Evaluating the Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors

Adrenomedullin (AM) is widely expressed in a variety of tumor types and was shown to be mitogenic for many human cancer cell lines in vitro whereas several in vivo studies have also highlighted its strong contribution to neoplastic angiogenesis. VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. It was previously shown that AM blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. To reveal these mechanisms, techniques such as endothelial cell culture, Western blot analysis, immunoprecipitation, RNA interference, SHP-2 inhibitors, permeability assay, Matrigel plug assay in vitro and in vivo, and in vivo tumor xenograft growth were used in this study. For statistical analysis, non-parametric analysis Kruskal-Wallis followed by Bonferroni test was performed using XLSTAT Software throughout the whole manuscript. In this article, it is demonstrated that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM- receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts.

Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature. This data, together with the reported observation that SHP-2 negative mutant ECs failed to organize themselves into a highly vascularized network in the yolk sac of mouse embryos (23), support the role of SHP-2 as an important player in the signaling transduced by AM to regulate pro-angiogenic and vascular stabilizing activities in endothelial cells.