The stable FOXP2-knockdown MCF-7 cells were selected and harvested for protein and total RNA preparation. that FOXA2 inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. In this study, by identifying FOXA2-interacting proteins from FOXA2-pull-down cell lysates with Mass Spectrometry Analysis, we found that FOXP2 interacted with FOXA2. After confirming the conversation between FOXP2 and FOXA2 through Co-IP and immunofluorescence assays, we showed a correlated expression of FOXP2 and FOXA2 existing Rabbit Polyclonal to LMTK3 in clinical breast cancer samples. The overexpression of FOXP2 attenuated the mesenchymal phenotype whereas the stable knockdown of FOXP2 promoted EMT in breast cancer cells. Even though FOXP2 was believed to act as a transcriptional repressor in most cases, we found that FOXP2 could activate the expression of tumor suppressor PHF2. Meanwhile, we also found that FOXP2 could endogenously bind to the promoter of E-cadherin and KDU691 activate its transcription. This transcriptional activity of FOXP2 relied on its interaction with FOXA2. Furthermore, the stable knockdown of FOXP2 enhanced the metastatic capacity of breast cancer cells (11). Thus, we intend to identify FOXA2-interacting proteins from FOXA2-pulled down cell lysates with Mass Spectrometry Analysis in current studies. Interestingly, transcription factor FOXP2, another member of the KDU691 FOX transcription factor family, has been found to interact with FOXA2. The FOX transcription factor family is widely distributed in various eukaryotes and contains more than 40 mammalian members, which possess a conserved DNA binding domain (DBD) known as Forkhead box/winged helix domain (12). The chromatin immune-precipitation experiment identifies the candidate FOXP2-binding sequence CAAATT as the most probable target for FOXP2 binding in chromatin (13). FOXP2 has been shown to both promote and more often inhibit the transcription of target genes (14). FOXP2 can interact with the co-repressors, KDU691 such as C-terminal binding protein-1 (CtBP-1) that mediates transcriptional repression primarily through KDU691 recruitment of histone deacetylases HDAC1/2 (15), to confer its transcriptional repressive properties (16, 17). An increasing amount of evidence supports the repressor role of FOXP2 upon the transcription of its target genes, such as SRPX2/uPAR complex (18) and DLL3 (19), which are involved in oncogenic progression of different types of cancers. On the other hand, FOXP2 has also been reported to activate the transcription of genes, such as the protein-tyrosine kinase SYK that is described as a tumor suppressor in breast cancer cells (20). This transcriptional activation of FOXP2 is often explained by the differential affinity of FOXP2 for DNA binding sites or by the cofactors that interact with FOXP2. While FOXP2 has first been reported to participate in speech and language development and neuronal development (21, 22), the expression of FOXP2 is observed in multiple adult tissues, such as heart, lung, liver, ovaries, and gut (23, 24). A growing number of evidences have linked FOXP2 to multiple cancers and its dysregulation may play a main role throughout cancer initiation and progression (25), even though it may act as either a tumor-suppressor or a tumor-stimulator depending on the type of cancers studied. For example, its expression is down-regulated in breast cancer (26), hepatocellular carcinoma (27), and gastric cancers (28), in which FOXP2 plays roles as a tumor-suppressor. Conversely, overexpressed FOXP2 has been found in lymphomas (29), neuroblastomas (30), and prostate cancers (31), implicating a pro-oncogenic role of FOXP2 in these cancers. These differences may suggest alternative and tissue-specific roles for FOXP2 as a tumor suppressor or as an oncogene, depending on activated signaling pathways in certain types of cancer. The strong evidence of FOXP2 as a tumor-suppressor role comes from a breast cancer study, in which silencing FOXP2 through miRNA-mediated FOXP2 repression promotes cancer stem cell traits and metastasis in breast cancer cells (32). In the current study, we identified that FOXP2 interacted with FOXA2, and the expression of FOXP2 was strongly correlated with the epithelial phenotype of breast cancer cells. The stable knockdown of FOXP2 expression promoted the mesenchymal phenotype of KDU691 breast cancer cells, while the overexpression of FOXP2 inhibited the EMT of breast cancer cells. We confirmed that FOXP2 alone could activate the expression of tumor suppressor PHF2. Meanwhile, FOXP2 could endogenously bind to the promoter of E-cadherin and activate E-cadherin transcription, relying on its interaction with FOXA2..