Regulation Mechanism Of Breast Carcinoma Metastasis By DACH1

In recent years, breast cancer is the most common malignancy and the first leading cause of cancer death in females worldwide, which seriously affected on the women’s lives and health. Most cancer related deaths are caused by highly metastatic tumours. Aberrant activation of epithelial-mesenchymal transition (EMT) has been implicated in this process. However, the specific mechanism of transcriptional repression involved in EMT remains largely unknown. Recent studies have indicated a key role for dachshund homologue 1 (DACH1), also known as cell fate determination factor, in tumourigenesis, particularly those of the breast, prostate, ovarian, brain and lung. The role of DACH1 in the inhibition of oncogene-induced cellular migration and metastasis is well established in breast cancer cells. However, the molecular mechanism by which DACH1 regulating metastasis and EMT remains largely unknown. Therefore, we investigated the DACH1 expression is associated with EMT, and discussed the regulated molecular mechanism of DACH1 inhibits zinc finger protein SNAI1-mediated EMT and represses breast carcinoma metastasis. The main works of the paper are as follows:1. In this study, significant correlation between the expression levels of DACH1 and the morphology of breast cancer cells was observed, which used phase-contrast microscopic images assays. Transwell, scratch wound-healing, cell colony scattering, western blot and immunofluorescence, scratch wound-healing and transwell assays demonstrated that overexpression of DACH1 in ZR-75-30 cells induced a shift toward epithelial morphology and cell-cell adhesion, as well as increased the expression of the epithelial marker E-cadherin and Cytokeratin 18, decreased the expression of the mesenchymal markers N-cadherin and Vimentin, and suppressed cell migration and invasion. In contrast, silencing DACH1 in MCF-7 and T47D cells disrupted the epithelial morphology and cell-cell contact, reduced the expression of E-cadherin and Cytokeratin 18, increased the expression of the mesenchymal markers N-cadherin and Vimentin, and induced cell migration and invasion.2. RT-PCR and reporter-gene assays indicated that DACH1 could promoted the E-cadherin transcription, resulting in its increase mRNA level. Reporter-gene and chromatin-immunoprecipitation assays demonstrated that DACH1 increased the activity of E-cadherin promoter via an E-box-dependent manner, and specifically interacted with E-box elements of E-cadherin promoter. The DNA-binding domain (DBD) of DACH1 was not important in the regulation of E-cadherin, suggesting other factors may be involved in coordinating the interaction between DACH1 and the E-box of E-cadherin.3. Co-immunoprecipitation, GST-Pulldown and mammalian two-hybrid system showed that DACH1 specifically interacted with SNAI1, but did not interact with SNAI2. We used immunofluorescence assays showed DACH1 and SNAI1 were colocalized in the nucleus.4. ChIP-reChIP assays indicated that DACH1 interacted with SNAI1 to form a complex, which could bind to the E-box on the E-cadherin promoter. Chromatin-immunoprecipitation, western blot and reporter-gene assays showed that DACH1 changed the level of E-cadherin-Luc reporter activity, the invasiveness and the expression of E-cadherin via SNAI1-dependent manner. In addition, co-immunoprecipitation assays DACHl reduced the interaction between SNAI1 and HDAC1, suggesting DACH1 interacted with SNAI1 and inhibited the recruitment of HDAC1 to SNAI1, effectively suppressing the transcriptional activity of SNAI1, and eventually leading to the activation of E-cadherin.5. Furthermore, the level of DACH1 also correlated with the extent of metastasis in a mouse model. DACH1 significantly decreased the metastasis and growth of 4T1/Luc cells in BALB/c mice. Analysis of tissue samples taken from human breast cancers showed a significant correlation between the expression of DACHl and E-cadherin in SNAI1-positive breast cancer.Collectively, our data identified a new mechanistic pathway for the regulation of EMT and metastasis of breast cancer cells, one that is based on the regulation of E-cadherin expression by direct DACH1-SNAI1 interaction. Hopefully, better insights into the regulation of DACH1-mediated repression of cancer metastasis from future investigation may form the basis for designing better therapies that specifically target the metastatic stage of breast cancer.