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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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β2AR-induced invadopodia formation is dependent on Src and increases tumor cell invasion in a three-dimensional collagen matrix. a Representative time series images acquired by confocal microscopy showing the effect of formotorol or isoproterenol (Iso) ± 0.05 μM ICI-118551 (ICI) on development of cell protrusions. Time in hours is indicated. b Graphical representation of track measurements is shown for 20 cells in each condition. c Quantification of cell displacement from the point of origin. d, e Representative maximum intensity projections of LifeAct-GFP+ MDA-MB-231 cells treated with 0.5 μM PP2 ± 0.5 μM formoterol and examined for active invadopodia. Inset shows GFP+ green puncta (active invadopodia that have degraded underlying matrix) or yellow puncta (inactive invadopodia where GFP+ puncta have not degraded the underlying red fluorescent matrix). Scale bar: 10 μm, or 5 μm for inset panels. e The percentage of invadopodia positive cells was quantified. N >80 cells per condition. f Graphical representation of track measurements after cells were embedded in 1 mg/ml collagen matrix and treated with 0.5μΜ formoterol ± PP2 Src inhibitor. N = 20 cells per condition. Error bars: SEM. *p <0.05 and **p <0.01
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Fig4: β2AR-induced invadopodia formation is dependent on Src and increases tumor cell invasion in a three-dimensional collagen matrix. a Representative time series images acquired by confocal microscopy showing the effect of formotorol or isoproterenol (Iso) ± 0.05 μM ICI-118551 (ICI) on development of cell protrusions. Time in hours is indicated. b Graphical representation of track measurements is shown for 20 cells in each condition. c Quantification of cell displacement from the point of origin. d, e Representative maximum intensity projections of LifeAct-GFP+ MDA-MB-231 cells treated with 0.5 μM PP2 ± 0.5 μM formoterol and examined for active invadopodia. Inset shows GFP+ green puncta (active invadopodia that have degraded underlying matrix) or yellow puncta (inactive invadopodia where GFP+ puncta have not degraded the underlying red fluorescent matrix). Scale bar: 10 μm, or 5 μm for inset panels. e The percentage of invadopodia positive cells was quantified. N >80 cells per condition. f Graphical representation of track measurements after cells were embedded in 1 mg/ml collagen matrix and treated with 0.5μΜ formoterol ± PP2 Src inhibitor. N = 20 cells per condition. Error bars: SEM. *p <0.05 and **p <0.01

Mentions: These findings suggest that β2AR signaling induces an invasive phenotype that favors tumor cell invasion in three-dimensional environments. To investigate this, MDA-MB-231 breast cancer cells were embedded in collagen matrix and cell locomotion was tracked over time. Under control conditions, cells remained rounded with little displacement from the position of origin (Fig. 4a, b). Treatment with β2AR-selective agonist formoterol induced cancer cells to elongate and form protrusions (Fig. 4a) and increased cell displacement over time (Fig. 4b, c), indicating that β2AR signaling is sufficient to induce invasion of MDA-MB-231 breast cancer cells. Treatment with isoproterenol similarly induced cellular protrusion formation and these effects were blocked by the β2AR-selective antagonist ICI-118551, confirming a role for β2AR signaling in these effects on cell invasion through a three-dimensional collagen matrix. By identifying differential effects of βAR signaling between cell movement on two-dimensional surfaces compared with migration in three-dimensional environments, these findings may reconcile seemingly inconsistent observations for the effects of endogenous neurotransmitters and βAR-selective agonists on tumor cell migration [30–33]. Because movement in three-dimensional environments reflects processes that are required for tumor cell intravasation and extravasation during metastasis, these findings suggest a cellular mechanism for observations that βAR signaling drives breast cancer progression [12, 13].Fig. 4


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-induced invadopodia formation is dependent on Src and increases tumor cell invasion in a three-dimensional collagen matrix. a Representative time series images acquired by confocal microscopy showing the effect of formotorol or isoproterenol (Iso) ± 0.05 μM ICI-118551 (ICI) on development of cell protrusions. Time in hours is indicated. b Graphical representation of track measurements is shown for 20 cells in each condition. c Quantification of cell displacement from the point of origin. d, e Representative maximum intensity projections of LifeAct-GFP+ MDA-MB-231 cells treated with 0.5 μM PP2 ± 0.5 μM formoterol and examined for active invadopodia. Inset shows GFP+ green puncta (active invadopodia that have degraded underlying matrix) or yellow puncta (inactive invadopodia where GFP+ puncta have not degraded the underlying red fluorescent matrix). Scale bar: 10 μm, or 5 μm for inset panels. e The percentage of invadopodia positive cells was quantified. N >80 cells per condition. f Graphical representation of track measurements after cells were embedded in 1 mg/ml collagen matrix and treated with 0.5μΜ formoterol ± PP2 Src inhibitor. N = 20 cells per condition. Error bars: SEM. *p <0.05 and **p <0.01
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Fig4: β2AR-induced invadopodia formation is dependent on Src and increases tumor cell invasion in a three-dimensional collagen matrix. a Representative time series images acquired by confocal microscopy showing the effect of formotorol or isoproterenol (Iso) ± 0.05 μM ICI-118551 (ICI) on development of cell protrusions. Time in hours is indicated. b Graphical representation of track measurements is shown for 20 cells in each condition. c Quantification of cell displacement from the point of origin. d, e Representative maximum intensity projections of LifeAct-GFP+ MDA-MB-231 cells treated with 0.5 μM PP2 ± 0.5 μM formoterol and examined for active invadopodia. Inset shows GFP+ green puncta (active invadopodia that have degraded underlying matrix) or yellow puncta (inactive invadopodia where GFP+ puncta have not degraded the underlying red fluorescent matrix). Scale bar: 10 μm, or 5 μm for inset panels. e The percentage of invadopodia positive cells was quantified. N >80 cells per condition. f Graphical representation of track measurements after cells were embedded in 1 mg/ml collagen matrix and treated with 0.5μΜ formoterol ± PP2 Src inhibitor. N = 20 cells per condition. Error bars: SEM. *p <0.05 and **p <0.01
Mentions: These findings suggest that β2AR signaling induces an invasive phenotype that favors tumor cell invasion in three-dimensional environments. To investigate this, MDA-MB-231 breast cancer cells were embedded in collagen matrix and cell locomotion was tracked over time. Under control conditions, cells remained rounded with little displacement from the position of origin (Fig. 4a, b). Treatment with β2AR-selective agonist formoterol induced cancer cells to elongate and form protrusions (Fig. 4a) and increased cell displacement over time (Fig. 4b, c), indicating that β2AR signaling is sufficient to induce invasion of MDA-MB-231 breast cancer cells. Treatment with isoproterenol similarly induced cellular protrusion formation and these effects were blocked by the β2AR-selective antagonist ICI-118551, confirming a role for β2AR signaling in these effects on cell invasion through a three-dimensional collagen matrix. By identifying differential effects of βAR signaling between cell movement on two-dimensional surfaces compared with migration in three-dimensional environments, these findings may reconcile seemingly inconsistent observations for the effects of endogenous neurotransmitters and βAR-selective agonists on tumor cell migration [30–33]. Because movement in three-dimensional environments reflects processes that are required for tumor cell intravasation and extravasation during metastasis, these findings suggest a cellular mechanism for observations that βAR signaling drives breast cancer progression [12, 13].Fig. 4

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