The Role Of Inhibitor Of YAP1 In Triple Negative Breast Cancer With Taxol-based Chemoresistance

Objective:1.To investigate the expression of YAP1 protein in clinical triple-negative breast cancer patients with taxol-based chemotherapy drug resistance,and to analyze the survival and prognosis of patients with different expression of YAP1.2.The effect of YAP1 inhibitor(Verteporfin)on the migration,autophagy and apoptosis of paclitaxel-resistant triple-negative breast cancer cells was confirmed by in vitro experiments,as well as the sensitization effect on the paclitaxel drugs.3.Through in vivo experiments,YAP1 inhibitor(Verteporfin)can partially reverse the drug resistance of paclitaxel in triple negative breast cancer transplantation model in nude mice.Methods:1.By The Cancer Genome Atlas(TCGA)database different frequencies(amplification,mutation,fusion and deep deletion)of YAP1 gene in breast cancer tissues were analyzed.Immunohistochemical staining was used to detect the expression level of YAP1 protein in tissues of paclitaxel-resistant patients with triple-negative breast cancer and its clinical correlation(including expression in tissues of triple-negative breast cancer and survival curve analysis).2.CCK-8 assay was used to detect the effect of YAP1 protein on the proliferation of triple-negative breast cancer cell lines in MDA-MB-231 and MDA-MB-231paclitaxel-resistant cell lines.The expression of YAP1 protein in MDA-MB-231 and MDA-MB-231 paclitaxel-resistant cell lines was detected by RT-PCR,Western-blot and cellular immunofluorescence.3.CCK-8 assay was used to detect the effect of YAP1 inhibitor(verteporfin)on cell proliferation in MDA-MB-231 paclitaxel-resistant cell lines.The effect of verteporfin on the migration and invasion of MDA-MB-231 paclitaxel-resistant cells was detected by scratch test and Transwell test.Changes in E-cadherin,vimentin,SOX2 CD44 and CD133 EMT pathways and autophagy related protein levels in MDA-MB-231 paclitaxel-resistant cells treated with verteporfin were detected by Western-blot.4.Annexin V/PI double staining(flow cytometry)was used to determine the effect of verteporfin on the apoptosis of triple-negative breast cancer cells with paclitaxel-resistant.Changes of apoptosis-related protein Bcl2 and BAX levels were detected by Western-blot.5.To establish a BALB/c immunodeficient mice model of triple negative breast cancer transplantation tumor.Verteporfin in vivo effect on drug resistance of triple negative breast cancer was tested by tumor size and weight measurement in nude mice.Immunohistochemistry was used to detect the expression of YAP1,Ki67 and E-cadherin related proteins.6.By transfection of small interfering RNA(si RNA),the expression of gene YAP1 was down-regulated to further detect the changes of cell migration,autophagy and apoptosis in vivo and in vitro.Results:1.Based on the cancer genome atlas data(http://www.cbioportal.org)for patients with “breast cancer”(TCGA,TCGA 2015,TCGA Pan Can and TCGA Pub)on the CBio website,the amplification and deep deletion of YAP1 gene was found in a subset of breast cancer patients.YAP1 overexpression was associated with the early relapse in TNBC patients after taxol-based chemotherapy(P=0.0119,Gehan-Breslow-Wilcoxon test).Furthermore,RT-PCR analysis demonstrated a significant higher level of YAP1 gene expression in TNBC patients than that in non-TNBC patients(P<0.0001).This result was confirmed in immunohistochemistry study that increased nuclear YAP1 staining was exhibited in TNBC tissues compared to normal tissues(P<0.0001).2.In the in vitro experiment,we first established the MDA-MB-231 taxol resistance cell line(231R,9.2 fold),and then this cell line was tested in the following experiment in vitro.Next,we investigated the RNA and protein level of YAP1 in MCF7,MDA-MB-231 and MDA-MB-231/taxol in vitro.The YAP1 was overexpression in MDA-MB-231 with taxol-resistance comparing to other two breast cancer cell lines(P<0.0001).These data suggested that YAP1 was upregulated in triple negative breast cancer with taxol resistance in vitro.3.The survival rate was higher in MDA-MB-231 than that in MDA-MB-231/taxo cells(P<0.0001).High inhibition of migration and survival of MDA-MB-231 cells was seen in Verteporfin+Taxol cells when incubated with Taxol,Verteporfin or combination of Verteporfin and Taxol.In addition,Verterporfin or YAP1 si RNA decreased SOX2,CD44 and CD133 expression of autophagy and enhanced Ecadherin and decreased Vimentin expression in the EMT pathway.4.When MDA-MB-231 paclitaxel-resistant cell lines were treated with YAP1 inhibitor or YAP1 si RNA,we found that the apoptosis rate of MDA-MB-231/taxo resistant cell lines was significantly increased(P<0.0001).In addition,intracellular Bcl2 expression was inhibited and Bax expression was increased in the YAP1 inhibitor treated group.5.Verteporfin significantly reduced the volume of paclitaxel-resistant tumors in mice.In addition,we observed that when the YAP1 protein was knockout,verteporfin reduced the expression of Ki67 protein and increased the expression of EcadherinConclustion:1.YAP1 expression was up-regulated in triple-negative breast cancer patients and was associated with early recurrence after paclitaxel treatment.2.The expression of YAP1 protein was up-regulated in paclitaxel-resistant triple negative breast cancer cells.3.The YAP1 inhibitor verteporfin may inhibit the migration of cells and regulate autophagy or EMT pathways in triple-negative breast cancer.4.In vitro,the YAP1 inhibitor verteporfin significantly enhanced the apoptosis of taxol-resistant TNBC cells.5.YAP1 inhibitor verteporfin reversed drug resistance to paclitaxel therapy for TNBC tumors in vivo.