Robo1 promotes angiogenesis in hepatocellular carcinoma through the Rho family of guanosine triphosphatases’ signaling pathway
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Robo1 is a member of the Robo immunoglobulin superfamily of proteins, and it plays an important role in angiogenesis and cancer. In this study, we investigate the role of roundabout 1 (Robo1) in tumor angiogenesis in hepatocellular carcinoma (HCC). Firstly, the relationship between Robo1 expression on tumors and patient’s survival and endothelial cells in tumor blood vessels and patient’s survival was studied. Secondly, Robo1 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). Cell proliferation, motility, and tube formation were compared in HUVEC with different Robo1 expression. Also, HUVECs with different Robo1 expression were mixed with HCCLM3 and HepG2 hepatoma cells and then implanted in a nude mouse model to examine the effects of Robo1 in endothelial cells on tumor growth and angiogenesis. Cell motility-related molecules were studied to investigate the potential mechanism how Robo1 promoted tumor angiogenesis in HCC. The disease-free survival of the patients with high Robo1 expression in tumoral endothelial cells was significantly shorter than that of those with low expression (P = 0.021). Overexpression of Robo1 in HUVECs resulted in increased proliferation, motility, and tube formation in vitro. In the implanted mixture of tumor cells and HUVECs with an increased Robo1 expression, tumor growth and microvessel density were enhanced compared with controls. Robo1 promoted cell division cycle 42 (Cdc42) expression in HUVECs, and a distorted actin cytoskeleton in HUVECs was observed when Robo1 expression was suppressed. In conclusion, Robo1 promoted angiogenesis in HCC mediated by Cdc42.
KeywordsHepatocellular carcinoma Angiogenesis Roundabout receptors 1 Rho GTPase
Human umbilical vein endothelial cell
Tumor endothelial cell
Ras homolog family member
This study was jointly supported by the grants from the National Major Science and Technology Project (2012ZX10002012-004, 2013ZX10002007), the National Natural Science Foundation of China (No. 81101805, No. 81472224), and the Shanghai Health Bureau Research Project (No. 2009Y007). We thank Ke Qiao from Key Laboratory of Medical Molecular Virology, Ministry of Education and Public Health, Shanghai Medical School, Fudan University, for technical expertise in confocal microscopy.
Conflicts of interest
JYA, ZTC, YYZ, and LQK performed the experimental work. NZ, BGY, HC, and DMG participated in the experiments. HCS participated in its design and coordination. The manuscript was written by HCS, JYA, and CTZ.
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