The Mechanism Between Lysyl Oxidase And Vascular Endothelial Growth Factor Driving Epithelial Mesenchymal Transition In Human Breast Cancer In Hypoxia

Research background: Breast cancer is the most frequently diagnosed cancer among women worldwild with high motality,which is a major cause of death during the period of progression.Evidence indicates that the hypoxic microenvironment plays an significant role in progression and metastasis of breast cancer.About 25–40% of invasive breast cancers exhibit in hypoxic regions.Hypoxia microenvironment widely exists in solid tumor tissues,in which hypoxia inducible factor 1(HIF-1)is activated and regulate the expression of a series of downstream genes,including lysyl oxidase(LOX)and vascular endothelial growth factor(VEGF).Studies have shown that intra-tumoral hypoxia has negative implications for survival of breast cancer patients,independent of prognostic parameters.Hypoxic tumors are associated with a more aggressive phenotype,increasing risk of metastasis,resistance to radiotherapy and chemotherapy,and induced cancer immune suppression.Epithelial mesenchymal transition(EMT)defines characteristics of migration and invasion of breast cancer cells during the progression of cancer.EMT is a biologic process by which epithelial cells attain a mesenchymal phenotype,allowing a polarized epithelial cell,which normally interacts with basement membrane via its basal surface,promoting them to break free from the primary tumor site.EMT undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype,which includes enhanced migratory capacity,invasiveness,elevated resistance to apoptosis,and greatly increased production of ECM components.Moreover,EMT also plays a significant roles in hypoxia.It plays an important role in embryonic development,chronic inflammation,tissue remodeling,cancer metastasis and a variety of fibrotic diseases.EMT is characterized by cellular and molecular changes that include loss of cell-to-cell adhesion,which involves E-cadherin in adherent junctions and claudins in tight junctions,of the mesenchymal protein vimentin,N-cadherin,fibronectin,Matrix metalloproteinase,reorganization of the cytoskeleton to acquire a more spindle-like morphology,and increased motility that involves dynamic actin microfilament networks.Multiple signaling pathways involved in the process of EMT regulation.,which include TGF-β/Smad signaling,Wnt/β-catenin signaling,Ras-MAPK signaling,PI3K/AKT signaling,Src/Rho kinase and so on.Hypoxia can influence the related genes expression of EMT,which further inducing stem cell phenotype.EMT is characterized by cellular and molecular changes that include loss of cell-to-cell adhesion,which involves E-cadherin in adherent junctions and claudins in tight junctions,upregulation of the mesenchymal protein vimentin,reorganization of the cytoskeleton to acquire a more spindle-like morphology,and increased motility that involves dynamic actin microfilament networks.During the process of metastasis,epithelial cells leaving from the primary tumor site,adhering and invasiving stromal site,entering the blood circulation and spreading to distant tissues and organs,where proliferated and formed secondary tumors.EMT has been regarded as the possible first step in the complex process of metastasis.Metastasis is one of the leading cause of death from cancer,with the result of metastasis has once occurred,the lesions will spread to the whole body.More and more research has showed that hypoxic microenvironment can promote the metastasis of tumor cells.50–60% of solid tumors in hypoxia and/or anoxic zone and hypoxic environment can induce intracellular gene expression reprogramming,which eventually lead to cell invasion behavior.HIF-1 provokes EMT through up-regulating EMT-associated transcription factors or repressors,activating the EMT associated signaling pathways,modulating EMT associated inflammatory cytokines,as well as regulating other pathways like epigenetic regulators [72].Evidence indicated that HIF-1induces EMT through the transcriptional control of E-cadherin,SNAIL,ZEB1,TWIST and TCF3(transcription factor 3).We focus on human breast cancer,targeting the mechanism between LOX and VEGF driving EMT in hypoxia,with the purpose of finding the new therapy target to breast cancer.Methods: Cells were divided into 2 groups: Control group(-Co Cl2)(0μmol/L),Cobalt chloride treatment group(+Co Cl2)(50μmol/L,100μmol/L,200μmol/L,400μmol/L)。(1)Wound healing assay measured the move ability of MCF-7 and MDA-MB-231 cells.Cells were scraped and allowed to migrate to sub-confluence in serum-free medium for 48 hours.The cell migration images were photographed at 0,24 and 48 hours following scraping.We examined the effect of Co Cl2 induced invasion by Boyden chamber assay.The system was incubated for 24 hours or 48 hours and then photographed.(2)We detected the intracellular protein content of MCF-7 and MDA-MB-231 cells by Western blot.Detection index include: hypoxia incude factor –HIF,matrix metalloproteinases MMP-9,epithelial maker—E-cadherin,mesenchymal maker—-Vimentin,key molecule of PI3K-AKT signaling—PI3K,AKT.(3)ELISA technology detected extracellular secreted protein content of vascular endothelial growth factor-VEGF and lysyl oxidase-LOX.(4)Immunoprecipitation analysis detected the interreaction between endothelial growth factor-VEGF and lysyl oxidase-LOXResults:(1)According to the results of scratch and transwell assay,hypoxia microenvironment endowed MCF-7 and MDA-MB-231 cells higher migration and invasion ability.(2)The detection of EMT marker revealed that:In hypoxia,compared with the control group,matrix metalloproteinases MMP-9 and mesenchymal maker—Vimentin are upregulation,epithelial maker—E-cadherin is down-regulation.The key target of PI3k-AKT pathway PI3 k and AKT are also upregulation,which indicate hypoxia inducing EMT via acting of PI3k-AKT pathway.(3)According to the results of westernblot and ELISA:In hypoxia,VEGF and LOX are overexpression simultaneous.Basing on the evidences about the important role between hypoxia microenvironment and EMT facilitating the metastasis of tumor,LOX act as a crucial point of HIF downstream,moreover,VEGF plays important role in the receptor protein tyrosine kinase,which can incuce partial or full EMT.there have some relationship between LOX and VEGF driving EMT in hypoxia to facilitate the metastasis of breast cancer.(4)The results of Immunoprecipitation analysis further confirmed there has some synergic effect between LOX and VEGF inducing EMT of breast cancer in hypoxia.Conclusion: hypoxia microenviroment induce the invasion and migration of breast cancer cell lines of MCF-7 and MDA-MB-231,which allow them to require more ability in terms of metastasis through PI3K-AKT pathway.In hypoxia,LOX and VEGF are over-expression in breast cancer at the same time.Moreover,hypoxia inducing EMT facilitate the invasion and metastasis of breast cancer via the synergistic effect of LOX and VEGF.