Angiogenesis is a hallmark of cancer cell malignancy. The role of the RHO family GTPase RHOG in angiogenesis in vascular endothelial cells has recently been elucidated. However, the regulation of RHOG during this process, as well as its cross-talk with other RHO GTPases, have yet to be fully examined. In this study, we found that siRNA-mediated depletion of RHOG strongly inhibits tube formation in vascular endothelial cells (ECV cells), an effect reversed by transfecting dominant active constructs of CDC42 or RAC1 in the RHOG-depleted cells. We also found CDC42 to be upstream from RAC1 in these cells. Inhibiting either Phosphatidyl inositol (3) kinase (PI3K) with Wortmannin or the mitogen-activated protein kinase extracellular-regulated kinase (MAPK ERK) with U0126 leads to the inhibition of tube formation. While knocking down either RHO, GTPase did not affect p-AKT levels, and p-ERK decreased in response to the knocking down of RHOG, CDC42 or RAC1. Recovering active RHO GTPases in U0126-treated cells also did not reverse the inhibition of tube formation, placing ERK downstream from PI3K-RHOG-CDC42-RAC1 in vascular endothelial cells. Finally, RHOA and the Rho activated protein kinases ROCK1 and ROCK2 positively regulated tube formation independently of ERK, while RHOC seemed to inhibit the process. Collectively, our data confirmed the essential role of RHOG in angiogenesis, shedding light on a potential new therapeutic target for cancer malignancy and metastasis.
As the tumor size increases, cancer cells stimulate neighboring vascular endothelial cells to proliferate and form new blood vessels in order to irrigate the tumor. This is through the secretion of vascular endothelial growth factors (VEGF) by the cancer cells that stimulate angiogenesis in vascular endothelial cells. The Rho GTPase family of proteins regulates VEGF secretion by cancer cells and regulates angiogenesis in vascular endothelial cells. Our study aims to elucidate the role of different isoforms of Rho GTPases in Astrocytoma cell expression of VEGF and blood vessels formation in vascular endothelial cells. The role of Rho GTPases in angiogenesis signaling was first examined in vascular endothelial cells. In ECV cells, the knock down of RhoA, RhoC and RhoG reduced the blood tubes formation. Knocking down StarD13, a RhoA and Cdc42 GAP, led to increase in angiogenesis and blood vessel formation. Second, the role of RhoA and RhoC on VEGF levels of expression on Astrocytoma cells was studied; results showed that RhoA regulated VEGF expression more efficiently than RhoC. The role of the PI3K pathway and VEGF secretion in SF268 Astrocytoma cells in response to EGF stimulation or in response to starvation was examined. The study showed that as the cancer cells are deprived from their physiological conditions for a longer period of time as they express more VEGF, MMP-2 and MMP-9. In addition, PI3K pathway is activated after EGF stimulation in tumor cells. In conclusion, this study will explicate the cross-talk between Astrocytoma cells and vascular endothelial cells and the pathways responsible for angiogenesis development
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