Blockade of IL-10 Signaling Ensures Mifamurtide Efficacy in Metastatic Osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of the bone, highly aggressive and metastasizing, and it mainly affects children and adolescents. The current standard of care for OS is a combination of surgery and chemotherapy. However, these treatment options are not always successful, especially in cases of metastatic or recurrent osteosarcomas. For this reason, research into new therapeutic strategies is currently underway, and immunotherapies have received considerable attention. Mifamurtide stands out among the most studied immunostimulant drugs; nevertheless, there are very conflicting opinions on its therapeutic efficacy. Here, we aimed to investigate mifamurtide efficacy through in vitro and in vivo experiments. Our results led us to identify a new possible target useful to improve mifamurtide effectiveness on metastatic OS: the cytokine interleukin-10 (IL-10). We provide experimental evidence that the synergic use of an anti-IL-10 antibody in combination with mifamurtide causes a significantly increased mortality rate in highest-grade OS cells and lower metastasis in an in vivo model compared with mifamurtide alone. Overall, our data suggest that mifamurtide in combination with an anti-IL-10 antibody could be proposed as a new treatment protocol to be studied to improve the outcomes of OS patients.

ß3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma.

Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via ß-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of ß3-adrenergic receptor (ß3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the ß3-AR modulation influenced the host immune system response against tumor. Results demonstrated that ß3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, ß3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that ß3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

Role of ß3-Adrenergic Receptor in Bone Marrow Transplant as Therapeutical Support in Cancer.

ß3-adrenergic receptor (ß3-AR) is the last ß-adrenoceptor subtype identified. ß3-AR is widely expressed and regulates numerous physiological processes, and it is also a potential target for the treatment of many diseases, including cancers. For some types of cancers, bone marrow transplant (BMT) represents a valid therapeutic support, especially in the case of the necessity of high-dose chemotherapy and radiotherapy. For a successful BMT, it is necessary that a donor's hematopoietic stem cells (HSCs) correctly reach the staminal niche in the recipient's bone marrow (BM) and contribute to restore normal hematopoiesis in order to rapidly repopulate BM and provide all the healthy blood cells of which the patient needs. Generally, it takes a long time. Control and accelerate homing and engraftment of HSCs could represent a helpful approach to avoid the complications and undesirable effects of BMT. The evidence that the ß-adrenergic system has a role in the BM can be found in different studies, and this leads us to hypothesize that studying this field could be interesting to meliorate the most critical aspects of a BMT. Here, we collected the data present in literature about the role of ß3-AR in the BM with the purpose of discovering a possible utility of ß3-AR modulation in regulating HSC trafficking and hematopoiesis.

ß2-and ß3-Adrenergic Receptors Contribute to Cancer-Evoked Pain in a Mouse Model of Osteosarcoma via Modulation of Neural Macrophages.

The mechanisms involved in the development and maintenance of cancer pain remain largely unidentified. Recently, it has been reported that ß-adrenergic receptors (ß-ARs), mainly ß2-and ß3-ARs, contribute to tumor proliferation and progression and may favor cancer-associated pain and neuroinflammation. However, the mechanism underlying ß-ARs in cancer pain is still unknown. Here, we investigated the role of ß1-, ß2-and ß3-ARs in a mouse model of cancer pain generated by the para-tibial injection of K7M2 osteosarcoma cells. Results showed a rapid tumor growth in the soft tissue associated with the development of mechanical allodynia in the hind paw ipsilateral to the injected site. In addition to reduce tumor growth, both propranolol and SR59230A, ß1-/ß2-and ß3-AR antagonists, respectively, attenuated mechanical allodynia, the number of macrophages and an oxidative stress by-product accumulated in the ipsilateral tibial nerve. The selective ß1-AR antagonist atenolol was able to slightly reduce the tumor growth but showed no effect in reducing the development of mechanical allodynia. Results suggest that the development of the mechanical allodynia in K7M2 osteosarcoma-bearing mice is mediated by oxidative stress associated with the recruitment of neural macrophages, and that antagonism of ß2-and ß3-ARs contribute not solely to the reduction of tumor growth, but also in cancer pain. Thus, the targeting of the ß2-and ß3-ARs signaling may be a promising therapeutic strategy against both tumor progression and the development of cancer-evoke pain in osteosarcoma.

Claisened Hexafluoro Inhibits Metastatic Spreading of Amoeboid Melanoma Cells.

Metastatic melanoma is characterized by poor prognosis and a low free-survival rate. Thanks to their high plasticity, melanoma cells are able to migrate exploiting different cell motility strategies, such as the rounded/amoeboid-type motility and the elongated/mesenchymal-type motility. In particular, the amoeboid motility strongly contributes to the dissemination of highly invasive melanoma cells and no treatment targeting this process is currently available for clinical application. Here, we tested Claisened Hexafluoro as a novel inhibitor of the amoeboid motility. Reported data demonstrate that Claisened Hexafluoro specifically inhibits melanoma cells moving through amoeboid motility by deregulating mitochondrial activity and activating the AMPK signaling. Moreover, Claisened Hexafluoro is able to interfere with the adhesion abilities and the stemness features of melanoma cells, thus decreasing the in vivo metastatic process. This evidence may contribute to pave the way for future possible therapeutic applications of Claisened Hexafluoro to counteract metastatic melanoma dissemination.

Peripheral Nerve Resident Macrophages and Schwann Cells Mediate Cancer-Induced Pain.

Although macrophages (MΦ) are known to play a central role in neuropathic pain, their contribution to cancer pain has not been established. Here we report that depletion of sciatic nerve resident MΦs (rMΦ) in mice attenuates mechanical/cold hypersensitivity and spontaneous pain evoked by intraplantar injection of melanoma or lung carcinoma cells. MΦ-colony stimulating factor (M-CSF) was upregulated in the sciatic nerve trunk and mediated cancer-evoked pain via rMΦ expansion, transient receptor potential ankyrin 1 (TRPA1) activation, and oxidative stress. Targeted deletion of Trpa1 revealed a key role for Schwann cell TRPA1 in sciatic nerve rMΦ expansion and pain-like behaviors. Depletion of rMΦs in a medial portion of the sciatic nerve prevented pain-like behaviors. Collectively, we identified a feed-forward pathway involving M-CSF, rMΦ, oxidative stress, and Schwann cell TRPA1 that operates throughout the nerve trunk to signal cancer-evoked pain. SIGNIFICANCE: Schwann cell TRPA1 sustains cancer pain through release of M-CSF and oxidative stress, which promote the expansion and the proalgesic actions of intraneural macrophages. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3387/F1.large.jpg.

Beta-Blockers and Berberine: A Possible Dual Approach to Contrast Neuroblastoma Growth and Progression.

The use of nutraceuticals during cancer treatment is a long-lasting debate. Berberine (BBR) is an isoquinoline quaternary alkaloid extracted from a variety of medicinal plants. BBR has been shown to have therapeutic effects in different pathologies, particularly in cancer, where it affects pathways involved in tumor progression. In neuroblastoma, the most common extracranial childhood solid tumor, BBR, reduces tumor growth by regulating both stemness and differentiation features and by inducing apoptosis. At the same time, the inhibition of ß-adrenergic signaling leads to a reduction in growth and increase of differentiation of neuroblastoma. In this review, we summarize the possible beneficial effects of BBR in counteracting tumor growth and progression in various types of cancer and, in particular, in neuroblastoma. However, BBR administration, besides its numerous beneficial effects, presents a few side effects due to inhibition of MAO A enzyme in neuroblastoma cells. Therefore, herein, we proposed a novel therapeutic strategy to overcome side effects of BBR administration consisting of concomitant administration of BBR together with ß-blockers in neuroblastoma.

ß3-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance.

ß-adrenergic signaling is known to be involved in cancer progression; in particular, beta3-adrenoreceptor (ß3-AR) is associated with different tumor conditions. Currently, there are few data concerning ß3-AR in myeloid malignancies. Here, we evaluated ß3-AR in myeloid leukemia cell lines and the effect of ß3-AR antagonist SR59230A. In addition, we investigated the potential role of ß3-AR blockade in doxorubicin resistance. Using flow cytometry, we assessed cell death in different in vitro myeloid leukemia cell lines (K562, KCL22, HEL, HL60) treated with SR59230A in hypoxia and normoxia; furthermore, we analyzed ß3-AR expression. We used healthy bone marrow cells (BMCs), peripheral blood mononuclear cells (PBMCs) and cord blood as control samples. Finally, we evaluated the effect of SR59230A plus doxorubicin on K562 and K562/DOX cell lines; K562/DOX cells are resistant to doxorubicin and show P-glycoprotein (P-gp) overexpression. We found that SR59230A increased cancer cell lines apoptosis especially in hypoxia, resulting in selective activity for cancer cells; moreover, ß3-AR expression was higher in malignancies, particularly under hypoxic condition. Finally, we observed that SR59230A plus doxorubicin increased doxorubicin resistance reversion mainly in hypoxia, probably acting on P-gp. Together, these data point to ß3-AR as a new target and ß3-AR blockade as a potential approach in myeloid leukemias.

Current Therapies and New Targets to Fight Melanoma: A Promising Role for the ß3-Adrenoreceptor.

Melanoma is one of the most aggressive types of cancer and the most deadly skin cancer. According to World Health Organization, about 132,000 melanoma skin cancers occur globally each year. Thanks to the efficacy of new therapies, life expectation has been improved over the last years. However, some malignant melanomas still remain unresponsive to these therapies. The ß-adrenergic system, among its many physiological roles, has been recognized as the main mediator of stress-related tumorigenic events. In particular, catecholamine activation of ß-adrenergic receptors (ß-ARs) affects several processes that sustain cancer progression. Among the ß-AR subtypes, the ß3-AR is emerging as an important regulator of tumorigenesis. In this review, we summarize data of different experimental studies focused on ß3-AR involvement in tumor development in various types of cancer and, particularly, in melanoma. Taken together, the preclinical evidences reported in this review demonstrate the crucial role of ß3-AR in regulating the complex signaling network driving melanoma progression. Therefore, a need exists to further disseminate this new concept and to investigate more deeply the role of ß3-AR as a possible therapeutic target for counteracting melanoma progression at clinical level.

ß3-Adrenoreceptor Blockade Induces Stem Cells Differentiation in Melanoma Microenvironment.

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 ß-adrenergic signaling in enhancing tumor growth through ß2-adrenoreceptors (ß2-ARs) has been confirmed in different cancer models, but the role played by the ß3-adrenergic receptor (ß3-AR) has recently emerged. Previous studies showed that ß3-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 ß3-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 ß3-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 ß3-AR induces mesenchymal stem cell (MSC) differentiation into adipocytes subtracting a potential renewal of the stem compartment by these cells. Here we demonstrate that ß3-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.

ß3-adrenoreceptor blockade reduces tumor growth and increases neuronal differentiation in neuroblastoma via SK2/S1P2 modulation.

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. ß3-adrenoreceptor (ß3-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 ß3-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 ß3-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.

Blueberry juice protects osteocytes and bone precursor cells against oxidative stress partly through SIRT1.

Oxidative stress and abnormal osteocyte apoptosis are often related to dysregulation of bone turnover and chronic bone loss, and so fruit and vegetables with high antioxidant potential may play an important role in the prevention and/or management of osteoporosis. Osteocytes are the main regulators of bone remodelling. For the first time, we demonstrate here that blueberry juice (BJ), obtained from Vaccinium myrtillus, rich in polyphenols, shows antioxidant and antiosteoclastogenic properties in MLO-Y4 osteocytes. We report that BJ prevents oxidative stress-induced apoptosis and reverses the increase in receptor activator of nuclear factor κB ligand and sclerostin expression, crucial factors for osteoclast activation and bone resorption. BJ is also able to prevent oxidative stress-induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), which are considered to be an important tool for cell therapy in bone disorders. No significant difference in preventing these events was observed between BJ and blueberry dry extract containing equal amounts of total soluble polyphenols. We have also shown that blueberry acts as both an antioxidant and an activator of sirtuin type 1, a class III histone deacetylase involved in cell death regulation and considered a molecular target for blocking bone resorption without affecting osteoclast survival. Overall, these novel data obtained in osteocytes and MSCs may help us clarify the mechanisms by which blueberry counteracts oxidative stress-induced damage in bone remodelling and osteogenesis at the cellular and molecular level. Our findings are consistent with the reported beneficial effects of blueberry on bone tissue reported in animal studies, which suggest that blueberry may be a useful supplement for the prevention and/or management of osteoporosis and osteogenic process.

ß3 -Adrenoceptor as a potential immuno-suppressor agent in melanoma.

BACKGROUND AND PURPOSE: Stress-related catecholamines have a role in cancer and ß-adrenoceptors; specifically, ß2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, ß3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which ß3 -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 ß3 -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 ß-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with ß-blockers (propranolol and SR59230A) and specific ß-adrenoceptor siRNAs targeting ß2 - or ß3 -adrenoceptors were used. KEY RESULTS: Only ß3 -, but not ß2 -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 ß3 -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 ß3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes. CONCLUSIONS AND IMPLICATIONS: Our data suggest that ß3 -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.

ß3-Adrenoreceptors Control Mitochondrial Dormancy in Melanoma and Embryonic Stem Cells.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that ß3-adrenergic receptor (ß3-AR) is involved in tumor progression, playing an important role in metastasis. Among ß-adrenergic receptors, ß3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. ß3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, ß3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that ß3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The ß3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific ß3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of ß3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.