The Effect And Mechanism Of ZEB1/LRIG2 Mediated By USP14 On Angiogenesis And Vascular Mimicry Formation In Breast Cancer

Objective:Breast cancer(BC)is the leading cause of cancer death and the second leading cause of cancer death among women worldwide.In recent years,the incidence of breast cancer has been increasing year by year.It is still urgent to explore the pathogenesis,diagnosis and treatment of breast cancer for the early diagnosis and improvement of therapeutic effect.Therefore,it is necessary to find potential markers for early diagnosis of breast cancer and potential targets for treatment to improve the survival rate and quality of life of patients.Ubiquitination is an essential posttranslational modification process in cells.deubiquitinating enzymes(DUBs)are proteases that bind to ubiquitin-labeled substrates or polyubiquitin-chains and reverse ubiquitination processes by hydrolyzing isopeptide bonds between lysine side chains or n-terminal methionines of ubiquitin and linear polyubiquitin-chains.Ubiquitin specific protease 14(USP14)is a type of DUBs,It is malregulated in hepatocellular carcinoma(HCC),epithelial ovarian cancer(EOC),prostate cancer(PC),and lung cancer,among others.At present,there are few studies on the role of USP14 in breast cancer,and the detailed mechanism of its involvement in regulating breast cancer progression remains to be further studied.Angiogenesis is an important process in the growth and spread of solid cancer and is expected to be an ideal target for BC treatment.Inhibition of angiogenesis will significantly inhibit the development and metastasis of malignant tumors.However,inhibition of angiogenesis obstructs oxygen supply to tumors,forming anoxic microenvironment and inducing vasculogenic mimicry(VM)formation.Thus,the prognosis of tumor patients is poor.Exploring the common targets involved in the regulation of VM formation and angiogenesis can provide new ideas for the development of anti-angiogenesis therapeutics.Zinc finger E-box binding homeobox 1(ZEB1)is located on Chr10p11.22,and its expression is associated with poor prognosis of breast cancer and plays a carcinogenic role in breast cancer.Regulation of its expression can regulate the progression of breast cancer.As a transcription factor of vascular endothelial growth factor(VEGF),ZEB1 promotes tumor angiogenesis by up-regulating VEGF expression.In addition,ZEB1 is a key of epithelial-mesenchymal transition(EMT)activator,promoting EMTs by regulating the transcription of various EMT-related factors,thus promoting tumor metastasis.EMT activation also facilitates VM formation.ZEB1 protein levels are regulated by posttranslational modification.This study speculated that USP14 might promote angiogenesis and vascular mimicry formation in breast cancer cells by stabilizing ZEB1.leucine-rich repeats and immunoglobulin like domains 2(LRIG2)are integrated membrane proteins belonging to the LRIG family of proteins,which play a role in promoting or suppressing cancer in a variety of tumors.In view of the role of ZEB1 as a transcription factor,this study used JASPAR to predict whether there is a potential binding site of ZEB1 in the LRIG2 promoter region,and found that there is a potential binding site of ZEB1 in the LRIG2 promoter region.This suggests that ZEB1 may regulate LRIG2 transcription.The first part of this study examined the expression,clinical significance and effect of USP14 on the function of breast cancer cells.The second part of this study examined the effect of USP14 on breast cancer angiogenesis and vascular mimicry formation,and further explored the mechanism of USP14-mediated ZEB1/LRIG2 in regulating breast cancer angiogenesis and vascular mimicry formation.In conclusion,this study provides a theoretical basis for the development of anti-angiogenesis and vascular mimicry combined therapy for breast cancer and the search for potential targets.Methods:In the first part of this study,differentially expressed genes in GSE22820,GSE33447 and GSE42568 were analyzed,and the deubiquitination enzyme USP14 in the shared gene was screened.USP14 expression was detected by qRT-PCR and Western blot from 24 breast cancer tissues and adjacent tissues.The expression of USP14 in different subtypes of breast cancer was analyzed by bioinformatics,and the correlation between USP14 expression and clinicopathological parameters such as grade,stage,phenotype and age of breast cancer patients was analyzed combined with clinical data.Kalan-Meier analyzed the correlation between USP14 and survival in patients with different subtypes of breast cancer.The predictive ability of USP14 expression in breast cancer was analyzed by ROC curve.USP14 expression was detected in normal mammary epithelial cells and breast cancer cell lines,and MCF-7 and T47D cells were selected for functional experiments.The effect of knockdown or overexpression of USP14 on the proliferation of breast cancer cells was detected by CCK-8 assay.The effect of knockdown or overexpression of USP14 on apoptosis of breast cancer cells was detected by flow cytometry.The effects of knockdown or overexpression of USP14 on migration and invasion of breast cancer cells were detected by scratch assay and Transwell assay.The effects of knockdown or overexpression of USP14 on apoptosis-related proteins and EMTrelated proteins in breast cancer cells were detected by Western blot.The breast cancer cells with USP14 deletion were injected subcutively into nude mice to establish the transplanted tumor model.The expression of Ki-67,a proliferative marker,in the transplanted tumor was detected by immunohistochemistry and apoptosis was detected by TUNEL assay to further verify the effect of USP14 deletion on the progression of breast cancer in vivo.In the second part of this study,HUVEC cells were first cultured with supernatant of breast cancer cell culture medium with USP 14 knockdown or overexpression,and the effect on the tube formation ability of HUVEC cells was observed.The effect of USP14 knockdown on angiogenesis of breast cancer cell graft was further detected in vivo,and the expression changes of endothelial vascular markers CD31 and CD34 in breast cancer cell graft tumor after USP 14 knockdown were detected by immunofluorescence.The effect of knockdown or overexpression of USP14 on tube formation of breast cancer cells was detected by duct formation assay.The effect of USP 14 knockdown on the formation of vascular mimicry in breast cancer cell transplantation was detected by PAS staining in vivo.Western blot was used to detect the changes of angiogenic-related proteins Ang2 and VEGFA,and vascular mimicry related proteins VE-cadherin,MMP2 and MMP9 in breast cancer cells after knockdown or overexpression of USP14.Differential expression genes in breast cancer samples with high and low expression of USP14 were analyzed using the TCGA database,and the genes LRIG2 and ZEB1 related to angiogenesis and vascular mimicry were screened.The changes of ZEB1 expression in breast cancer cells after knockdown or overexpression of USP 14 were detected by qRT-PCR and Western blot.The interaction between USP 14 and ZEB1 protein was detected by Co-IP assay.The USP 14knockout breast cancer cells were treated with CHX and MG132,respectively,and ZEB1 expression was detected by Western blot.The JASPAR online analysis software was used to predict whether there were potential binding sites of ZEB1 in the LRIG2 promoter region.The effect of overexpression of ZEB1 on LRIG2 expression in USP 14 knockout breast cancer cells was detected by Western blot.The changes of ZEB1 recruitment in LRIG2 promoter region after USP14 knockdown were detected by ChIP assay.The LRIG2 promoter double luciferase reporter gene plasmid was constructed,and the effect of USP14 knockdown on LRIG2 transcription was detected by double luciferase reporter assay.Breast cancer cells with USP14 knockdown were transfected with Lrig2-overexpressed lentivirus,and the effects of Lrig2-overexpressed LRIG2 on angiogenesis and vascular mimicry formation in breast cancer cells with USP14 knockdown were measured in vivo and in vitro.Results:In the first part of this study,it was found that USP14 was highly expressed in breast cancer tissues,and the expression level was higher in PR positive and ER positive breast cancer patients.The prognosis of breast cancer patients with high USP14 expression is poor,and USP14 has a certain accuracy in predicting breast cancer.USP14 expression is up-regulated in breast cancer cells.Knockdown USP14 inhibits proliferation,migration,invasion and EMT of breast cancer cells in vitro and promotes cell apoptosis.Conversely,overexpression of USP14 promotes proliferation,migration,invasion and EMT of breast cancer cells in vitro and inhibits cell apoptosis.USP14 knockdown also inhibited the growth of transplanted breast cancer cells.The second part of this study found that USP14 knockdown inhibited angiogenesis and vascular mimicry formation in breast cancer in vivo and in vitro.Conversely,overexpression of USP14 promotes angiogenesis and vascular mimicry formation in breast cancer in vivo and in vitro.The knockdown of USP14 also decreased the expression of Ang2 and VEGFA,and vascular mimicry related proteins VE-cadherin,MMP2 and MMP9 in breast cancer cells,whereas the overexpression of USP14 was the opposite.ZEB1 and LRIG2 are genes related to USP14 expression,angiogenesis and vascular mimicry formation.USP14 interacts with ZEB1 protein,knockdown USP14 reduces the half-life of ZEB1 protein in breast cancer cells,and inhibition of proteasome reverses the inhibitory effect of USP14 knockdown on ZEB1 protein expression in breast cancer cells.The LRIG2 promoter region has the potential binding site of ZEB1.Overexpression of ZEB1 increases the expression of LRIG2 in breast cancer cells that knockdown USP14,and knockdown USP14 reduces the recruitment of ZEB1 in LRIG2 promoter region and the luciferase activity of LRIG2 promoter.Overexpression of LRIG2 reversed the inhibitory effect of USP14 knockdown on angiogenesis and vascular mimicry in breast cancer.Conclusion:1.USP14 is highly expressed in breast cancer tissues and cells,and the expression level is higher in PR-positive and ER-positive breast cancer patients,and the prognosis of breast cancer patients with high USP14 expression is poor.2.USP14 promotes proliferation,migration and invasion of breast cancer cells in vitro and tumor growth in vivo,and inhibits cell apoptosis.3.USP14 binds and stabilizes ZEB1,promotes LRIG2 transcription by up-regulating ZEB1,and thus promotes breast cancer angiogenesis and vascular mimicry formation.