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Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

BACKGROUND AND PURPOSE: Stress‐related catecholamines have a role in cancer and β‐adrenoceptors; specifically, β(2)‐adrenoceptors have been identified as new targets in treating melanoma. Recently, β(3)‐adrenoceptors have shown a pleiotropic effect on melanoma micro‐environment leading to cancer progression. However, the mechanisms by which β(3)‐adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub‐population homeostasis. Understanding the mechanisms of cancer immune‐tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β(3)‐adrenoceptors in immune‐tolerance regulation. EXPERIMENTAL APPROACH: A mouse model of melanoma in which syngeneic B16‐F10 cells were injected in C57BL‐6 mice was used to evaluate the effect of β‐adrenoceptor blockade on the number and activity of immune cell sub‐populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β‐blockers (propranolol and SR59230A) and specific β‐adrenoceptor siRNAs targeting β(2)‐ or β(3)‐adrenoceptors were used. KEY RESULTS: Only β(3)‐, but not β(2)‐adrenoceptors, were up‐regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub‐populations including Treg, MDSC, and NK. SR59230A and β(3)‐adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub‐populations in the tumour mass, blood, and spleen. SR59230A and β(3)‐adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes. CONCLUSIONS AND IMPLICATIONS: Our data suggest that β(3)‐adrenoceptors are involved in immune‐tolerance, which opens the way for new strategic therapies to overcome melanoma growth. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors—New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc

Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

Although there is an increasing evidence that cancer stem cell (CSC) niches in the tumor microenvironment (TME) plays a crucial role in sustaining solid tumors progression, several molecular players involved in this regulation still remain unknown. The role of &beta, adrenergic signaling in enhancing tumor growth through &beta, 2-adrenoreceptors (&beta, 2-ARs) has been confirmed in different cancer models, but the role played by the &beta, 3-adrenergic receptor (&beta, 3-AR) has recently emerged. Previous studies showed that &beta, 3-AR promotes cancer growth through the activation of different stromal cells in the TME, and leads to melanoma malignancy progression through inflammation, angiogenesis, and immunotolerance. Here we show that in B16 melanoma-bearing mice, the pharmacological &beta, 3-AR blockade is able to reduce the expression of CSC markers, and to induce a differentiated phenotype of hematopoietic subpopulations in TME. In particular, cytofluorimetric analysis (FACS) of the tumor mass shows that &beta, 3-AR antagonist SR59230A promotes hematopoietic differentiation as indicated by increased ratios of lymphoid/hematopoietic stem cells (HSCs) and of myeloid progenitor cells/HSCs, and increases the number of Ter119 and natural killer (NK) precursor cells, and of granulocyte precursors, indicating active hematopoiesis within the tumor tissue. Moreover, pharmacological antagonism of &beta, 3-AR induces mesenchymal stem cell (MSC) differentiation into adipocytes subtracting a potential renewal of the stem compartment by these cells. Here we demonstrate that &beta, 3-AR blockade in the TME by inducing the differentiation of different stromal cells at the expense of stemness traits could possibly have a favorable effect on the control of melanoma progression.

Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress.