Estrogen Receptor ? Inhibits Breast Cancer Cells Migration And Invasion Through CLDN6-mediated Autophagy

Estrogen plays an important role in the progression and metastasis of hormone-dependent breast cancer.The effects of estrogen are primarily mediated through the estrogen receptor ? and estrogen receptor ?.The role of ER? in breast cancer has been extensively studied.The contribution of ER? to the tumorigenesis and progression of breast cancer is essential.ER? is currently the main target of endocrine therapy for breast cancer.ER? is the second estrogen receptor.ER? has the same structural domains as ER?,but its function is not exactly the same as ER?.The role of ER? in breast cancer remains elusive.Although a few studies claim that ER? expression promotes the invasion and metastasis of breast cancer and that high ER? level is linked with poor prognosis,multiple studies have demonstrated that ER? is an anti-oncogene in breast cancer.The levels of ER? were high in mammary epithelial tissues and decreased during tumor progression.In vitro studies showed that ER? expression inhibited the cell proliferation and the migratory and invasive properties of breast cancer cells.Nevertheless,the molecular mechanisms underlying the inhibitory effects of ER? on breast cancer remain unidentified and need to be explored.Recent studies have reported that ER? inhibits the proliferation of breast cancer cells by inducing autophagy.Autophagy plays a key role in the maintenance of cellular homeostasis.Dysregulation of autophagy has been implicated in cancers.Studies have shown that activation of ER? induces autophagy,leading to cell cycle arrest,which inhibits tumor cell proliferation.In glioblastoma cells,the absence of autophagy related proteins beclin1,atg5 or atg7 leads to decreased autophagy,which further promotes cell migration and invasion through epithelial-mesenchymal transition(EMT).However,few studies have reported that the inhibitory role of ER? on migration and invasion is directly related to the modulation of autophagy in breast cancer.Furthermore,the regulatory mechanism of ER? induced-autophagy is still unclear.In our recent studies,we found that CLDN6 induced autophagy,whereas the relationship between CLDN6-induced autophagy and breast cancer remains poorly investigated.Claudin 6(CLDN6)is a tight junction(TJ)protein.As an important part of TJs,CLDNs not only play important roles in the classic barrier and fence functions of TJs but are also involved in regulating cellular communication and signaling.The research group has been engaged in the research on the role of CLDN6 in breast cancer.We believe that CLDN6 is a breast cancer suppressor gene.It has been reported that CLDN6 induces apoptosis of breast cancer cells and inhibits proliferation,migration and invasion of tumor cells.Previous studies have shown that 17?-estradiol(E2)upregulates CLDN6 expression and hinders MCF-7 cell migration and invasion,but the molecular mechanisms are still unclear.In this study,we found that the expression of CLDN6 was increased and migration and invasion were hindered in both MCF-7 with positive ER? expression and MDA-MB-231 cells with negative ER? expression after E2 treatment.Thus,we supposed that this E2-induced effect was not ER?-dependent.Because ER? is expressed in both breast cancer cells,we speculate that ER? may play an important role in regulating the expression of CLDN6 in breast cancer cells.The possible mechanism is that ER? induces autophagy through CLDN6,which then affects the migration and invasion of breast cancer cells.The current study aimed to explore the regulatory role of ER? on CLDN6 expression in breast cancer cells and the related mechanism of ER?-induced autophagy and inhibition of migration and invasion of breast cancer cells.It provides theoretical and experimental basis for the mechanism study of ER? and CLDN6 as breast cancer suppressor genes and potential targets for breast cancer treatment.Methods:1.Regulatory effect and mechanism of ER? on the expression of CLDN6(1)Regulation of estrogen on CLDN6 expression in breast cancer cellsMCF-7(ER?+/ER?+/GPR30+),MDA-MB-231(ER?-/ER?+/GPR30-)and SK-BR-3(ER?-/ER?-/GPR30+)cells were treated with E2.The expression of CLDN6,ER? and ER? was measured using semiquantitative RT-PCR and western blot.MCF-7 and MDA-MB-231 cells were treated with a nonselective estrogen receptor antagonist ICI 182.780(ICI)and E2.The expression of CLDN6 was measured by q RT-PCR and western blot.Wound healing and Transwell assays were used to examine the migration and invasion of E2 on MCF-7 and MDA-MB-231 cells.(2)Regulation of ER? on CLDN6 expression in breast cancer cellsMDA-MB-231 cells were treated with diarylpropionitrile(DPN),a selective ER? agonist.The expression of CLDN6 was measured by semiquantitative RT-PCR and western blot.The expression and localization of CLDN6 were detected by immunofluorescence.The tight junction was detected by transmission electron microscopy.CLDN6 was silenced in DPN-treated cells by RNA interference(RNAi)technology.The migration and invasion abilities of DPN-treated cells were detected by wound healing and Transwell assays.Stable ER?-overexpressing cells lines were obtained by transfection with ER? plasmids,and the CLDN6 expression of DPN-treated ER?-overexpressing cells was detected by Western blot.PHTPP(a selective ER? antagonist)and RNAi technology were used to reduce the expression of ER?.The expression of CLDN6 in DPN-treated ER?-knockdown cells was detected by Western Blot.(3)Regulatory mechanism of ER? on CLDN6 expression in breast cancer cellsWestern Blot and immunofluorescence were used to detect the expression and localization of ER? in DPN-treated cells.Ch IP assay was used to detect the binding of ER? and Sp1 to the promoter region of CLDN6.Double luciferase reporter gene assays were used to detect the effect of ER? on the activity of the promoter region of CLDN6.2.Regulatory effect and mechanism of ER? on autophagy in breast cancer cells(1)The Regulatory effect of ER? on autophagy in breast cancer cellsEffects of DPN-activated ER? on autophagy of breast cancer cells were detected by transmission electron microscopy,immunofluorescence and western blot.The expression of LC3 B was detected by western blot and immunofluorescence in DPN-treated ER?-knockdown cells.Cells were treated by DPN and CQ or 3-MA,and the expression of LC3 B was detected by western blot,wound healing and Transwell assays were used to examine the migration and invasion.(2)The mechanism of ER? on autophagy in breast cancer cellsThe biomarkers of autophagy were detected by western blot in DPN-treated cells.CLDN6 was knocked down by RNAi technology in DPN-treated cells,and the biomarkers of autophagy were detected by western blot.Beclin1 was knocked down by RNAi technology in DPN-treated cells,and the biomarkers of autophagy were detected by western blot.Stable CLDN6-overexpressing cells lines were obtained by transfection with CLDN6 plasmids,and the biomarkers of autophagy were detected by Western blot.Beclin1 was knocked down by RNAi technology in CLDN6-overexpressing cells,and the biomarkers of autophagy were detected by western blot.The effect of CLDN6/beclin1 on breast cancer metastasis was detected by establishing lung metastasis mouse models which was through tail vein injection.Wound healing and Transwell assays were used to detect the effect of CLDN6/beclin1 on the migration and invasion of breast cancer cells.The expression of ZO-1 and UVRAG was detected by western blot in DPN-treated and CLDN6-overexpressing cells.The binding of UVRAG with ZO-1,beclin1 and CLDN6 and the binding of ZO-1 with UVRAG,beclin1 and CLDN6 were detected by Co-IP.3.The clinical correlation analyses of ER?,CLDN6 and beclin1 expression and prognosis in breast cancer patientsThe expression of ER?,CLDN6 and beclin1 in human breast cancer tissues was detected by immunohistochemistry.The relationship between the expression of ER?,CLDN6 and beclin1 and clinical indicators,and the expression of ER?,CLDN6 and beclin1 was analyzed.The relationships of ER?,CLDN6 and beclin1 with overall survival(OS)and disease free survival(DFS)were analyzed in kaplan-meier plotter database.Results:1.Regulatory effect and mechanism of ER? on the expression of CLDN6(1)Regulation of estrogen on CLDN6 expression in breast cancer cellsE2 upregulated the expression levels of CLDN6 m RNA and protein in MCF-7 and MDA-MB-231 cells,and the expression of CLDN6 in SK-BR-3 cells was unchanged with E2 treatment.Compared with E2 treatment alone,the expression of CLDN6 in E2 combined with ICI group was decreased.There was no change in ER? expression in E2-treated MCF-7 cells compared with untreated cells.The expression of ER? was increased in E2-treated MCF-7 and MDA-MB-231 cells.The migration and invasion abilities of MCF-7 and MDA-MB-231 cells treated with E2 were decreased compared with untreated cells.These results suggest that E2 regulates CLDN6 expression through ER?.(2)Regulation of ER? on CLDN6 expression in breast cancer cellsDPN upregulated the expression levels of CLDN6 mRNA and protein in MDA-MB-231 cells.The expression of CLDN6 on the cell membrane was increased and the tight junctions between cells were prominent in DPN-treated cells compared with the untreated cells.The migration and invasion abilities of DPN-treated cells were decreased compared with untreated cells.The migration and invasion abilities of DPN-treated CLDN6-knockdown cells were increased compared with DPN-treated empty vector group.The expression of CLDN6 was increased in DPN-treated ER?-overexpressing SK-BR-3 and MDA-B-231 cells compared with DPN-treated empty vector group.Compared with the control group,the expression of CLDN6 in DPN-treated ER?-knockdown cells was decreased.These results further suggest that DPN-activated ER? positively regulates the expression of CLDN6.(3)Regulatory mechanism of ER? on CLDN6 expression in breast cancer cellsCompared with untreated cells,the expression of ER? was increased in the nucleus and decreased in the cytoplasm of DPN-treated cells.ER? and Sp1 could bind to the promoter region of CLDN6.Compared with p GL3-CLDN6 cells,the fluorescence activity of DPN-treated p GL3-CLDN6 cells was increased.These results suggest that ER? regulates the expression of CLDN6 at the transcriptional level.CLDN6 is the target gene of ER?.2.Regulatory effect and mechanism of ER? on autophagy in breast cancer cells(1)The Regulatory effect of ER? on autophagy in breast cancer cellsCompared with untreated group,the number of autophagic vesicles was increased by electron microscopy,LC3 B labeled spots were increased by immunofluorescence,the ratio of LC3II/LC3 I was increased by western blot in DPN treated cells.In DPN-treated ER?-knockdown cells,the ratio of LC3II/LC3 I was decreased,and the numbers of LC3 B labeled spots were decreased compared with DPN-treated empty vector group.The ratio of LC3II/LC3 I in cells treated with CQ and DPN was higher than that of cells treated with DPN alone.The ratio of LC3II/LC3 I in cells treated with 3-MA and DPN was lower than that of cells treated with DPN alone.The migration and invasion abilities of cells treated with DPN combined with 3-MA were higher than that treated with DPN alone.These results suggest that ER? is involved in the early stage of autophagy formation,and ER?-induced autophagy inhibits the migration and invasion of breast cancer cells.(2)The mechanism of ER? on autophagy in breast cancer cellsCompared with untreated group,the expression of autophagy marker proteins beclin1,atg5,atg16 and LC3 II was increased in DPN-treated cells.Compared with DPN-treated empty vector group,the expression of autophagy marker proteins beclin1,atg5,atg16 and LC3 II was decreased in DPN-treated CLDN6-knockdown or beclin1-knockdown cells.Compared with empty vector group,the expression of autophagy marker proteins beclin1,atg5,atg16 and LC3 II was increased in CLDN6-overexpressing cells.Compared with empty vector group,the expression of autophagy marker proteins beclin1,atg5,atg16 and LC3 II was decreased in CLDN6-overexpressing beclin1-knockdown cells.Compared with untreated group,the migration and invasion abilities of DPN-treated cells were decreased.Compared with DPN-treated empty vector group,the migration and invasion abilities of DPN-treated beclin1-knockdown cells were increased.Compared with empty vector group,the migration and invasion abilities of CLDN6-overexpressing cells were decreased.Compared with CLDN6-overexpressing cells,the migration and invasion abilities of CLDN6-overexpressing beclin1-knockdown cells were increased.Compared with empty vector group,the number of mice with lung metastasis in CLDN6-overexpressing group was decreased.Compared with CLDN6-overexpressing cells,the number of mice with lung metastasis in CLDN6-overexpressing beclin1-knockdown group was increased.Compared with the respective control groups,the expression levels of ZO-1 and UVRAG were increased in both DPN-treated and CLDN6-overexpressing cells.Co-IP results showed that UVRAG could bind to ZO-1,beclin1 and CLDN6,and ZO-1 could bind to UVRAG,beclin1 and CLDN6,respectively.These results suggest that CLDN6/beclin1 mediated autophagy inhibits breast cancer cell metastasis.The complex of CLDN6 and ZO-1/UVRAG/beclin1 recruits other autophagy-related proteins to participate in the formation of autophagy.3.The clinical correlation analyses of ER?,CLDN6 and beclin1 expression and prognosis in breast cancer patientsIn breast cancer tissues,ER? expression was observed in the nucleus and the cytoplasm,CLDN6 was expressed in the cytoplasm and the plasma membrane and beclin1 was mainly expressed in the cytoplasm.The expression of ER? was associated with TNM stage.The expression of beclin1 was associated with tumors.No association existed between the expression of ER?,CLDN6 and beclin1 and patient age,lymph node metastasis or pathological grade.ER? and CLDN6 were positively correlated and that the expression of CLDN6 was positively correlated with beclin1 in breast cancer tissues.Breast cancer patients with high beclin1 expression had significantly longer OS than patients with low beclin1 expression,and patients with high ER?,CLDN6 and beclin1 expression levels showed longer DFS than the respective low expression groups.These results suggest that high expression of ER?,CLDN6 or beclin1 indicates a better prognosis for breast cancer patients.Conclusion:1.CLDN6 is the target gene of ER?.2.ER? inhibits the migration and invasion of breast cancer cells through CLDN6/beclin1-mediated autophagy.3.Breast cancer patients with high expression of ER?,CLDN6 and beclin1 have better prognosis.