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.

Cancer-associated fibroblasts (CAFs) play a role in tumour initiation and progression, possibly by inducing epithelial-to-mesenchymal transition (EMT), a series of cellular changes that is known to underlie the process of metastasis. The aim of this study was to determine whether CAFs and surrounding normal breast fibroblasts (NBFs) are able to induce EMT markers and functional changes in breast epithelial cancer cells. Matched pairs of CAFs and NBFs were established from fresh human breast cancer specimens and characterised by assessment of CXCL12 levels, a-smooth muscle actin (a-SMA) levels and response to doxorubicin. The fibroblasts were then co-cultured with MCF7 cells. Vimentin and E-cadherin expressions were determined in co-cultured MCF7 cells by immunofluorescence and confocal microscopy as well as by western blotting and quantitative PCR. Co-cultured MCF7 cells were also assessed functionally by invasion assay. CAFs secreted higher levels of CXCL12 and expressed higher levels of a-SMA compared with NBFs. CAFs were also less sensitive to doxorubicin as evidenced by less H2AX phosphorylation and reduced apoptosis on flow cytometric analysis of Annexin V compared with NBFs. When co-cultured with MCF7 cells, there was greater vimentin and less E-cadherin expression as well as greater invasiveness in MCF7 cells co-cultured with CAFs compared with those co-cultured with NBFs. CAFs have the ability to induce a greater degree of EMT in MCF7 cell lines, indicating that CAFs contribute to a more malignant breast cancer phenotype and their role in influencing therapy resistance should therefore be considered when treating breast cancer.

IntroductionFor 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.MethodsTo 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.Resultsβ-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the β2-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. β2-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 β2-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination.ConclusionThese findings provide insight into conditions that control tumor cell invasion by identifying signaling through β2-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using β-blockers to target β2-adrenoceptors to limit tumor cell dissemination and metastasis.

Activation of the sympathetic nervous system by stress increases breast cancer metastasis in vivo. Preclinical studies suggest that stress activates b-adrenoceptors (bARs) to enhance metastasis from primary tumors and that b-blockers may be protective in breast cancer. However, the subtype of bAR that mediates this effect, as well as the signaling mechanisms underlying increased tumor cell dissemination, remain unclear. We show that the b 2 AR is the only functionally relevant bAR subtype in the highly metastatic human breast cancer cell line MDA-MB-231HM. b 2 AR activation results in elevated cAMP (formoterol pEC 50 9.86 6 0.32), increased intracellular Ca 2+ (formoterol pEC 50 8.20 6 0.33) and reduced phosphorylated ERK (pERK; formoterol pIC 50 11.62 6 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 b 2 AR increased invasion (formoterol area under the curve [AUC] relative to vehicle: 1.82 6 0.36), which was dependent on the cAMP/ Ca 2+ loop (formoterol AUC in the presence of 2959-dideoxyadenosine 0.64 6 0.03, or BAPTA-AM 0.45 6 0.23) but independent of inhibition of basal pERK1/2 (vehicle AUC with U0126 0.60 6 0

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.