Functional Investigation Of Drug-resistant Cancer Cells-derived Exosomal EphA2 In Promoting The Metastasis Of Breast Cancer

Objective:Breast cancer is a malignant tumor with the highest morbidity and mortality in women.Chemotherapy still plays an important role in the treatment of advanced breast cancer.However,heterogeneity in the tumor leads to chemotherapy resistance,resulting in therapeutic failure.Drug-resistant cells display enhanced invasive and metastatic ability than drug-sensitive cells.The mixtures of diverse tumor subpopulations are subject to interactions among their components that modify growth,sensitivity to therapy,and even the ability to metastasize.Emerging evidence indicates that exosomes play important roles in intercellular communication by serving as vehicles for the transfer of various cellular constituents(for example,proteins and nucleic acids)between cells.Some previous studies have demonstrated that drugresistant cells can transfer a drug-resistant phenotype to drug-sensitive cells through exosome.However,there are few studies focus on the other effects of the drug-resistant cells derived exosomes on drug-sensitive cells.Methods:1.The exosomes of drug-resistant breast cancer cells were isolated.Then MDA-MB-468 and T47D cells were treated with the isolated exosomes,and the migration and invasion of treated cells were examined.Western blot assay was used to detect the EMT markers of MDA-MB-468 and T47D cells.2.Proteomic analysis was used to analyze the differential protein expression between exosomes derived from drug-resistant and sensitive breast cancer cells.3.The MDA-MB-468 and T47D cells were treated with exosomes carrying low or high level of EphA2 protein.Migration and invasion assay and in vivo mouse model were used to examine the effects of exosomal EphA2 on the metastasis of breast cancer.Western blot was used to detect the EMT markers of MDA-MB-468 and T47D cells.4.EphA2 variants,either lacking the EphA2-Ephrin A1 binding domain(EphA2-ΔL),or lacking the EphA2 effecting domain(EphA2-ΔS),were overexpressed in 293 T cells.After incubation with exosomes carrying different EphA2 variants,migration and invasion assay were used to detect the metastatic ability of the treated cells.Western blot was used to detect the EMT markers.5.By knocking down the expression of Ephrin-A1 in breast cancer cell lines,Transwell chamber test and Western blot assay were used to detect whether exosomal EphA2 promotes breast cancer cell metastasis through the reverse signal pathway.6.EphA2 inhibitor was used to inhibit the tyrosine kinase activity of EphA2.Transwell chamber test and Western blot test were used to detect whether exosomal EphA2 promotes breast cancer cell metastasis through Forward signaling pathway.7.PD98059 was used to inhibit the activity of Erk.Western blot assay and Transwell chamber test were used to detect whether Erk signal pathway was involved in the process of exosomal-EphA2 mediated metastasis enhancement of breast cancer cells.Results:1.Exosomes derived from drug-resistant cells contribute to the mobility of recipient breast cancer cells.Western blot assay showed that phosphorylation of Erk1/2 was significantly elevated in MDA-468 cells and T47D cells,while phosphorylation of Akt and STAT3 were not changed.Moreover,we found that higher expression of vimentin and lower expression of E-cadherin in drug-resistant cell exosome-treated T47D and MDA-468 cells compared to control cells.2.High throughput proteomic revealed that 295 proteins were enriched and 359 proteins were depleted in the MD-MB-468/EPR derived exosomes comparing with the MDA-MB-468 cells derived exosomes,Notably,EphA2 protein carrying by the MDMB-468/EPR cell derived exosomes was 2.671 times higher than MDA-MB-468 cell derived exosomes.3.Exosomes carrying low amount of EphA2 could no longer promote breast cancer cell migration and invasion.Western blot assay showed that the level of p-Erk1/2,Ecadherin and vimentin were changed invisibly.Thus,the EphA2 carrying exosomes could promote breast cancer cell migration and invasion in vitro and Vivo.4.Transwell assay showed that exosomes carrying EphA2-ΔS could promote the breast cancer cell migration and invasion as well as exosomes carrying EphA2.However,exosomes carrying EphA2-ΔL could not promote migration and invasion.Western blot assay also showed that higher expression of vimentin and lower expression of Ecadherin in T47D and MDA-468 cells which treated with exosome-carrying EphA2-ΔL comparing with control cells,and phosphorylation of Erk1/2 was significantly elevated.5.Transwell assay showed that the migration and invasion abilities of Ephrin-A1-KD cells were not increased comparing with control after incubated with exosomes derived from drug-resistant cells.Western blot assay also showed that the expression of Ecadherin,Vimentin and phosphorylation of Erk1/2 were changed invisibly.6.After inhibiting the phosphatase activity of EphA2,drug-resistant cell-derived exosomes can still promote the migration and invasion of breast cancer cells.7.Western blotting assay showed that PD98059 eliminated the phosphorylated Erk forms,as well as the increased expression of vimentin and decreased expression of Ecadherin in T47D and MDA-468 cells after treating with MDA-468/EPR-derived exosomes and MCF-7/ADR-derived-exosomes.Transwell assay showed that the migration and invasion abilities of cells pre-treated with PD98059 was not increased comparing with control.Conclusion:Our studies have shown that exosomes derived by drug-resistant cells can promote the migration and invasion of sensitive cells.Compared with sensitive cell-derived exosomes,EphA2 is highly expressed in drug-resistant cell-derived exosomes.The treatment of breast cancer sensitive cells with exosomes with low and high expression of EphA2 confirmed that EphA2 plays an important role in promoting the migration and invasion of breast cancer sensitive cells.In terms of mechanism,it has been found that EphA2 in drug-resistant breast cancer exosomes activates Erk signaling pathway in breast cancer sensitive cells through Eph-ephrin reverse signal transduction,resulting in decreased expression of E-cadherin and increased expression of Vimentin,and ultimately promotes the migration and invasion of breast cancer sensitive cells.In vivo experiments also confirmed that EphA2 plays an important role in the process of breast cancer cell metastasis promoted by drug-resistant exosomes of breast cancer cells.in exocrine promotes lung metastasis of tumor cells.