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

Creed SJ, Le CP, Hassan M, Pon CK, Albold S, Chan KT, Berginski ME, Huang Z, Bear JE, Lane JR, Halls ML, Ferrari D, Nowell CJ, Sloan EK - Breast Cancer Res. (2015)

Bottom Line: However, little is known about the conditions that favor invadopodia formation.The effects were selectively mediated by the β2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate 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.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. sjcreed@hotmail.com.

ABSTRACT

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.

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Related in: MedlinePlus

β2AR signaling induces invadopodia formation. a LifeAct-GFP+ MDA-MB-231 cells were plated on Alexa-568-labelled gelatin and active invadopodia were identified by confocal microscopy as LifeAct-GFP+ puncta colocalized with degraded gelatin (loss of red fluorescence; see inset). Representative images are shown. Scale bar: 20 μm, or 5 μm for inset panel. b The frequency of invadopodia-positive cells was determined in cells treated with isoproterenol (Iso). c, d Cells were treated with 0.5 μM Iso ± 0.05 μM propranolol (Prop), and c the frequency of invadopodia-positive cells was determined or d the number of invadopodia per cell was determined (N >130 cells per treatment). e Cells were treated with Iso ± β1AR-selective antagonist CGP-20712A (CGP) or β2AR-selective antagonist ICI-118551 (ICI) and the effect on invadopodia formation was quantified. f, g Cells were treated with Iso, β1AR-selective agonist xamoterol, or β2AR-selective agonist formoterol and f the effect on invadopodia was quantified. g Representative confocal sections are shown. Inset shows GFP+ active invadopodia on degraded matrix (loss of red fluorescence). Scale bar: 20 μm, or 5 μm for inset panels. N > 80 cells per treatment unless otherwise stated. Experiments were conducted in triplicate. Error bars: SEM. *p <0.05, **p <0.01 and ***p <0.001
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Fig2: β2AR signaling induces invadopodia formation. a LifeAct-GFP+ MDA-MB-231 cells were plated on Alexa-568-labelled gelatin and active invadopodia were identified by confocal microscopy as LifeAct-GFP+ puncta colocalized with degraded gelatin (loss of red fluorescence; see inset). Representative images are shown. Scale bar: 20 μm, or 5 μm for inset panel. b The frequency of invadopodia-positive cells was determined in cells treated with isoproterenol (Iso). c, d Cells were treated with 0.5 μM Iso ± 0.05 μM propranolol (Prop), and c the frequency of invadopodia-positive cells was determined or d the number of invadopodia per cell was determined (N >130 cells per treatment). e Cells were treated with Iso ± β1AR-selective antagonist CGP-20712A (CGP) or β2AR-selective antagonist ICI-118551 (ICI) and the effect on invadopodia formation was quantified. f, g Cells were treated with Iso, β1AR-selective agonist xamoterol, or β2AR-selective agonist formoterol and f the effect on invadopodia was quantified. g Representative confocal sections are shown. Inset shows GFP+ active invadopodia on degraded matrix (loss of red fluorescence). Scale bar: 20 μm, or 5 μm for inset panels. N > 80 cells per treatment unless otherwise stated. Experiments were conducted in triplicate. Error bars: SEM. *p <0.05, **p <0.01 and ***p <0.001

Mentions: To investigate the role of βAR signaling in invadopodia formation, LifeAct-GFP+ MDA-MB-231 cells were plated on a fluorescent gelatin matrix and the effect of β-agonist isoproterenol on matrix degradation was evaluated (Fig. 2a). While actin-rich puncta were identified in the entire population, only 16 % (± 4 %) of tumor cells produced active invadopodia as defined by degradation of underlying matrix fluorescence (Fig. 2a, b). Treatment with isoproterenol led to an increase in the frequency of invadopodia-positive cells, with a maximum increase of 2.5-fold in response to 0.5 μM isoproterenol (Fig. 2b). In addition to increasing the frequency of invadopodia-positive cells in the population (Fig. 2b, c), isoproterenol also increased the average number of invadopodia per cell by 2.7-fold (Fig. 2d). Treatment with the nonselective β-blocker propranolol blocked the effect of isoproterenol on both the frequency of invadopodia-positive cells and the number of invadopodia per cell (Fig. 2c, d). This confirms that isoproterenol signals via βAR to increase invadopodia formation. To confirm the βAR subtype that mediates this effect, MDA-MB-231 cells were treated with selective antagonists and the effect on invadopodia was evaluated. β2AR-selective antagonist ICI-118551 blocked the increase in invadopodia formation in response to isoproterenol, whereas there was no effect of β1AR-selective antagonist CGP-20712A (Fig. 2e). To determine whether β2AR signaling is sufficient to induce invadopodia formation, cells were treated with selective βAR agonists. β2AR-selective agonist formoterol – but not β1AR-selective agonist xamoterol – induced invadopodia formation (Fig. 2f, g), confirming a key role for β2AR signaling in the formation of invadopodia in these cells.Fig. 2


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

Creed SJ, Le CP, Hassan M, Pon CK, Albold S, Chan KT, Berginski ME, Huang Z, Bear JE, Lane JR, Halls ML, Ferrari D, Nowell CJ, Sloan EK - Breast Cancer Res. (2015)

β2AR signaling induces invadopodia formation. a LifeAct-GFP+ MDA-MB-231 cells were plated on Alexa-568-labelled gelatin and active invadopodia were identified by confocal microscopy as LifeAct-GFP+ puncta colocalized with degraded gelatin (loss of red fluorescence; see inset). Representative images are shown. Scale bar: 20 μm, or 5 μm for inset panel. b The frequency of invadopodia-positive cells was determined in cells treated with isoproterenol (Iso). c, d Cells were treated with 0.5 μM Iso ± 0.05 μM propranolol (Prop), and c the frequency of invadopodia-positive cells was determined or d the number of invadopodia per cell was determined (N >130 cells per treatment). e Cells were treated with Iso ± β1AR-selective antagonist CGP-20712A (CGP) or β2AR-selective antagonist ICI-118551 (ICI) and the effect on invadopodia formation was quantified. f, g Cells were treated with Iso, β1AR-selective agonist xamoterol, or β2AR-selective agonist formoterol and f the effect on invadopodia was quantified. g Representative confocal sections are shown. Inset shows GFP+ active invadopodia on degraded matrix (loss of red fluorescence). Scale bar: 20 μm, or 5 μm for inset panels. N > 80 cells per treatment unless otherwise stated. Experiments were conducted in triplicate. Error bars: SEM. *p <0.05, **p <0.01 and ***p <0.001
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Fig2: β2AR signaling induces invadopodia formation. a LifeAct-GFP+ MDA-MB-231 cells were plated on Alexa-568-labelled gelatin and active invadopodia were identified by confocal microscopy as LifeAct-GFP+ puncta colocalized with degraded gelatin (loss of red fluorescence; see inset). Representative images are shown. Scale bar: 20 μm, or 5 μm for inset panel. b The frequency of invadopodia-positive cells was determined in cells treated with isoproterenol (Iso). c, d Cells were treated with 0.5 μM Iso ± 0.05 μM propranolol (Prop), and c the frequency of invadopodia-positive cells was determined or d the number of invadopodia per cell was determined (N >130 cells per treatment). e Cells were treated with Iso ± β1AR-selective antagonist CGP-20712A (CGP) or β2AR-selective antagonist ICI-118551 (ICI) and the effect on invadopodia formation was quantified. f, g Cells were treated with Iso, β1AR-selective agonist xamoterol, or β2AR-selective agonist formoterol and f the effect on invadopodia was quantified. g Representative confocal sections are shown. Inset shows GFP+ active invadopodia on degraded matrix (loss of red fluorescence). Scale bar: 20 μm, or 5 μm for inset panels. N > 80 cells per treatment unless otherwise stated. Experiments were conducted in triplicate. Error bars: SEM. *p <0.05, **p <0.01 and ***p <0.001
Mentions: To investigate the role of βAR signaling in invadopodia formation, LifeAct-GFP+ MDA-MB-231 cells were plated on a fluorescent gelatin matrix and the effect of β-agonist isoproterenol on matrix degradation was evaluated (Fig. 2a). While actin-rich puncta were identified in the entire population, only 16 % (± 4 %) of tumor cells produced active invadopodia as defined by degradation of underlying matrix fluorescence (Fig. 2a, b). Treatment with isoproterenol led to an increase in the frequency of invadopodia-positive cells, with a maximum increase of 2.5-fold in response to 0.5 μM isoproterenol (Fig. 2b). In addition to increasing the frequency of invadopodia-positive cells in the population (Fig. 2b, c), isoproterenol also increased the average number of invadopodia per cell by 2.7-fold (Fig. 2d). Treatment with the nonselective β-blocker propranolol blocked the effect of isoproterenol on both the frequency of invadopodia-positive cells and the number of invadopodia per cell (Fig. 2c, d). This confirms that isoproterenol signals via βAR to increase invadopodia formation. To confirm the βAR subtype that mediates this effect, MDA-MB-231 cells were treated with selective antagonists and the effect on invadopodia was evaluated. β2AR-selective antagonist ICI-118551 blocked the increase in invadopodia formation in response to isoproterenol, whereas there was no effect of β1AR-selective antagonist CGP-20712A (Fig. 2e). To determine whether β2AR signaling is sufficient to induce invadopodia formation, cells were treated with selective βAR agonists. β2AR-selective agonist formoterol – but not β1AR-selective agonist xamoterol – induced invadopodia formation (Fig. 2f, g), confirming a key role for β2AR signaling in the formation of invadopodia in these cells.Fig. 2

Bottom Line: However, little is known about the conditions that favor invadopodia formation.The effects were selectively mediated by the β2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate 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.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. sjcreed@hotmail.com.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus