VEGF Stimulation Mediates The Activation Of Cdc42 To Promote Polarized Migration Of Colorectal Cancer Cells

BACKGROUND&OBJECTIVEColorectal cancer(CRC)is the fourth most common malignancies in China.Up to 80%of patients die from tumor metastasis,because it can lead to destruction of extensive struction and function of multiple organs.Tumor cells and the surrounding microenvironment jointly participated in invasion and metastasis of CRC.Therefore,it is particularly important to explore the interaction between tumor cell and microenvironment and direction migration of tumor cells for anti-tumor metastases therapy.Vascular endothelial growth factor(VEGF),an active factor with various biologic features,is secreted by the tumor cells and stromal cells.It has been indicated that VEGF are participated in endothelial cell migration,neoangiogenesis.Cell division cycle 42(Cdc42),a guanine trinucleotide(GTP)enzyme,expresse in cell membranes and cell plasma.It consists of two forms,active and inactive variants.When cells are stimulated by the external or internal signals,it can lead to the interconversion of active and inactive variants and alteration of downstream signaling pathways.It has been proven that the combination of VEGF and Neuropilin1(NRP1)activates Cdc42,and subsequently promotes the budding of endothelial cells in the blood vessels and angiogenesis.We have found that expression of prohibitin(PHB)in cancerous glands of CRC was eccentric and concentric via immunohistochemistry(IHC).The concentric distribution of PHB expressed in the lumenal margin of cancerous gland,maintaining the top-bottom polarity of the tumor cells,while eccentric distribution of PHB is referred to facing the interstitial side,showing the front-rear polarity of the cancer cells VEGF could induce PHB to the leading edge of CRC cells,showing front-rear polarity.In addition,interaction of PHB and Cdc42 was found in CRC cells.VEGF can induce redistribution of Cdc42 in CRC cells,and extracellular VEGF can promote direction.In general,this paper mainly contains the following content.The molecular mechanism of redistribution of Cdc42 in CRC cells and how does VEGF induce directional migration of CRC cells are still unknown.METHODS1.CRC cells(LS174T?SW480)were co-cultured with interstitial cells(HUVEC?PBMC).Then,we analysis the expression level of VEGF in supernatant via enzyme-linked immunosorbent assay.2.The activated Cdc42(GTP-Cdc42)was prepared by Cdc42 activation kit.Western blot was used to analysis the expression level of GTP-Cdc42.3.Using membrane fractionation experiment to detect the changes of Cdc42 protein in the cell membrane.4.Immunofluorescence assay was used to observe the fluorescence intensity of colorectal cancer cells under different treating conditions.5.Using conventional scratch tests and immunofluorescence assay to determine redistribution of Cdc42 in colorectal cancer cells.6.CO-Immunoprecipitation(CO-IP)verifies the interaction between NRP-1(VEGF receptor)and Cdc42 protein.7.Conventional scratch tests and transwell experiments was used to investigate invasive ability of CRC cells.RESULTS1.Under the condition of VEGF stimulation,we found that expression of activated Cdc42 increased in CRC cells.Similary,the expression of Cdc42 increased in the membrane of CRC cells.Compared to control group without VEGF,the proportion of membranous expression of Cdc42 was significantly increased in the injury direction.This results demonstrated the redistribution of Cdc42 in CRC cells.2.In order to analysis how does VEGF induce redistribution of Cdc42,we investigated the expression level of VEGF-related receptor(VEGFR1/VEGFR2/NRP-1/NRP-2).Consequently,hight expression of NRP-1 was found in CRC cells.VEGF receptor NRP-1 knockdown resulted in decreased expression of activated Cdc42 in CRC cells as well as membranous expression of Cdc42 under the condition of VEGF stimulation.Similary,the proportion of membranous expression of Cdc42 was significantly decreased in the injury direction.3.The results of immunofluorescence assay showed that when the expression of activated Cdc42 was increased,the expression of F-actin was obviously increased,with the feature of elongating and increased filamentous pseudopodia.When the expression of activated Cdc42 increased,the expression of phophorylated Cofilin also increased.Phosphorylated Cofilin can inhibin depolymerization of F-actin and strengthen stability of F-actin.4.We further investigated the biological characteristics of CRC cells.As a result,when the expression of cytoskeletal proteins increases,the migration distance was increased and the invasive ability of CRC cells enhanced.CONCLUSION1.VEGF mediates the activation and redistribution of Cdc42 in CRC cells.2.NRP-1,a membrane-anchored receptor of VEGF,mediates the activation and redistribution of Cdc42.3.Activated Cdc42 promotes the remodeling of cytoskeleton of CRC cells4.Remodeling of cytoskeleton of CRC cells by VEGF stimutation promotes the directional migration of colorectal cancer cells.