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Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus

αAM and αAMR induce phosphorylation of Tyr416Src in vitro(A & B) Src becomes phosphorylated on Tyr416 in response to αAM and αAMR treatment. αAM (70 μg/ml) (A) and αAMR (70 μg/ml) (B) increased Tyr416 Src phosphorylation in HUVECs in a time-dependent manner. β-actin was used as a loading control. (C) Src inhibitor impaired αAM-induced VE-cadherin phosphorylation. HUVECs were preincubated with increasing concentrations of SU6656 as indicated and then treated with αAM (70 μg/ml) for 6 h. VE-cadherin and pY731-VE-cadherin were detected by Western blotting. β-actin was used as a loading control. (D) αAM and αAMR promote VE-cadherin/Src association. VE-cadherin was immunoprecipitated from HUVECs treated with IgG-control (70 μg/ml), AM (10−7 M), αAMR (70 μg/ml), and αAM (70 μg/ml). Immunoblotting was used to reveal VE-cadherin, Src and pY416-Src.
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Figure 6: αAM and αAMR induce phosphorylation of Tyr416Src in vitro(A & B) Src becomes phosphorylated on Tyr416 in response to αAM and αAMR treatment. αAM (70 μg/ml) (A) and αAMR (70 μg/ml) (B) increased Tyr416 Src phosphorylation in HUVECs in a time-dependent manner. β-actin was used as a loading control. (C) Src inhibitor impaired αAM-induced VE-cadherin phosphorylation. HUVECs were preincubated with increasing concentrations of SU6656 as indicated and then treated with αAM (70 μg/ml) for 6 h. VE-cadherin and pY731-VE-cadherin were detected by Western blotting. β-actin was used as a loading control. (D) αAM and αAMR promote VE-cadherin/Src association. VE-cadherin was immunoprecipitated from HUVECs treated with IgG-control (70 μg/ml), AM (10−7 M), αAMR (70 μg/ml), and αAM (70 μg/ml). Immunoblotting was used to reveal VE-cadherin, Src and pY416-Src.

Mentions: Previous studies suggest that Src kinases play a significant role in cadherin regulation [20, 37]. To determine whether phosphorylation of the VE-cadherin Tyr731 can be mediated by Src, we assessed the effects of αAM and αAMR on the phosphorylation of Src. The data demonstrate that Src was phosphorylated on Tyr416 in response to αAM and αAMR treatment revealing the strong control of AM on Src kinases (Figures 6A, 6B). No increase in total Src protein was observed, suggesting that the phosphorylation patterns are independent of the changes in the overall protein expression (Figures 6A, 6B). When HUVECs were pre-treated with Src inhibitor, SU6656 as indicated, αAM-induced VE-cadherin phosphorylation after 6 h was inhibited in a dose-dependent manner (Figure 6C). The same data were obtained with αAMR (not shown). We conclude that Src is required for αAM and αAMR-induced VE-cadherin phosphorylation on Tyr731. Furthermore, we demonstrate that αAM and αAMR promote Src-VE-cadherin association, meanwhile AM decreases the Src-VE-cadherin association (Figure 6D). VE-cadherin immunoprecipitates from AM, αAM, αAMR, and IgG-control-treated HUVECs were immunoblotted with anti-Src antibody. The presence of Src was detected in each immunoprecipitate and demonstrates a clear increase of Src association to VE-cadherin in αAM and αAMR-treated HUVECs when compared to IgG-control (Figure 6D). In contrast, treatment with AM reduces VE-cadherin/Src association (Figure 6D). These findings reveal that AM blockade with αAM or αAMR activates Src kinase and promotes VE-cadherin/Src association leading to the pTyr731VE-cadherin and thus may account for the well known role that Src kinases play in VE-cadherin-mediated cell-cell junctional activity [23].


Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

αAM and αAMR induce phosphorylation of Tyr416Src in vitro(A & B) Src becomes phosphorylated on Tyr416 in response to αAM and αAMR treatment. αAM (70 μg/ml) (A) and αAMR (70 μg/ml) (B) increased Tyr416 Src phosphorylation in HUVECs in a time-dependent manner. β-actin was used as a loading control. (C) Src inhibitor impaired αAM-induced VE-cadherin phosphorylation. HUVECs were preincubated with increasing concentrations of SU6656 as indicated and then treated with αAM (70 μg/ml) for 6 h. VE-cadherin and pY731-VE-cadherin were detected by Western blotting. β-actin was used as a loading control. (D) αAM and αAMR promote VE-cadherin/Src association. VE-cadherin was immunoprecipitated from HUVECs treated with IgG-control (70 μg/ml), AM (10−7 M), αAMR (70 μg/ml), and αAM (70 μg/ml). Immunoblotting was used to reveal VE-cadherin, Src and pY416-Src.
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Related In: Results  -  Collection

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Figure 6: αAM and αAMR induce phosphorylation of Tyr416Src in vitro(A & B) Src becomes phosphorylated on Tyr416 in response to αAM and αAMR treatment. αAM (70 μg/ml) (A) and αAMR (70 μg/ml) (B) increased Tyr416 Src phosphorylation in HUVECs in a time-dependent manner. β-actin was used as a loading control. (C) Src inhibitor impaired αAM-induced VE-cadherin phosphorylation. HUVECs were preincubated with increasing concentrations of SU6656 as indicated and then treated with αAM (70 μg/ml) for 6 h. VE-cadherin and pY731-VE-cadherin were detected by Western blotting. β-actin was used as a loading control. (D) αAM and αAMR promote VE-cadherin/Src association. VE-cadherin was immunoprecipitated from HUVECs treated with IgG-control (70 μg/ml), AM (10−7 M), αAMR (70 μg/ml), and αAM (70 μg/ml). Immunoblotting was used to reveal VE-cadherin, Src and pY416-Src.
Mentions: Previous studies suggest that Src kinases play a significant role in cadherin regulation [20, 37]. To determine whether phosphorylation of the VE-cadherin Tyr731 can be mediated by Src, we assessed the effects of αAM and αAMR on the phosphorylation of Src. The data demonstrate that Src was phosphorylated on Tyr416 in response to αAM and αAMR treatment revealing the strong control of AM on Src kinases (Figures 6A, 6B). No increase in total Src protein was observed, suggesting that the phosphorylation patterns are independent of the changes in the overall protein expression (Figures 6A, 6B). When HUVECs were pre-treated with Src inhibitor, SU6656 as indicated, αAM-induced VE-cadherin phosphorylation after 6 h was inhibited in a dose-dependent manner (Figure 6C). The same data were obtained with αAMR (not shown). We conclude that Src is required for αAM and αAMR-induced VE-cadherin phosphorylation on Tyr731. Furthermore, we demonstrate that αAM and αAMR promote Src-VE-cadherin association, meanwhile AM decreases the Src-VE-cadherin association (Figure 6D). VE-cadherin immunoprecipitates from AM, αAM, αAMR, and IgG-control-treated HUVECs were immunoblotted with anti-Src antibody. The presence of Src was detected in each immunoprecipitate and demonstrates a clear increase of Src association to VE-cadherin in αAM and αAMR-treated HUVECs when compared to IgG-control (Figure 6D). In contrast, treatment with AM reduces VE-cadherin/Src association (Figure 6D). These findings reveal that AM blockade with αAM or αAMR activates Src kinase and promotes VE-cadherin/Src association leading to the pTyr731VE-cadherin and thus may account for the well known role that Src kinases play in VE-cadherin-mediated cell-cell junctional activity [23].

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus