The Mechanism Of C-MET And EGFR Family Synergistically Active STAT3 To Promote The Proliferation Of Drug-Resistant Breast Cancer

Objectives:Breast cancer is the most common malignant tumor among women throughout the world,and chemotherapy is still the main method for treatment.However,the primary or secondary drug-resistant is an important reason that seriously affects the treatment efficacy.The process of acquiring toleranc is often accompanied by the reprogramming of intracellular gene expression.In addition to the expression of up-regulation of drug-resistant genes,cancer cells often evolve new driver genes and activate multiple signaling pathways to maintain cell survival and proliferation.Therefore,it is important to identify the compensatory new genes and their key molecular mechanisms of drug resistance and provide new targets for clinical treatment.In this study,si RNA knockdown technique and a series of biological experiments were used to analyze the effects of EGFR family members and C-MET on the proliferation ability of drug-resistant cells and the specific molecular mechanisms.In addition,the combined application of the pan-EGFR inhibitor and the C-MET inhibitor was verified by Combination Index and xenograft tumor model.Methods:1.SK-BR-3 and MDA-MB-468 cell lines resistant to epirubicin were constructed.The IC₅₀ was detected by CCK8 assay,and the changes of BCRP and P-gp were detected by q RT-PCR and Western Blot.2.RNA-seq and high-throughput proteomics analysis were performed on drug-resistant cells and parent cells to look for differential genes and activated signaling pathways,and CCK8 assay was used to detect the sensitivity of drug-resistant cells to inhibitors.The relationship between differential genes and prognosis of breast cancer patients was analyzed by public websites.3.si RNA was used to knock down the expression of EGFR,HER3 or C-MET in drug-resistant cells,and the effects on cell proliferation were detected by CCK8 assay and colony formation assay.Colony formation assay was used to detected the effect of inhibitor on drug-resistant cells colony formation.4.Co-Immunoprecipitation assay and Western Blot assay were used to detect the interaction between EGFR,HER3 and C-MET.After HER3 or C-MET was inhibited by si RNA or inhibitors,CCK8 assay was used to detect the effects of HGF or NRG1on SK/EPI cells proliferation.5.GSEA analysis was performed on high-throughput omics data,CCK8 assay and colony formation assay were used to detect the effect of STAT3 signaling pathway on cell proliferation.Knock down the expression of EGFR,HER3 or C-MET,the changes of downstream signaling pathways were detected by Western Blot to explore the mechanism.6.STAT3 was knocked down by si RNA or inhibited by Stattic in drug-resistant cells,and the changes of C-MET were detected by q RT-PCR and Western Blot.Ch IP assay was used to detect whether STAT3 directly bound to the MET promoter.The luciferase reporter plasmid containing the sequence of the MET promoter was constructed.The dual luciferase reporter assay was used to detect the effect of STAT3on the activity of the MET promoter.7.The synergistic effect of AZD-8931 and Capmatinib was detected by CCK8 assay and drug combination index analysis,and the effect of the combined application of the two drugs on cell proliferation was detected by EDU test.8.In vivo,the synergistic effect of AZD-8931 combined with Capmatinib on proliferation was verified by xenograft tumor model.Results:1.After long time gradient induction,breast cancer drug-resistant cells SK/EPI and468/EPI were successfully constructed,with drug-resistant index greater than 5and expressing P-gp and BCRP.2.High-throughput sequencing results showed that in SK/EPI drug-resistant cells,the EGFR signal was down regulated and C-MET signal was increased.The drug-resistant cells were less sensitive to AZD-8931 and more sensitivity to Capmatinib.Breast cancer patients with high expression of EGFR,HER3,or C-MET have a worse prognosis.3.After the expression of C-MET,EGFR or HER3 was knocked down in drug-resistant cells,the proliferation ability and clone formation ability of cells were decreased.Inhibition of C-MET or EGFR family with inhibitors reduced the clonal formation of drug-resistant cells.4.There was no direct interaction between C-MET and EGFR or HER3 in the two drug-resistant cells.HGF still promoted the proliferation of SK/EPI cells after silencing HER3 or inhibiting by AZD-8931.NRG1 still promoted the proliferation of SK/EPI cells after the down-regulated expression of C-MET.5.GSEA analysis indicated that STAT3 signaling pathway was activated in drug-resistant cells.Downregulated expression of EGFR or HER3 or C-MET in drug-resistant cells resulted in a significant decrease in STAT3 phosphorylation.Knockdown STAT3 expression in drug-resistant cells decreased proliferation and plate clone formation.6.When STAT3 expression was knocked down by si RNA,the expression of C-MET was decreased at both m RNA and protein levels in drug-resistant cells.Ch IP results demonstrated that a physical interaction was exist between STAT3 and MET promoter.Dual luciferase reporter assay confirmed that the MET promoter in drug-resistant cells was more active than the parental cells.The activity of MET promoter of468/EPI cells was decreased after knocking down STAT3 or inhibiting by Stattic.Overexpression of STAT3 resulted in increased C-MET expression at both m RNA and protein levels,and the luciferase activity was also significantly elevated.7.Combined index analysis confirmed the synergistic effects between AZD-8931 and Capmatinib.The Ed U results showed that the combination of AZD-8931 and Capmatinib inhibited cell proliferation more than the single drug.8.In vivo,AZD-8931 and Capmatinib significantly inhibited tumor growth comparing with the control group and the monotherapy group.Conclusion:This study demonstrated that epirubicin-resistant breast cancer cells showed significant differences in the expression of EGFR family members and C-MET,as well as abnormal activation of C-MET signaling pathways comparied with parental cells.However,the sensitivity of drug-resistant cells to AZD-8931 decreased.Further studies demonstrated that EGFR family members and C-MET maintain cell survival by regulating the proliferation of drug-resistant cells.However,no direct interaction was found between EGFR,HER3 and C-MET.These RTKs played an independent role in promoting the proliferation of drug-resistant cells.In terms of mechanism,first,EGFR family members and C-MET promoted the proliferation of drug-resistant cells through the STAT3 signaling pathway.Second,STAT3 could bind to the MET promoter to regulate C-MET expression at the transcriptional level,forming a C-MET/STAT3 loop to promote the proliferation of drug-resistant cells.In addition,in vivo and in vitro studies demonstrated that the combination of Capmatinib and AZD-8931synergically inhibits the proliferation of drug-resistant breast cancer cells.These results provide reliable evidence and scientific support for Capmatinib and AZD-8931in the treatment of advanced breast cancer.