Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor‐dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions

BACKGROUND: Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia‐induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. METHODS: The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF‐7 cells using Western blot analysis, real‐time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF‐7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild‐type p53 (wt‐p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. RESULTS: Activated RhoA down‐regulated p53 protein, which increased VEGF expression in hypoxic MCF‐7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin‐mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF‐7 cells and interacted with each other physically. Furthermore, nutlin‐3, a specific MDM2 inhibitor, was capable of reducing activated RhoA‐induced p53 protein stability and attenuating VEGF accumulation. In an MCF‐7‐HUVEC coculture system, nutlin‐3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt‐p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt‐p53 expression, and high VEGF expression. CONCLUSIONS: The current data suggested that activated RhoA promotes VEGF expression and hypoxia‐induced angiogenesis through the up‐regulation of MDM2 to decrease p53 stability. Cancer 2012. © 2012 American Cancer Society. The current data suggest that activated ras homolog gene family, member A (RhoA) promotes vascular endothelial growth factor (VEGF) expression and hypoxia‐induced angiogenesis through the up‐regulation of the p53 binding protein homolog murine double minute 2 (MDM2) to d