independent prognostic factors associated with overall survival (OS) or disease-free survival. The present study demonstrated that USP33 expression was significantly downregulated in glioma tissues. Lower expression of USP33 was associated with a poorer patient disease-free survival and overall survival. In vitro studies revealed that overexpression of USP33 significantly inhibited the migration ability of glioma cells. Mechanistically, USP33 inhibits glioma cell migration by regulating the function of Slit/Robo signaling pathway. Downregulation of USP33 is associated with poor patient survival of glioma. USP33 inhibits glioma cell migration by Slit/Robo signaling pathway. This mechanism may be applied for development of targeting therapy especially for the high-grade glioma.

Abstract 3440: Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells

Abstract Breast cancer is the world’s leading cause of cancer mortality among women. Despite advances in both diagnosis and treatment a still large number of women die because of metastatic breast cancer. Research demonstrated a strong link between chronic stress and weakened immune system leaving the body prone to disease like cancer. Indeed, psychological stress has been shown to enhance tumor growth and progression. In addition, stress hormones, such as norepinephrine and epinephrine, are implicated in the growth, invasiveness and metastasis of cancer. Therefore, targeting the beta-adrenergic receptors in cancer chemoprevention may offer new promises for the design of therapeutic strategies. For that reason, we determine the cellular processes involved in beta-2 adrenergic receptor (ADRB2) mediated effects on human breast cancer cells in cultures. We particularly focused on the impact of the inhibition of ADRB2 on cell proliferation, migration and invasion of the triple negative breast cancer cell line MDA-MB-231. In order to determine the effects of loss of expression of the gene, we suppressed ADRB2 by knock down, using CRISPR technology. We also used pharmacologically suppression of ADBR by employing a general beta receptor blocker; propranolol. Cell proliferation, migration and invasion assays were performed to define a degree of aggressiveness to the cells phenotype after the knock down or pharmacological suppression of ADRB2 in comparison to control cells. Also, Western Blot analysis on characteristics proteins of the epithelial-mesenchymal transition (EMT) was used to further characterize the phenotype. PCR Array expression studies were done to analyze modifications of genes expression profile and signaling cascades in the ADRB2 knocked down cells. We found that the knocking down by CRISPR of the ADRB2 receptors or, pharmacologically suppressing ADRB2 reduces proliferation, migration and colony formation of MDA-MB-231 cells. Additionally, ADRB2 knock down increased expression of epithelial proteins (beta-catenin and E-cadherin) while decreased mesenchymal markers (vimentin and N-cadherin). The mRNA expression study using a PCR Array targeting cancer stem cell genes shows that the inhibition of ADRB2 significantly modifies signaling involved in cell movement, invasion, migration and proliferation such as Wnt/beta-catenin signaling, ILK signaling and Notch signaling. Within these signaling pathways, the expression of several genes such as ILK, MAP2K6, GSK3B and JUN seem to be critical as they are under-expressed. These data provide preliminary understanding of the possible connection between the beta-2 adrenergic receptor system and signaling pathways use by the tumor cells to grow and acquire a more aggressive phenotype. Also, some genes are identified as new targets that may be valuable for developing effective treatment of advanced breast cancer. (Supported by NIH grant R01 CA20863201) Citation Format: Benedicte Rousseau, Ananya Tirupathur, Sengottuvelan Murugan, Dipak K. Sarkar. Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells [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 3440.

Abstract # 3124 The effect of acute and sustained beta2-adrenergic receptor stimulation on the function of Natural Killer cells

Epinephrine is a neurotransmitter, which is produced by the adrenal glands and adrenergic neurons. It plays a crucial role during acute stress in the so-called fight-or-flight response. Additionally, epinephrine can act on the immune system. The number of lymphocytes in peripheral blood increases after exposure to epinephrine. More importantly, the outcome of cancer therapies and major surgery can be influenced by epinephrine-mediated effects on the immune system. Natural Killer (NK) cells are innate lymphoid cells that are involved in the control of viral infection and tumors. NK cells respond to epinephrine mainly through beta2-adrenergic receptor (beta2AR) signaling, which leads to increased cAMP levels in the cytoplasm. Our data show that beta2AR stimulation affects signal transduction of activating NK cell receptors. As a result, acute exposure of NK cells to epinephrine reduces NK cell cytotoxicity and production of interferon-gamma. More importantly, epinephrine blocks the affinity increase of the adhesion protein LFA-1, which can be induced through the stimulation of NK cell activating receptors. This suggests that beta2AR signaling interferes with LFA-1 mediated NK cell adhesion. Interestingly, chronic beta2AR exposure completely prevented the inhibitory effects of acute epinephrine stimulation on NK cell functions. Therefore, acute and chronic exposure to epinephrine can have very different effects on the function of immune cells, which may explain some effects of acute and chronic stress on the immune system.

Bidirectional Role of β2-Adrenergic Receptor in Autoimmune Diseases.

Disorder of the sympathetic nervous system (SNS) is closely related to the pathogenesis of various autoimmune diseases (ADs). Catecholamine triggered beta2-adrenergic receptor (β2-AR) signaling is important in creating a bidirectional response in the progression of ADs due to factors including diverse expression patterns, single nucleotide polymorphisms (SNPs), biased signals, and desensitization of β2-AR, as well as different subtypes of Gα binding to β2-AR. In this review, we summarize the actions of β2-AR signaling in regulating the functions of immunocytes and in the pathogenesis of ADs, and the application of β2-AR agonists or antagonists in treating major types of ADs is also discussed. We suggest that restoring the immune balance via a soft regulation of the expression or activation of β2-AR is one of the promising therapeutic strategies for systematic ADs.

Sympathetic neurotransmission in spleens from aging Brown-Norway rats subjected to reduced sympathetic tone

Senescence of innate and adaptive responses and low-grade inflammation (inflammaging) hallmarks normal aging, which increases vulnerability to infectious diseases, autoimmunity and cancer. In normal aging, sympathetic dysregulation contributes to the dysregulation of innate and adaptive immunity and inflammaging. Sympathetic innervation of immune cells in secondary immune organs regulates immune responses. Previously in Fischer 344 (F344) rats, we reported an age-related increase in sympathetic tone and sympathetic dysfunction in beta-adrenergic receptor (AR) signaling of splenic lymphocytes that contributes to immune senescence, although the responsible mechanisms remains unexplored. In this study, we extend our previous findings using the much longer-lived Brown-Norway (BN) rats, whose behavior and immune response profile differ strikingly from F344 rats. Here, we investigated whether increased sympathetic nerve activity (SNA) in the aging spleen contributes to age-related sympathetic neuropathy and altered neurotransmission in splenic lymphocytes in BN rats. Fifteen-month male BN rats received 0, 0.5 or 1.5 μg/kg/day rilmenidine intraperitoneally for 90 days to lower sympathetic tone. Untreated young and age-matched rats controlled for effects of age. We found that elevated SNA in the aging BN rat spleen does not contribute significantly to sympathetic neuropathy or the aging-induced impairment of canonical β-AR signal transduction. Despite the rilmenidine-induced increase in β-AR expression, splenocyte c-AMP production was comparable with age-matched controls, thus dampening nerve activity had no effect on receptor coupling to adenylate cyclase. Understanding how aging affects neuroimmune regulation in healthy aging rodent models may eventually lead to strategies that improve health in aging populations vulnerable to immunosenescence and low-grade systemic inflammation.

A novel adenylyl cyclase type 5 inhibitor that reduces myocardial infarct size even when administered after coronary artery reperfusion

We developed a novel adenylyl cyclase type 5 (AC5) inhibitor, C90, that reduces myocardial infarct size even when administered after coronary reperfusion. This is key, since it is not practical to administer a drug to a patient with myocardial infarction before revascularization, and is one reason why so many prior drugs, which reduced infarct in experimental animals, failed in clinical trials. C90 is the most potent AC5 inhibitor, as exhibited by its IC50 value for AC5 inhibition, which was 5 times lower than the next most potent AC5 inhibitor. C90 reduced cAMP in response to forskolin in wild type mice by 42%, but no longer reduced cAMP in response to forskolin in mice with disruption of AC5, indicating that the mechanism of C90 was specific for AC5 inhibition. Compared with vehicle treatment, C90 reduced infarct size by 64% at a dose of 0.6 mg/kg. Thus, C90 is a novel, selective and potent AC5 inhibitor that reduces infarct size, when delivered after coronary artery reperfusion, rendering it potentially clinically useful. It also reduces beta-adrenergic receptor signaling, which will provide additional benefit to patients with coronary artery disease or heart failure.

The Development of Compartmentation of cAMP Signaling in Cardiomyocytes: The Role of T-Tubules and Caveolae Microdomains.

3′-5′-cyclic adenosine monophosphate (cAMP) is a signaling messenger produced in response to the stimulation of cellular receptors, and has a myriad of functional applications depending on the cell type. In the heart, cAMP is responsible for regulating the contraction rate and force; however, cAMP is also involved in multiple other functions. Compartmentation of cAMP production may explain the specificity of signaling following a stimulus. In particular, transverse tubules (T-tubules) and caveolae have been found to be critical structural components for the spatial confinement of cAMP in cardiomyocytes, as exemplified by beta-adrenergic receptor (β-ARs) signaling. Pathological alterations in cardiomyocyte microdomain architecture led to a disruption in compartmentation of the cAMP signal. In this review, we discuss the difference between atrial and ventricular cardiomyocytes in respect to microdomain organization, and the pathological changes of atrial and ventricular cAMP signaling in response to myocyte dedifferentiation. In addition, we review the role of localized phosphodiesterase (PDE) activity in constraining the cAMP signal. Finally, we discuss microdomain biogenesis and maturation of cAMP signaling with the help of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Understanding these mechanisms may help to overcome the detrimental effects of pathological structural remodeling.

Non-selective beta blockers inhibit angiosarcoma cell viability and increase progression free- and overall-survival in patients diagnosed with metastatic angiosarcoma.

Patients with metastatic angiosarcoma undergoing chemotherapy, radiation, and/or surgery experience a median progression free survival of less than 6 months and a median overall survival of less than 12 months. Given the aggressive nature of this cancer, angiosarcoma clinical responses to chemotherapy or targeted therapeutics are generally very poor. Inhibition of beta adrenergic receptor (β-AR) signaling has recently been shown to decrease angiosarcoma tumor cell viability, abrogate tumor growth in mouse models, and decrease proliferation rates in preclinical and clinical settings. In the current study we used cell and animal tumor models to show that β-AR antagonism abrogates mitogenic signaling and reduces angiosarcoma tumor cell viability, and these molecular alterations translated into patient tumors. We demonstrated that non-selective β-AR antagonists are superior to selective β-AR antagonists at inhibiting angiosarcoma cell viability. A prospective analysis of non- selective β-AR antagonists in a single arm clinical study of metastatic angiosarcoma patients revealed that incorporation of either propranolol or carvedilol into patients' treatment regimens leads to a median progression free and overall survival of 9 and 36 months, respectively. These data suggest that incorporation of non-selective β-AR antagonists into existing therapies against metastatic angiosarcoma can enhance clinical outcomes.