| CLDNs are the main components of tight junctions of epithelial cells.CLDN6 is a member of the CLDNs family,which has barrier and fence functions.The carboxyl end of CLDN6 contains a PDZ domain binding motif(PBM).CLDN6 connects proteins containing the PDZ domain through its PBM,regulating intracellular signaling pathways to affect the malignant phenotype of cancer.Previously,we have cloned CLDN6 as a candidate suppressor gene of breast cancer from COP rat mammary epithelial cells for the first time.We found that CLDN6 was low expressed in breast cancer,and CLDN6 overexpression inhibited breast cancer metastasis,but the specific mechanism is still unclear.Recently,autophagy has been considered as one of the key mechanisms to inhibit cancer metastasis.Autophagy is a lysosome-dependent catabolic process induced by stress conditions such as starvation,infection and hypoxia,and plays an important role in maintaining cellular homeostasis.Autophagy inhibits tumor metastasis through a variety of pathways,including degrading EMT-related proteins,inhibiting Rho GTPase activity,and reducing tumor necrosis and immune cell infiltration.In our previous study,we found that ERβ inhibited breast cancer cell migration and invasion through CLDN6mediated autophagy.However,the mechanism of autophagy regulation by CLDN6 in breast cancer cells still needs to be further explored.The actin cytoskeleton plays a crucial role in autophagy.The actin cytoskeleton is mainly formed by different combinations of actin filaments(F-actin).It serves as a scaffold to stabilize and promote the bending of the autophagosome membrane,or formed branched networks on the surface of vesicles to drive the fusion of mature autophagosomes and lysosomes.In previous mRNA microarray,we observed that differential genes enriched in the regulation of actin cytoskeleton in CLDN6overexpressing breast cancer cells,which indicated that CLDN6 may mediate autophagy through actin cytoskeleton.However,the mechanism remains unclear.The activation of Arp2/3 complex is crucial for the formation of the actin cytoskeleton.Nucleation-promoting factors(NPF s)are the activator of Arp2/3 complex.NPFs and Arp2/3 complex can be recruited to autophagosomes and promote actin cytoskeleton assembly on the membrane,thereby regulating different stages of autophagy.WASP-interacting protein(WIP)is the protein interacting with WASP,a member of NPFs,and regulates the activation of the Arp2/3 complex or the stability of F-actin,and further affects actin cytoskeleton assembly.Using mRNA sequencing we found WIP expression was upregulated in CLDN6-overexpressing breast cancer cells.Jasper websites predicted that c-Jun can bind to the promoter region of WIP.C-Jun is an important transcription factor,which can be phosphorylated by JNK to enhance its transcriptional activity,participating in cell growth,apoptosis,and autophagy.The PDZ binding motif regulates the activation of JNK/c-Jun pathway.Thus,we speculated that CLDN6 may activate the JNK/c-Jun pathway through its PDZ binding motif,inducing autophagy through WIP-mediated actin cytoskeleton,thereby inhibiting the metastasis of breast cancer.This study aims to explore the role of CLDN6 in inhibiting breast cancer metastasis through autophagy,reveal the molecular mechanism of CLDN6 regulating autophagy through WIP-mediated actin cytoskeleton,and provide the theoretical and experimental basis for the precision therapy of breast cancer metastasis.Methods:MCF-7 and MDA-MB-231 cell lines with CLDN6 overexpression were constructed previously,and were used in the following studies after verifying the expression efficiency of CLDN6:1.Effect of CLDN6 on migration and invasion of breast cancer cells through autophagy(1)Effect of CLDN6 on migration and invasion of breast cancer cellsWound healing assay and Transwell migration assay were used to detect cell migration;Transwell invasion assay was used to detect cell invasion.(2)Effect of CLDN6 on autophagy of breast cancer cellsTransmission electron microscopy was used to observe the number of autophagosomes;IF was used to observe LC3 positive puncta;Western blot was used to detect the expression of autophagy-related proteins.(3)Role of autophagy in CLDN6 regulating migration and invasion of breast cancer cellsCLDN6-overexpressing breast cancer cells were treated with 3-MA to inhibit the early stage of autophagy,and with CQ to inhibit the late stage of autophagy,respectively;Western blot was used to detect the expression of autophagy-related proteins;Wound healing assay and Transwell migration assay were used to detect cell migration;Transwell invasion assay was used to detect cell invasion.2.Mechanism of CLDN6 regulating autophagy through WIP-mediated actin cytoskeleton(1)Role of actin cytoskeleton in CLDN6-induced autophagyPhalloidin staining was used to observe the structure and distribution of F-actin;IF was used to detect colocalization of LC3 and p-Arp3;CLDN6-overexpressing cells treated with the Arp2/3 inhibitor CK666;IF was used to observe LC3 positive puncta;Western blot was used to detect the expression of autophagy-related proteins.(2)Effect of CLDN6 on autophagy through WIP-mediated actin cytoskeleton①Effect of CLDN6 on WIP expression:RT-PCR and Western blot were used to detect WIP expression.②Role of WIP in CLDN6 regulating migration and invasion of breast cancer cells:MCF-7/CLDN6-shWIP and MDA-MB-231/CLDN6-shWIP cell lines were constructed using lentivirus transfection to stably knockdown WIP in CLDN6overexpressing cells;Western blot was used to identify the transfection effect;Wound healing assay and Transwell migration assay were used to detect cell migration;Transwell invasion assay was used to detect cell invasion.③ Role of WIP-mediated actin cytoskeleton in CLDN6 regulating autophagy:In MCF-7/CLDN6-shWIP and MDA-MB-231/CLDN6-shWIP cells,the structure and distribution of F-actin was detected by phalloidin staining;transmission electron microscopy was used to observe the number of autophagosomes;IF was used to observe LC3 positive puncta;Western blot was used to detect the expression of autophagyrelated proteins;IF was used to detect colocalization of WIP and LC3;Co-IP was used to detect the combination of WIP and LC3;Jasplakinolide,an actin depolymerization inhibitor,was used to treat MCF-7/CLDN6,MDA-MB-231/CLDN6,MCF-7/CLDN6shWIP,and MDA-MB-231/CLDN6-shWIP cells,and Western blot was used to detect the expression of autophagy-related proteins.(3)Molecular Mechanism of WIP regulation by CLDN6① Effect of CLDN6 on WIP expression through c-Jun:The correlation between c-Jun and WIP expression was analyzed by TCGA;ChIP assay was used to detect the combination of c-Jun and the WIP promoter region;dual luciferase reporter assay was used to detect the effect of CLDN6 on the activity of the WIP promoter region;Western blot was used to detect the expression of c-Jun and p-c-Jun;MCF-7/CLDN6-shJUN and MDA-MB-23 1/CLDN6-shJUN cell lines were constructed using lentivirus transfection to stably knockdown c-Jun in CLDN6-overexpressing cells;RT-PCR and Western blot were performed to detect transfection effect and WIP expression.②Effect of CLDN6 on c-Jun activation through JNK:Western blot was used to detect the expression of JNK and p-JNK;MCF-7/CLDN6-shJNK and MDA-MB231/CLDN6-shJNK cell lines were constructed using lentivirus transfection to stably knockdown JNK in CLDN6-overexpressing cells,and Western blot was used to detect the expression of JNK,c-Jun,and WIP;CLDN6-overexpressing cells were treated with JNK inhibitor SP600125,and Western blot was used to detect the expression of p-JNK,p-c-Jun,and WIP.③CLDN6 interacted with JNK via its PBM:IF was used to detect the colocalization of CLDN6 and JNK;Co-IP was used to detect the combination of CLDN6 and JNK;MDA-MB-231/CLDN6ΔPBM cell line was constructed previously to stably overexpress CLDN6 mutant with PBM deleted,and IF was used to detect colocalization of CLDN6ΔPBM and JNK;Co-IP was used to detect the combination of CLDN6ΔPBm and JNK;Western blot was used to detect the expression of JNK,c-Jun,p-c-Jun and WIP.(4)Feedback regulation of CLDN6 expression by JNK/c-Jun pathwayThe binding sites where c-Jun may bind to the CLDN6 promoter region was verified by ChIP assay;RT-PCR and Western blot were used to detect the expression of CLDN6 in MCF-7/CLDN6-shJUN and MDA-MB-231/CLDN6-shJUN cells;Western blot was used to detect the expression of CLDN6 in MCF-7/CLDN6-shJNK and MDAMB-231/CLDN6-shJNK cells.3.In vivo experiments to detect the inhibitory effect of CLDN6 on breast cancer metastasis via WIP-mediated autophagy(1)Role of WIP in CLDN6 regulating metastasis and autophagy of breast cancer in BALB/cA-nu miceBALB/cA-nu mice were injected with breast cancer cells through the tail vein to establish the lung metastasis model.The experiment was divided into three groups:MDA-MB-231/Vector,MDA-MB-231/CLDN6 and MDA-MB-231/CLDN6-shWIP.The lung metastatic nodules were examined macroscopically;the lung metastatic area was determined by HE staining;IHC staining was used to detect the expression of CLDN6,WIP,and LC3 in lung metastatic tissues.(2)Role of autophagy in CLDN6 regulating breast cancer metastasis in B ALB/cAnu miceBALB/cA-nu mice were injected with breast cancer cells through the tail vein to establish the lung metastasis model.The experiment was divided into three groups:MDA-MB-231/Vector,MDA-MB-231/CLDN6 and MDA-MB-231/CLDN6+CQ.The lung metastatic nodules were examined macroscopically;the lung metastatic area was determined by HE staining.4.Expression of CLDN6,WIP and LC3 in human breast cancer tissues and their relationship with clinicopathologic parametersThe mRNA expression of CLDN6,WIP and LC3 in breast cancer tissues was analyzed using the GEO database;the protein expression of CLDN6,WIP and LC3 in human breast cancer tissue microarray was detected by IHC,and their relationship with the clinicopathological parameters of breast cancer patients was analyzed;the correlation between the expression of CLDN6,WIP,and LC3 was analyzed.Results:1.Effect of CLDN6 on migration and invasion of breast cancer cells through autophagy(1)Effect of CLDN6 on migration and invasion of breast cancer cellsCLDN6 overexpression significantly reduced cell migration rate,and the number of migrated and invasive cells.These results indicated that CLDN6 inhibited the migration and invasion abilities of breast cancer cells.(2)Effect of CLDN6 on autophagy of breast cancer cellsCLDN6 overexpression increased the number of autophagosomes,the percentage of LC3 puncta-positive cells,and LC3Ⅱ/Ⅰ ratio,but decreased p62 expression.These results suggested that CLDN6 induced autophagy in breast cancer cells.(3)Role of autophagy in CLDN6 regulating migration and invasion of breast cancer cellsCLDN6-overexpressing cells treated with 3-MA significantly reduced LC3Ⅱ/Ⅰratio and increased p62 expression,indicating that 3-MA inhibited the early stage of autophagy.CLDN6-overexpressing cells treated with CQ cannot affect LC3Ⅱ/Ⅰ ratio but significantly increased p62 expression,indicating that CQ inhibited the late stage of autophagy.3-MA and CQ treatment significantly increased the cell migration rate and the number of migrated and invasive cells.These results suggested that CLDN6 inhibited the migration and invasion abilities of breast cancer cells through autophagy.2.Mechanism of CLDN6 regulating autophagy through WIP-mediated actin cytoskeleton(1)Role of actin cytoskeleton in CLDN6-induced autophagyIn the control group,F-actin bundles were distributed along the cell edges,while F-actin was distributed mainly in the cytoplasm around the nucleus in the CLDN6overexpressing group.These results indicated that CLDN6 promoted the rearrangement of actin cytoskeleton.LC3 was co-localized with p-Arp3 in the cytoplasm;CK666 treatment significantly decreased the percentage of LC3 puncta-positive cells and LC3Ⅱ/Ⅰ ratio,but significantly increased p62 expression.These results suggested that CLDN6 regulated autophagy through the actin cytoskeleton.(2)Effect of CLDN6 on autophagy through WIP-mediated actin cytoskeleton①Effect of CLDN6 on WIP expression:CLDN6 overexpression significantly increased the mRNA and protein expression of WIP.These results indicated that CLDN6 upregulated the expression of WIP.②Role of WIP in CLDN6 regulating migration and invasion of breast cancer cells:WIP knockdown significantly increased the cell migration rate and the number of migrated and invasive cells in CLDN6-overexpressing cells.These results suggested that CLDN6 inhibited the migration and invasion abilities of breast cancer cells through WIP.③Role of WIP-mediated actin cytoskeleton in CLDN6 regulating autophagy:In the control group,F-actin aggregated in the cytoplasm around the nucleus,but this phenomenon was not observed after WIP knockdown.WIP knockdown significantly decreased the number of autophagosomes,the percentage of LC3 puncta-positive cells,and LC3Ⅱ/Ⅰ ratio,but significantly increased p62 expression;in CLDN6overexpressing cells,WIP co-localized with LC3 in the cytoplasm and interacted with LC3.Jaslakinolide treatment significantly increased LC3Ⅱ/Ⅰ ratio and reduced p62 expression in CLDN6-overexpressing cells;compared with the control group,there were no significant changes in LC3Ⅱ/Ⅰ ratio and p62 expression in the MCF-7/CLDN6shWIP and MDA-MB-231/CLDN6-shWIP cells treated with Jaslakinolide.These results suggested that CLDN6 regulated autophagy through WIP-mediated actin cytoskeleton.(3)Molecular Mechanism of WIP regulation by CLDN6①Effect of CLDN6 on WIP expression through c-Jun:Data from the TCGA demonstrated that WIP expression was positively correlated with c-Jun expression;CJun bound to the promoter region of WIP;compared with the control group,the fluorescence activity of CLDN6-overexpressing cells was significantly increased,while the fluorescence activity was significantly reduced after the binding site mutation;compared with the control group,the expression of c-Jun remained unchanged,while the expression of p-c-Jun significantly increased in CLDN6-overexpressing cells;compared with the control group,the mRNA and protein expression of c-Jun and WIP were significantly reduced in MCF-7/CLDN6-shJUN and MDA-MB-231/CLDN6shJUN cells.These above results indicated that CLDN6 activated c-Jun to upregulate WIP expression at the transcriptional level.②Effect of CLDN6 on c-Jun activation through JNK:CLDN6 overexpression significantly increased the expression of JNK and p-JNK;compared with the control group,the expression of JNK,c-Jun,and WIP was significantly reduced in the MCF7/CLDN6-shJNK and MDA-MB-231/CLDN6-shJNK cells;SP600125 treatment significantly decreased the expression of p-JNK,p-c-Jun,and WIP in CLDN6overexpressing cells.These results suggested that CLDN6 activated c-Jun via JNK.③CLDN6 interacted with JNK via its PBM:CLDN6 co-localized with JNK at the cell edge and interacted with JNK;no colocalization and interaction between CLDN6ΔPBM and JNK were observed;CLDN6ΔPBM failed to regulate the expression of JNK,c-Jun,p-c-Jun and WIP.These results suggested that CLDN6 activated JNK/cJun pathway via its PDZ binding motif.(4)Feedback regulation of CLDN6 expression by JNK/c-Jun pathwayC-Jun bound to the promoter region of CLDN6;The expression of CLDN6 was reduced after c-Jun knockdown or JNK knockdown.These results indicated that JNK/cJun pathway feedback regulated CLDN6 expression.3.In vivo experiments to detect the inhibitory effect of CLDN6 on breast cancer metastasis via WIP-mediated autophagy(1)Role of WIP in CLDN6 regulating metastasis and autophagy of breast cancer in BALB/cA-nu miceCompared with the MDA-MB-231/Vector group,the number of lung surface metastatic nodules and the area of lung metastasis in the MDA-MB-231/CLDN6 group were significantly reduced.Compared with the MDA-MB-231/CLDN6 group,the number of lung surface metastatic nodules and the area of lung metastasis were significantly increased in the MDA-MB-231/CLDN6-shWIP group.The expression of CLDN6,WIP,and LC3 in the MDA-MB-231/Vector group was negative;in the MDAMB-231/CLDN6 group,CLDN6 was mainly expressed in the cell membrane,while WIP and LC3 were mainly expressed in the cytoplasm;the expression of WIP and LC3 was negative in the MDA-MB-231/CLDN6-shWIP group.These results indicated that CLDN6 inhibited breast cancer metastasis in BALB/cA-nu mice through WIP,which is related to autophagy.(2)Role of autophagy in CLDN6 regulating breast cancer metastasis in B ALB/cAnu miceCompared with the MDA-MB-231/Vector group,the number of lung surface metastatic nodules and the area of lung metastasis in the MDA-MB-231/CLDN6 group were significantly reduced,while the number of lung surface metastatic nodules and the area of lung metastasis were significantly increased after CQ treatment.These results indicated that CLDN6 inhibited breast cancer metastasis in BALB/cA-nu mice through autophagy.4.Expression of CLDN6,WIP and LC3 in human breast cancer tissues and their relationship with clinicopathologic parametersThe expression of CLDN6 and WIP in breast cancer tissues was lower than that in normal adjacent tissues,and the expression of WIP and LC3 in metastatic tissues was lower than that in primary breast cancer tissues.CLDN6 is mainly expressed in the cell membrane,while WIP and LC3 are mainly expressed in the cytoplasm.Low expression of WIP was associated with lymph node metastasis.There was no significant relationship between the expression of CLDN6 and LC3 and patient pathological parameters.CLDN6 was positively correlated with WIP and LC3,and WIP was positively correlated with LC3 in breast cancer tissues.Conclusions:1.CLDN6 induced autophagy through WIP-mediated actin cytoskeleton,thereby inhibiting breast cancer metastasis.2.Through its PDZ-binding motif,CLDN6 activated the JNK/c-Jun pathway to upregulate WIP expression at the transcriptional level,and there was a positive feedback loop between CLDN6 and JNK/c-Jun.3.In breast cancer tissues,CLDN6 was positively correlated with WIP and LC3,and WIP was positively correlated with LC3;low expression of WIP was associated with lymph node metastasis. |
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