Molecular and cellular regulation of mammary epithelial dissemination

Dissemination of epithelial cancer cells represents the first step in metastatic spread. One postulated mechanism for dissemination is activation of an epithelial to mesenchymal transition (EMT), in which repression of the cell adhesion gene E-cadherin (E-cad) is considered the driving molecular event. We sought to test the sufficiency of single gene perturbations to induce dissemination out of primary mouse mammary epithelium. Deletion of E-cad disrupted simple architecture and morphogenesis but, contrary to expectation, rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cytokeratin+ epithelial cells. A core concept in EMT is that cells lose epithelial characteristics, such as tight cell-cell adhesion, and acquire mesenchymal characteristics to escape. However, we found that Twist1 did not significantly regulate epithelial-specific genes, such as E-cad. Rather, Twist1 induced dramatic transcriptional changes in extracellular compartment and cell-matrix adhesion genes. Surprisingly, Twist1+ cells disseminated with membrane-localized E-cad, and E-cad knockdown strongly inhibited Twist1-induced single cell dissemination. Dissemination can therefore occur through an innately epithelial migratory program. The integrity of the myoepithelium, the outer mammary epithelial layer, is the major diagnostic criterion used to distinguish in situ from invasive breast tumors. Interestingly, we found that constitutive Twist1 expression induced dissemination of both inner luminal and outer myoepithelial cells and abnormal myoepithelial ingression, resulting in gaps in myoepithelial coverage at the basal surface. We next developed mouse models to restrict Twist1 to distinct mammary lineages. Twist1 expression in the myoepithelial compartment induced cell autonomous myoepithelial dissemination. In contrast, Twist1 expression in the luminal compartment rarely resulted in dissemination. Using cell type-specific fluorescent reporters, we observed that normal myoepithelial cells appear to contain Twist1+ luminal cells protruding into the extracellular matrix. Myoepithelial cells display a similar response to invasive E-cad– luminal cell behavior. Taken together, our data supports the hypothesis that normal myoepithelial cells dynamically block luminal cell escape. We now seek to test the molecular basis of myoepithelial barrier function through knockdown of myoepithelial-specific genes important for contractility, cell-cell adhesion, and the intermediate filament network. We expect that perturbation of myoepithelial function will enable E-cad– and Twist1+ luminal cell dissemination