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

Structure-Based Design and Discovery of New M2 Receptor Agonists.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes

Beta2-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 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. Conclusion These 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

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

Structure-Based Design and Discovery of New M₂ Receptor Agonists

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M₂ receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds <b>3</b> and <b>17</b>), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound <b>28</b>, which was 4-fold more potent than its parent <b>3</b>. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to <b>28</b> or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes

Eigenstate Thermalization, Random Matrix Theory and Behemoths

The eigenstate thermalization hypothesis (ETH) is one of the cornerstones in our understanding of quantum statistical mechanics. The extent to which ETH holds for nonlocal operators is an open question that we partially address in this paper. We report on the construction of highly nonlocal operators, Behemoths, that are building blocks for various kinds of local and non-local operators. The Behemoths have a singular distribution and width w∼D−1 (D being the Hilbert space dimension). From them, one may construct local operators with the ordinary Gaussian distribution and w∼D−1/2 in agreement with ETH. Extrapolation to even larger widths predicts sub-ETH behavior of typical nonlocal operators with w∼D−δ, 0<δ<1/2. This operator construction is based on a deep analogy with random matrix theory and shows striking agreement with numerical simulations of non-integrable many-body systems