Smad4 In Blood Vessel Endothelial Cells Mediates Hematogenous Metastasis Of Ovarian Cancer

Objective: Smad4 is a critical co-smad in the TGF-?/Smad signaling pathway.The roles played by Smad4 inactivation in tumors highlighted it as a tumor-suppressor gene.Herein,we analyzed the role of vascular endothelial cell Smad4 expression in ovarian cancer metastasis.Methods: Kaplan-Meier plotter was used to evaluate the prognostic value of Smad4 in patients with ovarian cancer.A total of 14 normal ovarian tissues and 19 ovarian cancer tissues were collected.The expression of Smad4 in vascular endothelial cells was detected by immunohistochemistry colocalized with the blood vessel marker CD34.The Smad4 expression levels of ovarian cancer cell lines and human umbilical vein endothelial cells(HUVECs)were detected by western blot.Immunofluorescence was used to detect the cellular localization of Smad4 in vascular endothelial cells.The role of Smad4 in regulation of HUVECs migration and tube formation was investigated by transfection of si RNA in vitro,and the permeability was examined by co-culture with pericytes.The effects of endothelial cell Smad4 downregulation on angiogenesis and metastasis were detected in ovarian cancer xenograft mice.The microarray assay was performed to analyze the gene expression after downregulation of Smad4 in HUVECs.Results: High level of Smad4 m RNA was correlated with longer progression-free survival(PFS)in patients with ovarian cancer diagnosed at early(stage ? and ?)or late(stage ? and ?)stage.Compared with vascular endothelial cell in normal ovarian tissues,the expression of Smad4 protein was significantly decreased in ovarian cancer.Smad4 is expressed in ovarian tumor cell lines and HUVECs.Immunofluorescence analysis of Smad4 protein are distributed in the nuclear and cytoplasm in HUVECs.Endothelial cells with Smad4 deletion expression have reduced ability in migration and tube formation.Downregulation of Smad4 expression in vascular endothelial cells increased permeability when co-cultured with pericytes.In ovarian cancer xenografts,downregulation of Smad4 in vascular endothelial cells resulted in reduced angiogenesis and defective endothelial cell-mural cell contact.Tumor xenografts with Smad4-defcient HUVECs did not display a significant reduction in volume and tumor burden,but they were associated with a remarkable increase of spontaneous metastatic dissemination to the live.The microarray assay showed the decrease of Smad4 in HUVECs induced change in some genes such as FYN,PTPRM,and INSR.Conclusions: In this study,we identified that the m RNA levels of Smad4 in tumor tissue were associated with prognosis in ovarian cancer.Moreover,Smad4 expression is reduced in the vessel endothelial cells of ovarian cancer.The decrease expression of Smad4 in vascular endothelial cells can promote invasion and metastasis in ovarian cancer by damaging the vascular barrier composed of pericytes.These results indicate that Smad4 in vascular endothelial cells play an important role in ovarian cancer metastasis and can be used as potential targets for the treatment of ovarian cancer metastasis.