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.
Department of Oncology, Xijing Hospital, State Key Discipline of Cell Biology, Fourth Military Medical University, Xi'an, China.
Ji
J
Ma
Yan
Y
Xue
Wei
W
Cui
Yan
Y
Li
Qingli
Q
Zhao
Wenmin
W
Ye
Jin
J
Zheng
Yuanxiong
Y
Cheng
Yuguang
Y
Ma
Sen
S
Li
Tenglong
T
Han
Lu
L
Miao
Libo
L
Yao
Jian
J
Zhang
Wenchao
W
Liu
D000818:Animals; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D015687:Cell Hypoxia; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D018920:Coculture Techniques; D019610:Cytoprotection; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008875:Middle Aged; D009389:Neovascularization, Pathologic; D010879:Piperazines / Q000494:pharmacology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D025801:Ubiquitin / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D007093:Imidazoles; D010879:Piperazines; D025801:Ubiquitin; D042461:Vascular Endothelial Growth Factor A; C482205:nutlin 3; D051736:Proto-Oncogene Proteins c-mdm2; D020742:rhoA GTP-Binding Protein