Metastasis is a multistep process during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs. In a search for key regulators of metastasis in a murine breast tumor model, we have found that the transcription factor Twist, a master regulator of embryonic morphogenesis, plays an essential role in metastasis. Suppression of Twist expression in highly metastatic mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist results in loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT). In human breast cancers, high level of Twist expression is correlated with invasive lobular carcinoma, a highly infiltrating tumor type associated with loss of E-cadherin expression. These results establish a mechanistic link between Twist, EMT, and tumor metastasis.

SUMMARY Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem-cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct auto-regulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

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Summary Epithelial-Mesenchymal Transition (EMT) is implicated in converting stationary epithelial tumor cells into motile mesenchymal cells during metastasis. However, the involvement of EMT in metastasis is still controversial due to the lack of a mesenchymal phenotype in human carcinoma metastases. Using a spontaneous squamous cell carcinoma mouse model, we show that activation of the EMT-inducing transcription factor Twist1 is sufficient to promote carcinoma cells to undergo EMT and disseminate into blood circulation. Importantly, in distant sites, turning off Twist1 to allow reversion of EMT is essential for disseminated tumor cells to proliferate and form metastases. Our study demonstrates in vivo the requirement of “reversible EMT” in tumor metastasis and may resolve the controversy on the importance of EMT in carcinoma metastasis.

Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.