| The Wnt/?-catenin pathway is a canonical Wnt signal pathway,which controls a variety of critical cellular processes.The abnormal activation of this pathway is related to the metastasis of breast cancer and the decrease of overall survival rate.Wnt/?-catenin signaling is subject to regulation on multiple levels,including the production,release and post-translational modification of Wnt ligand,the stability of Wnt receptor,and the binding of antagonists such as CHIBBY(Cby)to Wnt and its receptors or ?-catenin.The inhibitory effect of Cby on this signal pathway can be eliminated by thyroid cancer-1(TC-1;also referred to as C8orf4).That is,up-regulated TC-1 enhances Wnt/?-catenin signal pathway by binding and inhibiting Cby,thus promoting the invasive biological behavior of cancer cells.Our previous high-throughput sequencing results suggested that the overexpression of oncogene TBC1D3 could up-regulate the level of TC-1 in human breast cancer MCF-7 cells,but it was not clear whether TBC1D3 affected the Wnt/?-catenin signal pathway and whether this pathway played a role in TBC1D3-induced migration of human breast cancer cells.Therefore,we carried out the current study.1.Objectives:To investigate the effect of TBC1D3 on Wnt/?-catenin signal pathway and its role in TBC1D3-induced migration of human breast cancer cells.2.Methods:(1).In order to study the effect of TBC1D3 overexpression on TC-1 expression and migration of human breast cancer cells,Flag-TBC1D3 was transfected into MCF-7 and BT-549 cells with Flag vector as control.The cells were treated with or without TNF-?inhibitor Pom,NF-?B inhibitor CAPE or FGFR inhibitor BGJ-398,respectively.TC-1m RNA and protein levels were detected by RT-PCR and Western blotting.Wound healing assay and Transwell assay were used to detect the migration of human breast cancer cells.(2).In order to study TC-1 mediates TBC1D3-induced migration of human breast cancer cells,four lentiviral vectors KD-TC-1(653),KD-TC-1(1512),KD-TC-1(1566)and KD-TC-1(1665),with knockdown TC-1 were constructed and infected with MCF-7 cells.The stable infected cells were screened with 1.5 ?g/ml puromycin,and the level of TC-1 protein in the cells was detected by Western blotting.Compared with Flag vector,TBC1D3 was overexpressed in knockdown TC-1stable infected cells.Wound healing assay and Transwell test were used to detect the migration of human breast cancer cells,respectively.(3).In order to study the effect of Wnt/?-catenin signal pathway on the migration of human breast cancer cells induced by TBC1D3 and TC-1,Flag-TBC1D3 was transfected into MCF-7,KD-TC-1(1566)-MCF-7 and BT-549 cells,respectively,with Flag vector as control.The m RNA and protein levels of MT1-MMP,c-Myc and Cyclin D1,which were downstream of Wnt/?-catenin pathway,were detected by RT-PCR and Western blotting,respectively.After the transfected cells were treated with XAV-939,a specific inhibitor of Wnt signal pathway,and SKL-2001,an agonist,the migration of human breast cancer cells was detected by Wound healing assay and Transwell test,respectively.(4).In order to study the relationship between the expression of TBC1D3,TC-1and?-catenin and the metastasis and recurrence of invasive breast cancer,the protein levels of TBC1D3,TC-1 and ?-catenin in invasive breast cancer tissues were detected by immunohistochemical staining in clinicopathological samples,respectively.CT,MRI,PET-CT and Bonescanning were used to analyze the correlation between metastasis and recurrence combined with correlation consistency test,ROC curve and 5-year progression-free survival(PFS).3.Results:(1).TBC1D3 promotes TC-1 expression and migration of human breast cancer cells in a TNF/NF-?B-dependent mannerHigh expression of TBC1D3 could stimulate the expression of TC-1 m RNA and proteinlevels in non-triple negative breast cancer MCF-7 cells and triple negative breast cancer BT549 cells,but the addition of either TNF ? inhibitor Pom or NF-?B inhibitor CAPE,but not FGFR inhibitor BGJ-3988,considerably reduced this effect of TBC1D3.Wound healing assay and Transwell assay showed that both TBC1D3 and TC-1 could promote the migration of MCF-7 cells and BT549 cells.(2).TC-1 mediates TBC1D3-induced migration of human breast cancer cells.Compared with the Flag vector,the overexpression of TBC1D3 and TC-1 could significantly increase the migration of control sh RNA-NC-MCF-7 cells,respectively.However,knockdown TC-1 can significantly inhibit TBC1D3-induced migration of MCF-7cells,suggesting that TC-1 mediates TBC1D3-induced migration of human breast cancer cells.(3).Wnt/?-catenin signal pathway mediates TBC1D3 and TC-1-induced migration of human breast cancer cells.The overexpression of TBC1D3 and TC-1 both could significantly increase the level of?-catenin protein in the cytoplasm and nucleus of MCF-7 and BT-549,and up-regulate the m RNA and protein levels of MT1-MMP,c-Myc and Cyclin D1,the target genes downstream of Wnt/?-catenin signal pathway.However,knockdown TC-1 not only decreased the level of?-catenin protein in cytoplasm and nucleus,and down-regulated the m RNA and protein levels of target genes MT1-MMP,c-Myc and Cyclin D1 downstream of Wnt/?-catenin signal pathway,but also reversed the accumulation of ?-catenin in cytoplasm and nucleus induced by TBC1D3,and significantly inhibited the stimulating effect of TBC1D3 on the expression of target genes downstream of Wnt/?-catenin signal pathway.It was suggested that TC-1 may mediate the activation of Wnt/?-catenin signal pathway induced by TBC1D3 in human breast cancer cells.Similar to knockdown TC-1,treatment of cells with XAV-939,a specific inhibitor of Wnt/?-catenin signal pathway,could also inhibit the accumulation of ?-catenin in the cytoplasm and nucleus of MCF-7 and BT-549 cells induced by TBC1D3,and make TBC1D3 lose the effect of stimulating the increased expression of MT1-MMP,c-Myc and Cyclin D1 genes downstream of Wnt/?-catenin signal pathway,which significantly inhibited the migration of MCF-7 and BT-549 cells induced by TBC1D3.It was suggested that Wnt/?-catenin signal pathway mediates the migration of human breast cancer cells induced by TBC1D3 and TC-1.(4).TBC1D3,TC-1 and ?-catenin are highly correlated with metastasis and recurrence of invasive breast cancer.The clinical samples of 36 patients with breast cancer were stained by immunohistochemistry.The results showed that TBC1D3 and TC-1,TBC1D3 and ?-catenin,and TC-1 and ?-catenin all had medium consistency(? = 0.471,0.421,0.501,respectively).A retrospective analysis of the data of these patients showed that breast cancer metastasis existed in 94% of TBC1D3-,80% of TC-1-and 84% of ?-catenin-positive patients,versus 50%of TBC1D3-,40% of TC-1-and 30% of ?-catenin-negative patients,representing a 1.9-,2.0-,and 2.8-fold greater odds of lymph node metastasis in the TBC1D3-,TC-1-,and?-catenin-positive population,respectively.Furthermore,the expression of TBC1D3,TC-1 or?-catenin made triple-negative breast cancer more likely to metastasize than non-triple-negative breast cancer.Receiver operating characteristic curve(ROC curve)analysis suggested that TBC1D3,TC1 and ?-catenin were more sensitive and more specific than ER,PR,HER2 and Ki-67 in the prediction of breast cancer metastasis.Finally,PFS assessment showed that breast cancer patients with positive TBC1D3,TC-1 and ?-catenin were more likely to relapse than those with negative TBC1D3,TC-1 and ?-catenin,respectively.4.Conclusion:(1)TBC1D3 promotes TC-1 expression in human breast cancer cells in a TNF/NF-?B dependent manner.(2)TC-1 mediates TBC1D3-induced migration of human breast cancer cells.(3)Wnt/?-catenin signaling pathway mediates human breast cancer cell migration induced by TBC1D3 and TC-1.(4)The expression of TBC1D3,TC-1 and ?-catenin protein is positively correlated with the metastasis and recurrence of human invasive breast cancer. |
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