| The tight junction(TJ)is located at the top of the cellular junction and consists of transmembrane proteins and cytoplasmic actin.Transmembrane proteins include Claudins(CLDNs),Occludins(OCLDs)and junctional adhesion molecules(JAMs).Of these,CLDNs are the most important tight junction proteins that function as barriers and fences.As a member of the CLDNs family,CLDN6 is a candidate breast cancer suppressor gene that has been cloned and identified by our group.It is now believed that abnormal expression of CLDN6 is involved in the proliferation,migration and invasion and drug resistance of a variety of tumor cells,including breast cancer.In our previous work,we found that CLDN6 was lowly expressed in breast cancer and overexpression of CLDN6 significantly inhibited breast cancer growth,but the mechanism was not yet clear.In order to meet the demands of rapid growth,tumor cells use aerobic glycolysis as the main glucose metabolic pathway for rapid energy provision and synthesis of metabolically required intermediates.Pre-metabolomic analysis revealed that lactate content was altered in MDA-MB-231 cells overexpressing CLDN6,suggesting that CLDN6 may regulate the aerobic glycolytic process in breast cancer cells.During tumor cell proliferation,the increase in aerobic glycolysis is mainly associated with an abnormal increase or activation of glycolytic transport proteins or related enzymes.The transcription factor c-MYC is one of MYC products involved in cell proliferation and metabolism,and regulates aerobic glycolysis in tumor cells by promoting the transcription of target genes such as glucose transporter 1(GLUT 1)and lactate dehydrogenase A(LDHA).A RNAseq screen revealed that c-MYC was significantly downregulated in MCF-7 cells overexpressing CLDN6.It was hypothesized that CLDN6 may inhibit breast cancer growth via c-MYC-mediated aerobic glycolysis,but the molecular mechanisms involved need to be further investigated in this study.CLDN6 is involved in the regulation of tumor development through its signaling function.On the one hand,the carboxyl terminus of CLDN6 contains a PDZ binding motif,which can interact with proteins containing the PDZ structural domain and participate in cell signaling;on the other hand,CLDN6 relies on the extracellular loop 2(ECL2)and Tyr196/200 to bind to and activate Src-family kinases(SFK)to regulate SFK-related signaling pathways.Recent studies have revealed that tight junctionassociated proteins such as CLDNs regulate the expression and localization of YAP/TAZ by binding to them.Cytoplasmically localized YAP/TAZ is degraded via the proteasome pathway,and nucleus localized YAP/TAZ binds to the transcriptional enhanced associate domain(TEAD)transcription factor to promote its transcriptional activity,while c-MYC is the target gene of TEAD.Analysis of the TCGA database showed that CLDN6 mRNA expression was negatively correlated with TAZ protein expression.Since TAZ contains a PDZ binding motif,we speculated that CLDN6 and TAZ may inhibit breast cancer growth by binding to proteins containing the PDZ structural domain,causing TAZ to remain in the cytoplasm and decrease its entry into the nucleus,thereby regulating c-MYC-mediated aerobic glycolysis.This paper aims to investigate the role and mechanism of CLDN6 in inhibiting breast cancer growth through c-MYC-mediated aerobic glycolysis,to provide a theoretical and experimental basis for CLDN6 to be used as a biological marker for breast cancer diagnosis and prognosis.Methods:MCF-7 and MDA-MB-231 cells(noted as MCF-7/CLDN6 and MDA-MB231/CLDN6 cells,respectively),which were overexpressed with CLDN6 and constructed by our group in the previous phase,were used for the following studies.1.Effect of CLDN6 on the proliferation and aerobic glycolysis of breast cancer cells(1)Effect of CLDN6 on the proliferation of breast cancer cells:EdU cell proliferation assay to detect DNA replication;CCK8 assay to detect cell viability;plate colony formation assay to detect colony formation ability.(2)Effect of CLDN6 on aerobic glycolysis of breast cancer cells:? Metabolomic analysis of MDA-MB-231/CLDN6 cells:Metabolomic assay(positive and negative ion mode)using UHPLC-QTOFMS platform,principal component analysis(PCA)and orthogonal partial least-squares-discriminant analysis(OPLA-DA)were used to analyze metabolites;differential metabolites were presented as volcano plots and subjected to hierarchical clustering analysis and KEGG analysis.? Role of CLDN6 in aerobic glycolysis in breast cancer cells:Glucose,lactate,and ATP assay kits to measure glucose uptake,lactate production,and ATP content respectively;Seahorse Cell Energy Metabolism Analyzer to measure ECAR and calculate glycolysis,glycolytic capacity,and glycolytic reserve.2.Mechanism of CLDN6 inhibition of breast cancer cell proliferation through cMYC-mediated aerobic glycolysis(1)Effect of CLDN6 on the expression of c-MYC and its target genes GLUT1 and LDHA:RT-PCR and Western blot for mRNA and protein expression of c-MYC,GLUT 1,and LDHA,respectively.(2)Role of c-MYC in the inhibition of proliferation and aerobic glycolysis in breast cancer cells by CLDN6:?Construction and characterization of MCF-7/CLDN6-c-MYC and MDAMB-231/CLDN6-c-MYC cell lines:c-MYC was stably overexpressed in MCF7/CLDN6 and MDA-MB-231/CLDN6 cells using lentiviral transfection,noted as MCF-7/CLDN6-c-MYC and MDA-MB-231/CLDN6-c-MYC cells,and the lentiviral infection efficiency was observed by fluorescence microscopy;RT-PCR and Western blot were performed to detect the mRNA and protein expression of c-MYC,respectively,to identify the transfection efficiency.?Role of c-MYC in the inhibition of breast cancer cell proliferation byCLDN6:EdU cell proliferation assay,CCK8 assay and plate colony formation assay were performed to detect DNA replication ability,cell viability and colony formation ability,respectively.? Role of c-MYC in the inhibition of aerobic glycolysis in breast cancer cells by CLDN6:mRNA and protein expression of GLUT1 and LDHA were detected by RT-PCR and Western blot,respectively;glucose,lactate and ATP assay kits were used to detect glucose uptake,lactate production and ATP content,respectively.? Role of c-MYC-mediated aerobic glycolysis in the inhibition of breast cancer cell proliferation by CLDN6:MCF-7/CLDN6-c-MYC and MDA-MB-231/CLDN6-cMYC cells were treated with the glycolysis inhibitor 2-Deoxyglucose(2-DG),using the EdU cell proliferation assay,the CCK8 assay and plate colony formation assay to detect DNA replication ability,cell viability and colony formation ability respectively.(3)Mechanisms of c-MYC regulation by CLDN6:? Effect of CLDN6 on TAZ expression and subcellular localization:TAZ and YAP protein expression was detected by Western blot;TAZ expression and subcellular localization were detected by IF.? The interaction of CLDN6 and TAZ:IF detection of CLDN6 co-localization with TAZ;Co-IP detection of CLDN6 binding to TAZ interactions;stable overexpression of CLDN6 mutant with a PDZ binding motif deletion(CLDN6?PBM)in MCF-7 and MDA-MB-231 cells using lentiviral transfection,noted as MCF7/CLDN6?PBM and MDA-MB-231/CLDN6?PBM cells,respectively;Western blot for CLDN6?PBM protein expression;IF for CLDN6?PBM expression and subcellular localization;Co-IP for interaction between CLDN6?PBM and TAZ.3.In vivo experiments to detect the inhibitory effect of CLDN6 on breast cancer growth via c-MYC(1)In vivo experiments to detect the effect of CLDN6 on breast cancer growth:10 female BALB/cA-nu mice were divided into the following 2 groups:MDA-MB231/Vec group and MDA-MB-231/CLDN6 group,subcutaneously injected with breast cancer cell suspension to establish a nude mouse xenograft tumor model.The volume of the transplanted tumor was measured and calculated,and the weight of the transplanted tumor was weighed;the morphology of the transplanted tumor was observed by HE staining;Ki67 expression in the transplanted tumor was detected by IHC and the cell proliferation index was calculated;CLDN6,c-MYC,GLUT1,and LDHA expression in the transplanted tumor were detected by IHC.(2)In vivo experiments to detect the role of c-MYC in the inhibition of breast cancer growth by CLDN6:10 female BALB/cA-nu mice were divided into 2 groups as follows:MDA-MB-231/CLDN6-Vec group and MDA-MB-231/CLDN6-c-MYC group,and a nude mouse xenograft tumor model was established by subcutaneous injection of breast cancer cell suspension.The volume of the transplanted tumor was measured and calculated,and the weight of the transplanted tumors was weighed;the morphology of the transplanted tumors was observed by HE staining;Ki67 expression in the transplanted tumors was detected by IHC,and cell proliferation index was calculated;c-MYC,GLUT1 and LDHA expression in the transplanted tumors were detected by IHC.4.Expression of CLDN6,TAZ and c-MYC in human breast cancer tissues and their clinical significance(1)Expression of CLDN6,TAZ,and c-MYC in human breast cancer tissues and their relationship with pathological parameters:The expression of CLDN6,TAZ,and c-MYC in human breast cancer tissue microarrays was detected by IHC,and the correlation between the three and their relationship with clinicopathological parameters of breast cancer patients were analyzed.(2)Relationship between CLDN6,TAZ,and c-MYC expression in human breast cancer tissues and patient prognosis:Kaplan-Meier plotter database to assess the relationship between CLDN6 expression,CLDN6 to TAZ expression ratio and CLDN6 to c-MYC expression ratio and overall survival(OS)and recurrence-free survival(RFS)of breast cancer patients,predicting the association of CLDN6,TAZ and c-MYC with prognosis in breast cancer patients.Results:1.Effect of CLDN6 on proliferation and aerobic glycolysis of breast cancer cells(1)Effect of CLDN6 on the proliferation of breast cancer cells:Compared with the control group,the DNA replication ability,cell viability,and colony formation ability of cells in the MCF-7/CLDN6 and MDA-MB-231/CLDN6 groups were significantly reduced.The above results indicated that CLDN6 inhibited the proliferation of breast cancer cells.(2)Effect of CLDN6 on aerobic glycolysis of breast cancer cells:? Metabolomic analysis of MDA-MB-231/CLDN6 cells:In the positive ion mode,there was a significant increase in 9 metabolites and a significant decrease in 29 metabolites in the MDA-MB-231/CLDN6 group.In the negative ion mode,there was a significant increase in 22 metabolites and a significant decrease in 23 metabolites in the MDA-MB-231/CLDN6 group,including lactate;purine metabolism,pyrimidine metabolism,glycine,serine and threonine metabolism,fatty acid biosynthesis metabolism,and pentose phosphate pathway were significantly altered in the MDA-MB-231/CLDN6 group.These results suggested that CLDN6 affected the metabolic pattern of breast cancer cells,in which intracellular lactate content was altered,and may be involved in regulating aerobic glycolysis in breast cancer cells.? Role of CLDN6 in aerobic glycolysis of breast cancer cells:Compared with the control group,cellular glucose uptake,lactate production,and ATP content were significantly reduced in MCF-7/CLDN6 and MDA-MB-231/CLDN6 groups;cellular glycolysis and glycolytic capacity were significantly reduced in MCF-7/CLDN6 and MDA-MB-231/CLDN6 groups;the glycolytic reserve of the MCF-7/CLDN6 group was significantly decreased,and the glycolytic reserve of the MDA-MB-231/CLDN6 group was decreased,but the difference was not significant.The above results indicated that CLDN6 inhibited aerobic glycolysis in breast cancer cells.2.Mechanism of CLDN6 inhibition of breast cancer cell proliferation through cMYC-mediated aerobic glycolysis(1)Effect of CLDN6 on the expression of c-MYC and its target genes GLUT1 and LDHA:mRNA and protein expression of c-MYC,GLUT1,and LDHA were significantly reduced in MCF-7/CLDN6 and MDA-MB-231/CLDN6 groups of cells compared to the control group.The above results indicated that CLDN6 inhibited the expression of c-MYC and its target genes GLUT1 and LDHA.(2)Role of c-MYC in the inhibition of proliferation and aerobic glycolysis of breast cancer cells by CLDN6:? Construction and characterization of MCF-7/CLDN6-c-MYC and MDAMB-231/CLDN6-c-MYC cell lines:the lentiviral infection efficiency was above 90%;compared with the control group,the mRNA and protein of c-MYC in MCF7/CLDN6-c-MYC and MDA-MB-231/CLDN6-c-MYC group cells expression were significantly increased.The above results indicated that the MCF-7/CLDN6-c-MYC and MDA-MB-231/CLDN6-c-MYC cell lines were successfully constructed.?Role of c-MYC in the inhibition of breast cancer cell proliferation by CLDN6:The DNA replication ability,cell viability,and colony formation ability of cells in MCF-7/CLDN6-c-MYC and MDA-MB-231/CLDN6-c-MYC groups were significantly increased compared with the control group.The above results indicated that CLDN6 inhibited the proliferation of breast cancer cells dependent on c-MYC.? Role of c-MYC in the inhibition of aerobic glycolysis in breast cancer cells by CLDN6:mRNA and protein expression of GLUT1 and LDHA were significantly increased in cells of MCF-7/CLDN6-c-MYC and MDA-MB-231/CLDN6-c-MYC groups compared with the control group;glucose uptake and lactate production were significantly increased,but ATP content was significantly decreased,which may be related to the altered cell proliferation caused by ATP depletion after c-MYC overexpression.The above results suggested that CLDN6 inhibited aerobic glycolysis in breast cancer cells via c-MYC.? Role of c-MYC-mediated aerobic glycolysis in the regulation of breast cancer cell proliferation by CLDN6:Compared with the control group,the DNA replication ability,cell viability,and colony formation ability of cells in the 2-DGtreated group were significantly reduced.These results suggested that CLDN6 relied on c-MYC-mediated aerobic glycolysis to inhibit the proliferation of breast cancer cells.(3)Mechanisms by which CLDN6 regulates c-MYC.? Effect of CLDN6 on TAZ expression and subcellular localization:Compared with the control group,TAZ protein expression was significantly reduced but its homologue YAP protein expression did not change significantly in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells;TAZ cytoplasmic localization was increased while cytosolic localization was reduced.The above results indicated that CLDN6 inhibited the expression and nuclear translocation of TAZ.?The interaction of CLDN6 and TAZ:In MCF-7/CLDN6 cells,CLDN6 and TAZ co-localized in the cytoplasm;in MDA-MB-231/CLDN6 cells,CLDN6 and TAZ co-localized in the cytoplasm and cell membrane;CLDN6 and TAZ bound to each other;compared with the control group,MCF-7/CLDN6?PBM and MDA-MB231/CLDN6?PBM groups showed significantly increased levels of CLDN6?PBM protein and CLDN6?PBM was located in the cell membrane and cytoplasm;CLDN6?PBM and TAZ interacted with each other.The above results indicated that CLDN6 did not depend on the PDZ binding motif to bind to TAZ.3.In vivo experiments to detect the inhibitory effect of CLDN6 on breast cancer growth via c-MYC(1)The effect of CLDN6 on breast cancer growth was examined in vivo:Significant reduction in transplanted tumors volume and weight formed by cells in the MDA-MB-231/CLDN6 group compared to the control group;the transplanted tumor tissues formed by the MDA-MB-231/CLDN6 group cells had smaller cells,smaller nuclei,lighter staining,and fewer mitotic figures;the number of Ki67-positive cells in the transplanted tumor tissues formed by the MDA-MB-231/CLDN6 group cells was significantly reduced.In the transplanted tumor tissues formed by the MDA-MB231/CLDN6 group cells,CLDN6 was mainly expressed in the cell membrane,cMYC was mainly expressed in the nucleus,GLUT1 was mainly expressed in the cell membrane and LDHA was mainly expressed in the cytoplasm,all of which stained brownish-yellow;the expression of c-MYC,GLUT1,and LDHA was significantly reduced.The above results indicated that CLDN6 inhibited breast cancer growth and the expression of c-MYC and its target genes in and transplanted tumor tissues in nude mice.(2)In vivo experiments examined the role of c-MYC in the inhibition of breast cancer growth by CLDN6:The volume and weight of tumors formed by the MDAMB-231/CLDN6-c-MYC group increased significantly compared with the control group;the heterogeneity of transplanted tumor formed by the MDA-MB231/CLDN6-c-MYC group cells was obvious,with deeper nuclear staining,increased nuclear schizophrenia and neovascularization observed;the number of Ki67-positive cells in transplanted tumor tissues formed by the MDA-MB-231/CLDN6-c-MYC group cells increased significantly;the expression of c-MYC,GLUT1 and LDHA increased significantly.The above results indicated that CLDN6 inhibited the growth of breast cancer in nude mice through c-MYC and was associated with aerobic glycolysis.4.Expression of CLDN6,TAZ,and c-MYC in human breast cancer tissues and their clinical significance(1)Expression of CLDN6,TAZ,and c-MYC in human breast cancer tissues and their relationship with pathological parameters:In human breast cancer tissues,CLDN6 was mainly expressed in the cell membrane,TAZ and c-MYC were mainly expressed in the nucleus,all of which showed brownish-yellow staining;CLDN6 was negatively correlated with TAZ and c-MYC in breast cancer tissues,but the difference was not significant,and c-MYC was positively correlated with TAZ;there was a significant positive correlation between c-MYC and TAZ;patients with high c-MYC expression were prone to lymph node metastasis,while there was no significant relationship between CLDN6 and TAZ expression and patients' pathological parameters.(2)Relationship between CLDN6,TAZ,and c-MYC expression in human breast cancer tissues and patients' prognosis:Breast cancer patients with higher CLDN6 expression,higher CLDN6 to TAZ expression ratio,and higher CLDN6 to c-MYC expression ratio had longer OS and RFS,so breast cancer patients with higher CLDN6 expression and lower TAZ and c-MYC expression had a better prognosis.Conclusions:1.CLDN6 may regulate c-MYC-mediated aerobic glycolysis via TAZ,which in turn inhibits breast cancer growth.2.Breast cancer patients with higher CLDN6 expression and lower TAZ and cMYC expression have a better prognosis. |
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