The Snail signaling branch downstream of the TGF-β/Smad3 pathway mediates Rho activation and subsequent stress fiber formation

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-(3 (TGF-(3). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fi bers. Here, we examined the TGF-(3 signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fi ber formation, and enhanced motility in cells stimulated with TGF-(3. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fi ber formation by TGF-(3, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-(3-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fi ber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-(3, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail- independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-(3.