β2-Adrenoceptors on tumor cells play a critical role in stress-enhanced metastasis in a mouse model of breast cancer

© 2016 The Authors Chronic stress accelerates metastasis – the main cause of death in cancer patients – through the activation of β-adrenoceptors (βARs). We have previously shown that β2AR signaling in MDA-MB-231HM breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express βAR, the role of β2AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of β2AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231HM breast cancer cells that are deficient in β2AR. β2AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of β2AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of β2AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for β2AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block β2AR on tumor cells to fully control metastatic progression

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations

G protein–coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal–regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations

Carvedilol blocks neural regulation of breast cancer progression in vivo and is associated with reduced breast cancer mortality in patients

© 2021 Elsevier Ltd Purpose: The sympathetic nervous system drives breast cancer progression through β-adrenergic receptor signalling. This discovery has led to the consideration of cardiac β-blocker drugs as novel strategies for anticancer therapies. Carvedilol is a β-blocker used in the management of cardiovascular disorders, anxiety, migraine and chemotherapy-induced cardiotoxicity. However, little is known about how carvedilol affects cancer-related outcomes. Methods: To address this, we investigated the effects of carvedilol on breast cancer cell lines, in mouse models of breast cancer and in a large cohort of patients with breast cancer (n = 4014). Results: Treatment with carvedilol blocked the effects of sympathetic nervous system activation, reducing primary tumour growth and metastasis in a mouse model of breast cancer and preventing invasion by breast cancer cell lines. A retrospective analysis found that women using carvedilol at breast cancer diagnosis (n = 136) had reduced breast cancer-specific mortality compared with women who did not (n = 3878) (5-year cumulative incidence of breast cancer deaths: 3.1% versus 5.7%; p = 0.024 and 0.076 from univariate and multivariable analyses, respectively) after a median follow-up of 5.5 years. Conclusions: These findings provide a rationale to further explore the use of the β-blocker carvedilol as a novel strategy to slow cancer progression

The β2‐adrenoceptor activates a positive cAMP‐calcium feedforward loop to drive breast cancer cell invasion

Activation of the sympathetic nervous system by stress increases breast cancer metastasis in vivo. Preclinical studies suggest that stress activates β-adrenoceptors (βARs) to enhance metastasis from primary tumors and that β-blockers may be protective in breast cancer. However, the subtype of βAR that mediates this effect, as well as the signaling mechanisms underlying increased tumor cell dissemination, remain unclear. We show that the β(2)AR is the only functionally relevant βAR subtype in the highly metastatic human breast cancer cell line MDA-MB-231HM. β(2)AR activation results in elevated cAMP (formoterol pEC(50) 9.86 ± 0.32), increased intracellular Ca(2+) (formoterol pEC(50) 8.20 ± 0.33) and reduced phosphorylated ERK (pERK; formoterol pIC(50) 11.62 ± 0.31). We demonstrate that a highly amplified positive feedforward loop between the cAMP and Ca(2+) pathways is responsible for efficient inhibition of basal pERK. Importantly, activation of the β(2)AR increased invasion (formoterol area under the curve [AUC] relative to vehicle: 1.82 ± 0.36), which was dependent on the cAMP/Ca(2+) loop (formoterol AUC in the presence of 2′5′-dideoxyadenosine 0.64 ± 0.03, or BAPTA-AM 0.45 ± 0.23) but independent of inhibition of basal pERK1/2 (vehicle AUC with U0126 0.60 ± 0.30). Specifically targeting the positive feedforward cAMP/Ca(2+) loop may be beneficial for the development of therapeutics to slow disease progression in patients with breast cancer.—Pon, C. K., Lane, J. R., Sloan, E. K., Halls, M. L. The β(2)-adrenoceptor activates a positive cAMP-calcium feedforward loop to drive breast cancer cell invasion

β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion

INTRODUCTION: For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion. METHODS: To characterize the molecular and cellular mechanisms of β-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of β-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis. RESULTS: β-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the β(2)-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. β(2)-adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo. Selective antagonism of β(2)-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination. CONCLUSION: These findings provide insight into conditions that control tumor cell invasion by identifying signaling through β(2)-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using β-blockers to target β(2)-adrenoceptors to limit tumor cell dissemination and metastasis