Description: IntroductionEpithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods: MYB, ZEB1 and CDH1 expression levels were manipulated by lentiviral small hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified by Western blotting, immunocytochemistry and qRT-PCR. Proliferation was assessed by bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed by qRT-PCR, cell morphology and colony morphology. Protein expression in human breast cancers was assessed by immunohistochemistry. ZEB1-MYB promoter binding and repression were determined by Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student's paired T tests, Mann--Whitney and repeated measures 2-way ANOVA tests determined statistical significance (P 〈 0.05). Results: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relationship between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphological changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB over-expressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 over-expression repressed MYB promoter activity. Conclusions: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relationship, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.