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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 β-catenin of vascular endothelial cells in U87 xenograftsU87 cells (2.5 × 106) were implanted s.c. into athymic (nu/nu) mice. After 2, 6, 11, and 16 days of treatment with αAM, αAMR or IgG control, animals were sacrificed and tumor xenografts were harvested. (A) microphotographs of serial sections from αAM, αAMR and IgG-control tumors treated for 6 days, were immunostained with vWF and pSer33/Ser37/Thr41 β-catenin antibodies. Sections showed a positive staining for vascular endothelial cells using vWF and pSer33/Ser37/Thr41 β-catenin antibodies. BV; Blood Vessels. (B) quantitative assessment of cell density of endothelial cells that stained positive for pSer33/Ser37/Thr41 β-catenin was conducted through microscope. MBF_Image J 1.43 U software was used for analysis. Values are mean ± SE (n = 6). Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
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Figure 8: αAM and αAMR induce phosphorylation of β-catenin of vascular endothelial cells in U87 xenograftsU87 cells (2.5 × 106) were implanted s.c. into athymic (nu/nu) mice. After 2, 6, 11, and 16 days of treatment with αAM, αAMR or IgG control, animals were sacrificed and tumor xenografts were harvested. (A) microphotographs of serial sections from αAM, αAMR and IgG-control tumors treated for 6 days, were immunostained with vWF and pSer33/Ser37/Thr41 β-catenin antibodies. Sections showed a positive staining for vascular endothelial cells using vWF and pSer33/Ser37/Thr41 β-catenin antibodies. BV; Blood Vessels. (B) quantitative assessment of cell density of endothelial cells that stained positive for pSer33/Ser37/Thr41 β-catenin was conducted through microscope. MBF_Image J 1.43 U software was used for analysis. Values are mean ± SE (n = 6). Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.

Mentions: To determine whether αAM and αAMR treatment of U87 tumor xenografts can induce phosphorylation of the β-catenin in endothelial cells of nascent vessels, groups of animals bearing U87 xenografts were treated i.p. with αAM or αAMR and sacrificed at different times. Histological examination of U87 tumors removed from animals after αAM and αAMR treatment for 2, 6, 11, and 16 days showed a markedly decreased vessel density when compared with the control IgG-treated group. Immunostaining analysis of serial sections of tumors after 6 days of treatment with vWF antibody demonstrates a clear disruption of blood vessels in tumors after 6 days treatment with αAM and αAMR (Figure 8A). In contrast, stable vascularisation can be observed in IgG-control treated tumors (Figure 8A).


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 β-catenin of vascular endothelial cells in U87 xenograftsU87 cells (2.5 × 106) were implanted s.c. into athymic (nu/nu) mice. After 2, 6, 11, and 16 days of treatment with αAM, αAMR or IgG control, animals were sacrificed and tumor xenografts were harvested. (A) microphotographs of serial sections from αAM, αAMR and IgG-control tumors treated for 6 days, were immunostained with vWF and pSer33/Ser37/Thr41 β-catenin antibodies. Sections showed a positive staining for vascular endothelial cells using vWF and pSer33/Ser37/Thr41 β-catenin antibodies. BV; Blood Vessels. (B) quantitative assessment of cell density of endothelial cells that stained positive for pSer33/Ser37/Thr41 β-catenin was conducted through microscope. MBF_Image J 1.43 U software was used for analysis. Values are mean ± SE (n = 6). Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: αAM and αAMR induce phosphorylation of β-catenin of vascular endothelial cells in U87 xenograftsU87 cells (2.5 × 106) were implanted s.c. into athymic (nu/nu) mice. After 2, 6, 11, and 16 days of treatment with αAM, αAMR or IgG control, animals were sacrificed and tumor xenografts were harvested. (A) microphotographs of serial sections from αAM, αAMR and IgG-control tumors treated for 6 days, were immunostained with vWF and pSer33/Ser37/Thr41 β-catenin antibodies. Sections showed a positive staining for vascular endothelial cells using vWF and pSer33/Ser37/Thr41 β-catenin antibodies. BV; Blood Vessels. (B) quantitative assessment of cell density of endothelial cells that stained positive for pSer33/Ser37/Thr41 β-catenin was conducted through microscope. MBF_Image J 1.43 U software was used for analysis. Values are mean ± SE (n = 6). Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
Mentions: To determine whether αAM and αAMR treatment of U87 tumor xenografts can induce phosphorylation of the β-catenin in endothelial cells of nascent vessels, groups of animals bearing U87 xenografts were treated i.p. with αAM or αAMR and sacrificed at different times. Histological examination of U87 tumors removed from animals after αAM and αAMR treatment for 2, 6, 11, and 16 days showed a markedly decreased vessel density when compared with the control IgG-treated group. Immunostaining analysis of serial sections of tumors after 6 days of treatment with vWF antibody demonstrates a clear disruption of blood vessels in tumors after 6 days treatment with αAM and αAMR (Figure 8A). In contrast, stable vascularisation can be observed in IgG-control treated tumors (Figure 8A).

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