5358

Epithelial-to-mesenchymal transition (EMT), important for cell movement during morphogenesis, is reactivated during malignant progression to enable invasion and metastasis. Transforming growth factor-β (TGF-β) induces EMT and stimulates cell migration in a variety of cancer cell types, including mammary carcinomas. Whereas the Snail zinc-finger transcription factors directly repress genes, such as E-cadherin, down-regulated during EMT, the interaction between Snail1/2 and TGF-β is unclear. We show that TGF-β (200 pM) induces Snail1 transcription within 30 minutes by 9-fold and 5.5-fold in the mouse mammary epithelial cell lines, NMuMG and Ras-transformed EpH4 (EpRas), respectively. Snail1 induction is sustained above basal levels for up to 48 hours concomitant with phenotypic conversion of the epithelial cells to a notable mesenchymal/fibroblastic phenotype including a time-dependent consistent decrease in E-cadherin and the tight junction protein, ZO-1, and an increase in N-cadherin and vimentin, as observed by immunofluorescence. By contrast, TGF-β decreases transcription of the homologue member Snail2 by 0.75-fold by one hour with incremental increases over time that remained slightly below basal levels by 48 hours. TGF-β signal transduction directly activates Smad transcription factors. Smad binding elements (SBE) were detected in the Snail1 promoter region suggesting a direct role for TGF-β signaling via Smads in Snail1 induction. Accordingly, TGF-β did not affect Snail1 or Snail2 expression in the Smad4-defective human breast carcinoma cells MDA-MB-468, and inhibitors of the p38 MAPK, PI3K and MEK1/2 pathways partially blocked TGF-β-regulated transcription of both genes. Therefore, TGF-β induces EMT via Smad4 signaling and cross-talk with additional intracellular pathways. Finally, stable over-expression of Snail1 and Snail2 in NMuMG cells mimicked the TGF-β-induced effects consistent with EMT including cell invasion through matrigel coated transwell chambers, down-regulation of ZO-1 and E-cadherin and, up-regulation of vimentin, fibronectin and N-cadherin. However, knock-down of gene transcription with Snail1 siRNA, but not Snail2 siRNA, blocked these TGF-β-induced effects. Therefore, whereas both Snail1 and Snail2 induce EMT and related alterations in markers of EMT, only Snail1 appears to mediate TGF-β induction of the mesenchymal phenotype consistent with its role in metastasis. Further, since TGF-β is present in high concentrations in many cancers and, as blocking Snail1 expression is sufficient to protect NMuMG cells from undergoing EMT induced by TGF-β/Smad signaling, Snail1 may be a rational target to prevent [TGF-β-induced] metastatic disease.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA