The Study Of FOXA2-interacting Transcription Factor FOXP2 In Regulating The Epithelial-mesenchymal Transition Of Breast Cancer Cells

Breast cancer is the most common malignant tumor and the first leadi ng cause of cancer death among women.Metastasis of breast cancer is one of the main reasons of for poor survival in patients.Tumor metastasis refers to the process of cancer cell s dissociating from the primary location of the tumor,entering the circulatory system,transferring to distant tissues and organs through blood circulation or lymphatic circulation,and proliferating at new locations to produce new tumor tissues.One of the important mechanisms regulating the invasive behavior of cancer cells is the epithelial-mesenchymal transition(EMT).Activation of EMT results in loss of cell polarity,destruction of intercellular connections,degradation of basement membranes,and reorganization of extracellular matrix.Breast cancer cells acquire stronger motility,migration and invasion ability through the EMT process,and the mesenchymal cells show significantly higher drug resistance at the same time.In our previous studies,transcription factor FOXA2 was confirmed to inhibit EMT in breast cancer cells by regulating the transcription of EMT-related genes and the stable overexpression of FOXA2 abolished breast cancer cell metastasis in vivo.Thus,we intend to identify FOXA2-interacting proteins from FOXA2-pulled down cell lysates with Mass Spectrometry Analysis in current studies.Based on previous studies,this paper demonstrates the role of FOXA2 and its interacting protein FOXP2 in the regulation of EMT-related gene expression,which is of great value for understanding the mechanism of breast cancer metastasis.In previous study,the 28 putative FOXA2-interacting proteins were identified by mass spectrometry.Among these proteins,the transcription factor FOXP2,a member of the FOX gene family,caught our attention.The breast cancer stu dies have shown that FOXP2 inhibits the stemness of tumor cells a nd impairs their metastasis ability.and other studies have suggested that FOXP2 is involved in the EMT process in breast cancer cells.In this study,we need to confirm the interaction between FOXP2 and FOXA2 in breast cancer cells.Next,we need to clari fy the role of FOXP2 in the EMT process of breast cancer cells,further investigate the molecular mechanism of transcriptional regulation in breast cancer EMT by FOXP2 and explore the function of FOXA2 and FOXP2 interaction in the expression regulation of EMT-related genes.Finally,a model of breast cancer metastasis in nude mice was established by injecting FOXP2-knockdown MCF-7 cells via the tail vein,to confirm the effect of FOXP2 on breast cancer cell metastasis in vivo.The following results were obtained:First,we confirmed the interaction between FOXA2 and FOXP2 in breast cancer cells through Co-IP experiment.The interaction of endogenous FOXA2 and FOXP2 was further confirmed with immunofluorescence assay,which showed that FOXA2 and FOXP2 co-localized in the nucleus of MCF7 cells.FOXA2 protein was divided to three regions including 1-165aa(the N-terminal transcription activation domain),166-324aa(the DNA binding domain),325-463aa(the C-terminal transcription activation domain),and FOXP2 protein was divided to three regions including1-196aa(the N-terminus),197-408aa(the zinc-finger/leucine zipper motif),409-633aa(the DNA binding domain plus the C-terminus).The 166-324 aa region of FOXA2 and the 197-408 aa region of FOXP2 were identified to participate in the interaction between the two proteins.Then we analyzed the levels of FOXP2 expression in the clinical breast cancer samples(n=394)from TCGA and found that the levels of FOXP2 in all four breast cancer subgroups(Basal,Her2,Lum A,a nd Lum B)were lower than that in the normal group.We observed a weak correlation between the expressions of FOXA2 and FOXP2 in the samples(R=0.14,p=0.007),while a moderate correlation between their expressions was further found in the subgroup of basal breast cancer especially.Then,we examined FOXP2 and EMT-related genes E-cadherin and Vimentin in the EGF-induced EMT in MCF-7.The results showed that FOXP2 expression level s were down-regulated during EMT process.Furthermore,we transfected mesenchymal cells MDA-MB-231 and MDA-MB-436 with a FOXP2-expression plasmid.We found that the overexpression of FOXP2 resulted in the elevation of E-cadherin levels and the reduction of Vimentin levels and decreased the migration ability of cells.These results suggest that FOXP2 inhibits the EMT process in breast cancer cells.Subsequently,we interfered with FOXP2 expression in MCF-7 and HCC-1937 cells to test whether FOXP2 was required for the epithelial phenotype of breast cancer cells.Consequently,the knockdown of FOXP2 also decreased the expression of E-cadherin and resulted in the increased expression of Vimentin in the cells.In addition,the migration ability of FOXP2-knockdown cells was dramatically strengthened when measured by Transwell invasion test.All the reults suggested that FOXP2 was required for maintaining the epithelial characteristics of breast cancer cells and was an important factor in inhibiting EMT process of breast cancer cells.Subsequently,we established the CTx-induced MET model of MDA-MB-468 cells,the FOXP2 expression was reduced during this CTx-induced MET progression.We noticed the correlation of the increased expression of FOXP2 and PHF2,a tumor suppressor that plays a key role in CTx-induced MET.The results of Ch IP,EMSA and dual luciferase reporter assay showed that FOXP2 could bind to the promoter regions of PHF2 and E-cadherin genes and activate their expression.Though EMSA experiments we confirmed that FOXA2 and FOXP2 bound together to the upsteam region of E-cadherin gene.The Dual luciferin reporter assay under various conditions jointly indicated that the activation of E-cadherin promoter by FOXP2 relied on the participation of FOXA2.Finally,FOXP2-knockdown MCF-7 cells were injected into nude mice via tail vein injection to establish an in vivo lung metastasis model,which showed the inhibition of FOXP2 expression promoted metastasis of breast cancer cells in vivo.To further analyze the effect of FOXP2 on the survival of breast cancer patients,the results confirmed that the FOXP2-improved survival in breast cancer patients was correlated with the high levels of FOXA2.In the current study,we identified that FOXP2 interacted with FOXA2,and the expression of FOXP2 was strongly correlated with the epithelial phenotype o f breast cancer cells.The stable knockdown of FOXP2 expression promoted the mesenchymal phenotype of breast cancer cells,while the overexpres sion of FOXP2 inhibited the EMT of breast cancer cells.We confirmed that FOXP2 alone could activate the expression of tumor suppressor PHF2.Meanwhile,FOXP 2 could endogenously bind to the promoter of E-cadherin and activate E-cadherin transcription,relying on its interaction with FOXA2.Furthermore,the stable knockdown of FOXP2 enhanced the metastatic capacity of breast cancer cells in vivo.Together,the results suggested that FOXP2 could inhibit EMT in breast cancer cells by activating transcription o f certain genes,such as E-cadherin and PHF2.