Functional Hierarchy and Cooperation of EMT Master Transcription Factors in Breast Cancer Metastasis

Several master transcription factors (TF) can activate the epithelial-to-mesenchymal transition (EMT). However, their individual and combinatorial contributions to EMT in breast cancer are not defined. We show that overexpression of EMT-TFs individually in epithelial cells upregulated endogenous SNAI2, ZEB1/2, TCF4, and TWIST1/2 as a result of positive feedback mediated in part by suppression of their negative regulator miRNAs miR200s/203/205. We identified TCF4 as a potential new target of miR200s. Expression of ZEB1/2 strongly correlated with the mesenchymal phenotype in breast cancer cells, with the CD24^-/CD44⁺ stemness profile, and with lower expression of core epithelial genes in human breast tumors. Knockdown of EMT-TFs identified the key role of ZEB1 and its functional cooperation with other EMT-TFs in the maintenance of the mesenchymal state. Inducible ZEB1+2 knockdown in xenograft models inhibited pulmonary metastasis, emphasizing their critical role in dissemination from primary site and in extravasation. However, ZEB1+2 depletion one-week after intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT-TFs and establish ZEB1 as a key master regulator of EMT and metastasis in breast cancer. IMPLICATIONS: The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors, whereas ZEB1 integrates the EMT master regulatory network and plays the major role in promoting EMT and metastasis.