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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 Ser33/Ser37/Thr41 β-catenin in pericytes in vivo and in HUVSMCs in vitro(A) tumor sections were evaluated by immunfluorescence for α-SMA (red) and pSer33/Ser37/Thr41-β-catenin (green) using anti-α-SMA and anti-pSer33/Ser37/Thr41-β-catenin antibodies in IgG-control, αAM, and αAMR-treated animals for 6 days. DAPI-stained nuclei are in blue. (B & C) western blot analysis showed that incubation of HUVSMCs in vitro for the indicated times with αAM and αAMR induces pSer33/Ser37/Thr41 β-catenin. β-actin was used as a loading control.
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Figure 9: αAM and αAMR induce phosphorylation of Ser33/Ser37/Thr41 β-catenin in pericytes in vivo and in HUVSMCs in vitro(A) tumor sections were evaluated by immunfluorescence for α-SMA (red) and pSer33/Ser37/Thr41-β-catenin (green) using anti-α-SMA and anti-pSer33/Ser37/Thr41-β-catenin antibodies in IgG-control, αAM, and αAMR-treated animals for 6 days. DAPI-stained nuclei are in blue. (B & C) western blot analysis showed that incubation of HUVSMCs in vitro for the indicated times with αAM and αAMR induces pSer33/Ser37/Thr41 β-catenin. β-actin was used as a loading control.

Mentions: Sections of αAM and αAMR-treated tumors showed vascular endothelial cells that stained with phospho-β-catenin antibody (Figure 8A). No staining for phospho-β-catenin can be observed in vascular endothelial cells in IgG-control section (Figure 8A). Quantification of phospho-β-catenin stained endothelial cells demonstrates a significant increase in αAM and αAMR-treated tumors after 2 and 6 days treatment when compared with IgG-control treated tumors (Figure 8B). No staining differences can be observed at days 11 and 16 that might be the results of phospho-β-catenin degradation after ubiquitination and proteasomal degradation (Figure 8B). Positive phospho-β-catenin staining can be observed for pericytes (α-SMA positive cells) (Figure 9A). Treatment of HUVSMC in vitro with αAM and αAMR induces phosphorylation of β-catenin at Ser33/Ser37/Thr41 (Figures 9B, 9C).


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 Ser33/Ser37/Thr41 β-catenin in pericytes in vivo and in HUVSMCs in vitro(A) tumor sections were evaluated by immunfluorescence for α-SMA (red) and pSer33/Ser37/Thr41-β-catenin (green) using anti-α-SMA and anti-pSer33/Ser37/Thr41-β-catenin antibodies in IgG-control, αAM, and αAMR-treated animals for 6 days. DAPI-stained nuclei are in blue. (B & C) western blot analysis showed that incubation of HUVSMCs in vitro for the indicated times with αAM and αAMR induces pSer33/Ser37/Thr41 β-catenin. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480698&req=5

Figure 9: αAM and αAMR induce phosphorylation of Ser33/Ser37/Thr41 β-catenin in pericytes in vivo and in HUVSMCs in vitro(A) tumor sections were evaluated by immunfluorescence for α-SMA (red) and pSer33/Ser37/Thr41-β-catenin (green) using anti-α-SMA and anti-pSer33/Ser37/Thr41-β-catenin antibodies in IgG-control, αAM, and αAMR-treated animals for 6 days. DAPI-stained nuclei are in blue. (B & C) western blot analysis showed that incubation of HUVSMCs in vitro for the indicated times with αAM and αAMR induces pSer33/Ser37/Thr41 β-catenin. β-actin was used as a loading control.
Mentions: Sections of αAM and αAMR-treated tumors showed vascular endothelial cells that stained with phospho-β-catenin antibody (Figure 8A). No staining for phospho-β-catenin can be observed in vascular endothelial cells in IgG-control section (Figure 8A). Quantification of phospho-β-catenin stained endothelial cells demonstrates a significant increase in αAM and αAMR-treated tumors after 2 and 6 days treatment when compared with IgG-control treated tumors (Figure 8B). No staining differences can be observed at days 11 and 16 that might be the results of phospho-β-catenin degradation after ubiquitination and proteasomal degradation (Figure 8B). Positive phospho-β-catenin staining can be observed for pericytes (α-SMA positive cells) (Figure 9A). Treatment of HUVSMC in vitro with αAM and αAMR induces phosphorylation of β-catenin at Ser33/Ser37/Thr41 (Figures 9B, 9C).

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