4C; Pearson’s r = 0.6190; P = 0.0006). Because loss of E-cadherin expression and increased invasiveness are hallmarks of EMT, we further analyzed the expression of mesenchymal markers, ACP-196 price such as vimentin, and the transcription factors, Snail1, Slug, zinc finger E-box binding homeobox 1 (ZEB1), and Smad-interacting protein 1 (SIP1)/ZEB2, which are described as transcriptional repressors of E-cadherin.25 qRT-PCR analysis revealed that Rnd3 silencing induced the mRNA expression of ZEB2, but not of ZEB1 or other EMT markers (Fig. 5A;

Supporting Fig. 4). Because ZEB1/2 expression is under the control of the miR-200 family that targets their 3′ untranslated regions (UTRs),26 we monitored miR-200b and miR-200c expression in Rnd3-silenced Hep3B cells. The expression of both miRNAs was significantly decreased upon Rnd3 silencing (Fig. 5B). Moreover, forced overexpression of miR-200b and/or miR-200c in hepatoma cells down-regulated ZEB1 and ZEB2 expression, leading to Palbociclib in vivo E-cadherin up-regulation and increased cell-cell

contacts (Supporting Fig. 5). Thus, Rnd3 knockdown induced a decrease in expression of the guardians of the epithelial phenotype, miR-200, and an increase in that of the EMT promoter, ZEB2, leading to E-cadherin repression. In a three-dimensional (3D) environment, individual cancer cells use a broad spectrum of migration and invasion mechanisms, which are dictated by the extracellular matrix (ECM) together with specific cell determinants. These include amoeboid and mesenchymal modes of movement, which are distinguished by their different usage of Rho GTPase-signaling pathways and distinct requirements selleck compound for extracellular proteolysis.27 Amoeboid cells show high levels of actomyosin contractility involving signaling through RhoA/ROCK,

and their movement is associated with deformation of the cell body through the ECM without proteolysis. In the mesenchymal-type movement, cells have an elongated morphology with Rac/Cdc42-induced protrusions at the leading edge, and this movement requires ECM proteolysis. We first attempted to discriminate between the two modes of invasion through the inhibition of matrix metalloproteinases (MMPs), whose activity is only required for the mesenchymal movement. The broad-spectrum MMP inhibitor, GM6001, did not decrease the invasion induced by Rnd3 depletion, suggesting that Rnd3-silenced cells invade the ECM without degrading it (Fig. 6A; Supporting Fig. 6A). Second, we analyzed the morphology of cells invading a thick type I collagen matrix.22 Although both control and Rnd3-silenced cells showed a rounded morphology, Rnd3-silenced cells were observed as isolated cells in the matrix and developed long actin-based protrusions, such as pseudopodia (Fig. 6B; Supporting Fig. 6B,C).