Chlorogenic Acid Inhibits Breast Cancer Metastasis And EMT Via Targeting LRP6

Objective This study aims to investigate the molecular mechanism of chlorogenic acid targeting LRP6 against breast cancer cells metastasis and EMT in vitro and in vivo.Methods Firstly,we explored the correlation between LRP6 expression level and clinicopathological factors of breast cancer,the relationship between overall survival and disease free survival of the patients,and the correlation between LRP6 expression level and EMT markers analyzed by bioinformatics databases.Then,LRP6 as a target for anti-metastatic breast cancer therapy,docking with natural products used Auto Dock Vina 1.1.2 software through computer virtual screening technology.Based on the score ranking and literature reports on anti-tumor activity,we screened out top 10 anti-metastatic candidate compounds.The target compounds were screened in vitro.The candidate compounds were preliminarily screened and observed by CCK-8 assay and cell morphology assay.To further determine the inhibitory effects of the target compound on breast cancer cell metastasis and EMT,wound-healing assay and Transwell assay were used to evaluate the ability of migration and invasion in breast cancer cells.RT-q PCR,WB and IF were used to detect the expression levels of target compound against EMT marker proteins E-cadherin,ZO-1,ZEB1,N-cadherin,Vimentin,Snail and Slug.The activity of MMP2 and MMP9 in breast cancer cells was detected by gelatin zymography.Knockdown or overexpression of LRP6 in breast cancer cells was used to verify the role of target compound against metastasis and EMT through LRP6.To elucidate the target compound directly targets LRP6 to exert anti-metastasis role in breast cancer,binding affinity of LRP6 with the target compound and Wnt ligand was examined by MST assay.Gene expression correlation analysis,PPI network,gene functional annotation,and signaling pathway enrichment of LRP6 in patients with breast cancer were analysized by bioinformatics databases.Effects of target compound on Wnt/β-catenin signaling and other signalings were detected by Western bloting.To further clarify the inhibitory effect of the target compound on breast cancer cell metastasis and EMT,the interaction between LRP6 with target compound and Wnt ligand was detected by Co-IP assay.In addition,we constructed xenograft tumor nude mice model and evaluated the inhibitory effect of the target compound on breast cancer cell metastasis in vivo.The expression levels of LRP6,E-cadherin,and Vimentin in tumor tissue were tested by IHC.Results Bioinformatics analysis indicated that the high level expression of LRP6 is significantly correlated with the expression status of ER~-,PR~-and HER2~-,status of lymph node metastasis,TNBC and Luminal A subtype breast cancer in patient pathological tissues.Patient OS closely associated with status of ER,HER2 and lymph node metastasis.LRP6 positively correlated with metastasis and expression of EMT marker proteins MMP2,Slug and ZEB1 in breast cancer patients.It suggested that LPR6 is one of the important factors for poor prognosis of patients,and may be contribute to breast cancer metastasis and EMT.Ten candidate compounds were preliminary screened out based on molecular docking scoring,among which CA was confirmed to exert a certain inhibitory effect on breast cancer cells metastasis.It was shown that CA significantly changed cellular morphology,inhibited the invasion and migration of MCF-7 cells,suppressed the activities of MMP2 and MMP9,increased the expression levels of EMT epithelial phenotypic marker proteins E-cadherin and ZO-1,and decreased the levels of mesenchymal phenotypic marker proteins N-cadherin,Vimentin,ZEB1,Snail and Slug.Knockdown the expression of LRP6 significantly inhibited the invasion,migration and EMT of MCF-7 cells.Overexpression of LRP6 dramatically promoted the invasion,migration and EMT of MCF-7 cells,while CA could attenuate the effect induced by overexpression of LRP6.MTS results showed that the binding affinity constant(Kd)between CA and LRP6 was 2.47±0.45μM,which indicated CA can directly bind to LRP6 in MCF-7 cells.We also found that LRP6 can directly bind to Wnt3a in the Wnt/β-catenin signaling pathway,but the affinity is weaker than that of CA.CA can down-regulate the expression levels of LRP6,p-LRP6 andβ-catenin in Wnt/β-catenin signaling pathway.LRP6 and Wnt3a can form LRP6/Wnt3a complex which could be dissociated due to CA competitively binding LRP6 with Wnt3a.In addition,we also detected the expression levels of TGF-βand Notch1 based on bioinformatics signal pathway enrichment analysis,and the results showed that the expression levels of TGF-βand Notch1 in MCF-7 cells were not affected by CA,indicating that CA inhibited breast cancer metastasis and EMT through Wnt/β-catenin signaling pathway rather than TGF-βsignaling pathway and Notch1 signaling pathway.To determine whether CA inhibits tumor progression and EMT in vivo,we established a nude mouse MCF-7 xenografts model.After treatment with CA,the tumor volume and weight of CA treated mice were significantly lower than untreated mice.CA significantly down-regulated the expression levels of LRP6 and Vimentin,up-regulated the levels of E-cadherin in tumor tissue.Conclusion This study proposed that CA inhibited the metastasis and EMT of breast cancer in vitro and in vivo,and the underlying mechanism may be related to inactiving Wnt/β-catenin signaling pathway by targeting LRP6.LRP6 is one of direct targets of CA in breast cancer cells,and CA may be developed as a potential EMT inhibitor to treat the metastatic breast cancer.