Knockdown Of EpCAM Suppresses Angiogenesis Of Breast Cancer Through JAK/STAT Signaling Pathway

Breast cancer is the most common malignant tumor in women and one of the three most common cancers in the world.The methods commonly used to treat breast cancer today include radiotherapy,chemotherapy,and surgery,as well as targeted therapy and hormone therapy.But these methods often cannot get satisfactory treatment effect,often accompanied by distant metastasis,metastasis after some concurrent diseases and recurrence.Therefore,it is of great clinical significance to further understand the pathogenesis of breast cancer and seek new treatment options for breast cancer.Epithelial cell adhesion molecules(EpCAM)is a type I single-transmembrane glycoprotein.Under normal physiological conditions,EpCAM is expressed to varying degrees in normal epithelial cells except squamous epithelium.Under pathological conditions,EpCAM is highly expressed in all adenocarcinomas,including breast cancer,and its overexpression is closely related to poor prognosis of breast cancer.There is increasing evidence that EpCAM contributes to the proliferation,migration,invasion and metastasis of breast cancer cells.EpCAM overexpression is negatively correlated with survival of breast cancer patients,making it a potential diagnostic and therapeutic target in oncology.Tumor microenvironment(TME)is made up of tumor cells,endothelial cells,fibroblasts and immune cells and the extracellular components,such as cytokines,growth factors and extracellular matrix,and so on,these cytokines accelerate the process of tumor by various means in the tumor microenvironment,and a large number of studies have found that the process of angiogenesis in tumor is closely related to the tumor microenvironment.During Tumor growth,Tumor cells mainly rely on adequate blood vessels to provide necessary nutrients and oxygen for Tumor cell proliferation.Tumor cells secrete Tumor angiogenesis factor(TAF)to initiate angiogenesis and induce Tumor metastasis,among which vascular endothelial growth factor(VEGF)plays a key role in Tumor angiogenesis.VEGF is a major mediator of angiogenesis and a major regulator of angiogenesis and metastasis during human carcinogenesis.The VEGF family includes five secreted proteins(VEGF-A,B,C and D)and placental growth factor(PIGF).The process of angiogenesis is highly dependent on VEGFA signaling.VEGFA is detected in most malignant tumors and plays a very important role in tumor angiogenesis.The previous work of this project has proved that down-regulation of EpCAM can inhibit the growth and metastasis of breast cancer cells and inhibit the adhesion ability of breast cancer cells.This project aims to study the effect of EpCAM on breast cancer cell angiogenesis and its regulatory mechanism,and explore new therapeutic strategies for breast cancer(new applications of breast cancer targets).First,EpCAM overexpressed plasmids and EpCAM interference sequences were transiently transfected into breast cancer cells(MCF-7,MDA-MB-231),and the effects of EpCAM on breast cancer cell proliferation were analyzed by CCK8 cell proliferation assay,immunofluorescence assay and western blot assay.The results showed that EpCAM overexpression increased Ki67 and PCNA protein expression,promoted the proliferation of breast cancer cells,down-regulated EpCAM decreased Ki67 and PCNA protein expression,and inhibited the proliferation of breast cancer cells.Breast cancer cells(MCF-7 and MDA-MB-231)were transfected with EpCAM overexpressed plasmids and EpCAM interference sequences,respectively,for 48 h.Western Blot results showed that the expression of Ki67 and VEGFA in HUVEC cells co-cultured with EpCAM-conditioned medium was significantly higher than that in the down-regulated EpCAM-conditioned medium group.Compared with the down-regulated EpCAM conditioned medium group,the expression levels of p-JAK2 and p-STAT3 in the JAK/STAT signaling pathway were significantly increased in HUVEC cells co-cultured with EpCAM overexpression conditioned medium.Using breast cancer EpCAM overexpression of HUVEC cells conditioned medium treatment after 48 hours into tube experiments,the results show that breast cancer EpCAM overexpression of conditioned medium enhanced HUVEC cell lumen formation ability,at the same time,we used the in vitro rat aortic ring in breast cancer conditioned medium(control group),EpCAM expression condition cut EpCAM medium and medium incubation nine days.The results showed that EpCAM medium could promote aortic budding in rats.However,the conditioned medium after the treatment of breast cancer cells with EpCAM interference sequence could significantly inhibit the proliferation of HUVEC cells,reduce the formation and density of microvascular and inhibit the ability of rat aorta to sprout.In summary,Down-regulation of EpCAM inhibits the expression of VEGFA in breast cancer through the JAK/STAT pathway,thereby inhibiting the angiogenesis of breast cancer.5-fluorouracil(5-FU)is a broad-spectrum anticancer drug.The preliminary work of this study found that down-regulation of EpCAM enhanced the chemical sensitivity of breast cancer cells to 5-FU,and the down-regulation of EpCAM enhanced the cytotoxic effect of 5-FU and promoted cell apoptosis,but the effect of down-regulation of EpCAM and 5-FU on the angiogenesis of breast cancer is still unknown.In this follow-up study,5-FU was applied to HUVEC,and the results showed that Ki67 and VEGFA expression levels were significantly decreased,and p-JAK2 and p-STAT3 expression levels in the JAK/STAT signaling pathway were decreased.When5-FU was combined with breast cancer down-regulated EpCAM conditioned medium on HUVEC,the results suggested that 5-FU further reduced Ki67 and VEGFA expression through the JAK/STAT pathway.5-FU and breast cancer cut EpCAM conditioned medium and specificity of JAK2 inhibitors Ruxolitinib combination in HUVEC 48 hours,the results indicated that the expression levels of p-JAK2 and p-STAT3 in the JAK/STAT signaling pathway were decreased,while the expression levels of Ki67 and VEGFA were decreased.Therefore,the down-regulation of EpCAMin breast cancer reduced the expression levels of Ki67 and VEGFA through the JAK/STAT signaling pathway involved in cell proliferation and angiogenesis,thereby reducing the angiogenic capacity of breast cancer.In summary,our results suggest that EpCAM down-regulation specifically targets VEGFA through the JAK/STAT signal transduction pathway to play a strong anti-angiogenic role in breast cancer,down-regulated EpCAM can not only inhibit the proliferation of breast cancer cells,but also play a greater role in the treatment of breast cancer as an anti-angiogenic therapy combined with 5-fu and other chemotherapy drugs.