Tight Junction Protein CLDN6 Enhances Human Breast Cancer Cells Chemoresistance Through GSTP1

Chemoresistance is a major obstacle to the treatment of breast cancer.Recently,it is generally believed that the abnormal expression of P-gp,GSTP1,Topo II,and Ki67 is an important prognostic indicator of chemoresistance in breast cancer,whereas the mechanism is still unclear.Therefore,the study on mechanism of chemoresistance is especially important for searching potential targets in the treatment of breast cancer.Our group had applied RNA-seq technology to screen differentially expressed genes between breast cancer multidrug resistance cell line MCF-7/MDR and MCF-7 cells.Occasionly,we found that CLDN6,as a member of CLDNs family expressed relatively higher in MCF-7/MDR cells.Hence,we speculated that CLDN6 may be involved in multidrug resistance of breast cancer.Recent years,researchers had reported that CLDNs family proteins mediated chemoresistance of tumor cells,in addition to participated in the regulation of biological characteristics.We also found that GSTP1 was relatively higher in CLDN6-overexpressed MCF-7 cells.Whereas whether CLDN6 is involved in the regulation of breast cancer resistance to chemotherapy drugs has not yet been reported.In this experiment,we aim to explore the role of CLDN6 in breast cancer chemoresistance,and to make it clear that whether CLDN6 affecting chemoresistance through GSTP1 in breast cancer,and to provide guidance in the selection of chemotherapy drugs for breast cancer and the development of new chemotherapeutic drugs.Methods 1.Effect and mechanism of CLDN6 on chemoresistance in breast cancer(1)Effect of CLDN6 on chemoresistance in breast cancer 1)In breast cancer cell line MCF-7,CLDN6 expression vector was constructed and lentiviral transduction was applied to acquire CLDN6 stablely expressed MCF-7 cells.Western blot was applied to detect the expression of P-gp,which is a marker of multidrug resistance,when overexpressed CLDN6 in MCF-7 cells.CCK8 assay was used to evaculate the role of CLDN6 on different chemotherapeutic drugs sensitivity.Effect of CLDN6 on the apoptosis when treated with ADM: DAPI staining and western blot assays.Western blot was used to detect the effect of CLDN6 on the expression of γH2AX,which is a marker of DNA damage when treated with ADM.2)In breast cancer multidrug resistance cell line MCF-7/MDR,tranfected with CLDN6 sh RNA and G418 was applied to screen CLDN6 stablely silenced MCF-7/MDR clones.Western blot was applied to detect the expression of P-gp when scilenced CLDN6 in MCF-7 cells.CCK8 assay was used to evaluate the effect of CLDN6 silencing on different chemotherapeutic drugs sensitivity.Effect of CLDN6 silencing on the apoptosis when treated with ADM: DAPI staining and western blot assays.Western blot was used to detect the effect of CLDN6 silencing on the expression of γH2AX when treated with ADM.(2)Mechanism of CLDN6 on chemoresistance in breast cancer 1)The role of GSTP1 in CLDN6 regulating chemoresistance in breast cancer(1)In CLDN6 overexpressed-MCF-7 cells,western blot was used to evaluate GSTP1 expression.GST activity assy was conducted to measure GST enzyme activity.GSTP1 sh RNA vector was constructed and lentiviral transduction was applied to acquire GSTP1 stablely silenced cells.CCK8 assay was conducted to evaluate the effect of GSTP1 silencing on different chemotherapeutic drugs sensitivity in CLDN6 overexpressed MCF-7 cells.Role of GSTP1 silencing on ADM induced-apoptosis: DAPI staining and western blot assays.Western blot was used to detect the effect of GSTP1 silencing on the expression of γH2AX.(2)In CLDN6 silencing-MCF-7/MDR cells,western blot was used to detect GSTP1 expression.GST activity assay was used to measure GST enzyme activity when CLDN6 silenced.GSTP1 expression vector was constructed and lentiviral transduction was applied to acquire GSTP1 stablely overexpressed cells,and western blot was applied to verify GSTP1 expression.GST activity assay detect GST enzyme activity when GSTP1 overexpressed.CCK8 assay was conducted to evaluate the effect of GSTP1 on different chemotherapeutic drugs sensitivity in CLDN6 silencing-MCF-7/MDR cells.Role of GSTP1 on ADM induced-apoptosis: DAPI staining and western blot assays.Western blot was used to detect the effect of GSTP1 on the expression of γH2AX.2)The role of p53 in CLDN6 regulating breast cancer cells chemoresistance through GSTP1In CLDN6 overexpressed-MCF-7 cells,western blot was used to detect p53 expression.TP53 sh RNA vector was constructed and lentiviral transduction was applied to acquire TP53 stablely silencing cells.RT-q PCR was used to measure the expression of TP53 and GSTP1 when TP53 silenced.Western blot was conducted to detect protein expression of GSTP1 when TP53 silenced.GST activity assay was used to measure GST enzyme avtivity.CCK8 assay was applied to evaluate the effect of TP53 silencing on different chemotherapeutic drugs sensitivity.Immunoprecipitation assay was used to determine the interaction between CLDN6 and p53.Nuclear and cytoplasmic protein extraction kit and western blot were conducted to evaluate the expression of p53 in nuclear and cytoplasmic protein of CLDN6 overexpressed-MCF-7 cells.3)In vivo effects of CLDN6 regulating breast cancer cells chemoresistance through GSTP1(1)Xenografts model in nude mice was conducted to measure the in vivo effect of CLDN6 on chemoresistance.Subcutaneous transplanted with CLDN6 overexpressed-MCF-7 cells and the negative control cells,and tail vein injection of 1mg/kg ADM.Describe the tumor growth curve by measuring the volume of the transplanted tumor.Weighed and compared the weight of transplated tumor.The expression of cleaved-caspase-9 and cleaved-PARP in the transplanted tumor were detected by western blot.(2)Xenografts model in nude mice was conducted to measure the in vivo effect of GSTP1 on CLDN6 enhancing chemoresistance.Subcutaneous transplanted with GSTP1 silencing cells and the control cells,and tail vein injection of 1mg/kg ADM.Describe the tumor growth curve by measuring the volume of the transplanted tumor.Weighed and compared the weight of transplated tumor.The expression of cleaved-caspase-9 and cleaved-PARP in the transplanted tumor were detected by western blot.4)The correlation of CLDN6 and GSTP1 expression in human breast cancer tissues Immunohistochemistry was used to detect CLDN6 and GSTP1 expression in 40 cases of human breast cancer tissues,and analyzed the correlation between CLDN6 and GSTP1 expression.2.Effect of CLDN6 on chemoresistance in breast cancer MDAMB231 cellsIn breast cancer cell line MDAMB231 which with mutated TP53 gene,lentiviral transduction was applied to acquire CLDN6 stablely overexpressed-MDAMB231 cells.CCK8 assay was applied to evaluate the effect of CLDN6 on ADM sensitivity.DAPI staining and western blot were used to measure ADM induced-apoptosis.Western blot was used to detect expression of apoptosis related proteins cleaved-caspase-3 and Bcl-2.Besides,western blot was applied to measure expression of proteins related to chemoresistance closely,like GSTP1,P-gp,BCRP and MRP.RT-q PCR and western blot were conducted to measure expression of EMT markers E-cadherin,N-cadherin,Vimentin and classic stem cell markers OCT4,SOX2 and Nanog.Western blot was used to detect expression of AF-6,p-ERK and ERK.Immunoprecipitation assay was used to determine the interaction between CLDN6 and AF-6 in CLDN6 overexpressed-MDAMB231 cells.ERK activator PMA was used and western blot to evaluate the effect of PMA on expression of EMT markers E-cadherin,Vimentin and stem cell markers OCT4,SOX2 and Nanog.CCK8 assay was applied to evaluate the effect of PMA on ADM sensitivity.Results 1.CLDN6 regulates GSTP1 and influences breast cancer cell chemoresistance through p53(1)CLDN6 enhances chemoresistance in breast cancer 1)In breast cancer cell line MCF-7 overexpressing CLDN6,CLDN6 increased the expression of P-gp in MCF-7 cells.The IC50 values of ADM,5-FU and DDP were increased.Compared with the control,CLDN6 partly inhibited ADM induced MCF-7 cells apoptosis.When treated with the same concentration of ADM,the ratio of apoptosis cells in CLDN6 overexpressed cells was relatively lower than the control,and CLDN6 inhibited ADM induced cleaved-caspase-9 and cleaved-PARP generation.2)In breast cancer multidrug resistance cells MCF-7/MDR silencing CLDN6,CLDN6 silencing downregulated P-gp expression in MCF-7/MDR cells.The IC50 values of ADM,5-FU and DDP were decreased.Compared with the control,silencing CLDN6 enhanced ADM induced-apoptosis.When treated with the same concentration of ADM,the ratio of apoptosis cells in CLDN6 silencing cells was relatively higher,and CLDN6 promoted ADM induced cleaved-caspase-9 and cleaved-PARP generation.The above results show that CLDN6 expression promotes chemoresistance in breast cancer cell MCF-7,and silencing CLDN6 reduces chemoresistance in breast cancer multidrug resistance cell MCF-7 / MDR.(2)CLDN6 enhances chemoresistance through GSTP1 regulated by p53 1)CLDN6 increases chemoresistance through GSTP1(1)Overexpressed CLDN6 increased GSTP1 expression in MCF-7 cells,and elevated GST enzyme activity.When silencing GSTP1 in CLDN6 overexpressed-MCF-7 cells,the IC50 values of ADM and DDP were decreased.ADM induced apoptosis was increased and ADM induced cleaved-caspase-9 and cleaved-PARP generation were increased further.Silencing GSTP1 increased ADM induced γH2AX expression further.(2)Silencing CLDN6 decreased GSTP1 expression in MCF-7/MDR cells,and reduced GST enzyme activity.When overexpressed GSTP1 in CLDN6 silencing-MCF-7/MDR cells,the IC50 values of ADM and DDP were increased,ADM induced-apoptosis and ADM induced cleaved-caspase-9 and cleaved-PARP were decreased.Overexpressed GSTP1 inhibited ADM induced γH2AX expression.The above results show that CLDN6 influence chemoresistance through GSTP1 in breast cancer cells.2)CLDN6 regulates GSTP1 and influences breast cancer cell chemoresistance through p53In the breast cancer cell MCF-7,CLDN6 overexpression upregulated p53 expression,silencing TP53 gene downregulated GSTP1 expression and decreased GST enzyme activity.Silencing TP53 gene reduces the IC50 values of ADM and 5-FU in CLDN6 overexpressed-MCF-7 cells.In CLDN6 overexpressed-MCF-7 cells,the combination between CLDN6 and p53 protein was detected.CLDN6 overexpression decreased the expression of p53 in the nuclear protein and increased the expression of p53 in the cytoplasmic protein,suggesting that CLDN6 expression induced subcellular translocalization of p53.This suggests that CLDN6 enhances chemorasistance by combining with p53 and affecting the translocation of p53 protein,and then p53 regulates GSTP1 expression and enzymatic activity and affects chemoresistance further in MCF-7 cells.At the same time,silencing TP53 gene reduced the resistance of 5-FU,indicating that in addition to GSTP1,there are other mechanisms involved in CLDN6 regulating chemoresistance in breast cancer cells.3)CLDN6 increases breast cancer ADM resistance in vivo(1)In the xenografts model in nude mice,subcutaneous transplanted with CLDN6 overexpressed-MCF-7 cells and the negative control cells,ADM inhibited growth of tumor cells and decreased the weight of transplated tumor compared with the control which treated without ADM.When treated with ADM,the growth inhibition of CLDN6 overexpressed MCF-7 cells xenografts was less than the negative control group,and the weight of CLDN6 overexpressed MCF-7 cells xenografts were relatively higher than the negative control group.The expression of cleaved-caspase-9 and cleaved-PARP in the CLDN6 overexpressed MCF-7 cells xenografts was relatively lower than the negative control group.(2)In the xenografts model in nude mice,subcutaneous transplanted with GSTP1 silencing cells and the control cells.When treated with ADM,the growth inhibition of GSTP1 silencing cells xenografts was stronger than the control group,and the weight of GSTP1 silencing cells xenografts were relatively less than the control group.The expression of cleaved-caspase-9 and cleaved-PARP in the CLDN6 overexpressed MCF-7 cells xenografts was relatively higher than the control.The above results revealed that CLDN6 promote breast cancer cell MCF-7 ADM resistance through GSTP1 in vivo,which is consistent with the results of in vitro experiments.4)CLDN6 expression positively correlated with GSTP1 expression in human breast cancer tissues(1)Among 40 cases of breast cancer,CLDN6 expression was detected in 30 cases,of which 24 cases of breast cancer tissue showed weak CLDN6 immunoreactivity,moderate in 4 cases and strong CLDN6 expression in 2 cases.CLDN6 mainly cell membrane staining.Among 40 cases of breast cancer,GSTP1 expression was observed in 25 cases,and of which 8 cases were moderate staining,17 cases showed GSTP1 weakly expression,GSTP1 expression mainly located in the nucleus.(2)There was a positive correlation between GSTP1 and CLDN6 expression in breast cancer tissues.2.CLDN6 enhances ADM resistance in breast cancer MDAMB231 cells In breast cancer MDAMB231 cells,CLDN6 overexpression increased ADM IC50 value.CLDN6 overexpression inhibited ADM induced-apoptosis: overexpression of CLDN6 decreased the ratio of ADM induced apoptosis,and overexpression of CLDN6 decreased the expression of cleaved-caspase-3 and increased the expression of Bcl-2 when treated with ADM.CLDN6 downregulated P-gp and BCRP,and CLDN6 did not affect the expression of GSTP1 and MRP1.CLDN6 decreased epithelial marker E-cadherin and increased the expression of mesenchymal markers N-cadherin and Vimentin,increased the expression of OCT4,SOX2 and Nanog.Overexpressed CLDN6 upregulated the expression of AF-6 and inhibited the expression of p-ERK.The combination between CLDN6 and AF-6 protein was detected in CLDN6 overexpressed-MDAMB231 cells.When PMA activated ERK,the expression of E-cadherin was increased,and the expression of Vimentin,OCT4,SOX2 and Nanog were decreased.PMA decreased the IC50 value of ADM in CLDN6 overexpressed MDAMB231 cells,increased the expression of cleaved-caspase-3 and decreased the expression of anti-apoptotic protein Bcl-2.These results suggest that CLDN6 enhacing ADM resistance in MDAMB231 cells through AF-6/ERK signaling,inducing EMT and increasing stem cell-like characteristics.Conclusion 1.CLDN6 promotes chemoresistance through GSTP1 regulated by p53 in MCF-7 cells.2.There was a positive correlation between CLDN6 and GSTP1 expression in breast cancer tissues.3.CLDN6 enhances ADM resistance through AF-6/ERK signaling pathway in MDAMB231 cells,independent of GSTP1.