Phosphatidylinositol 3-kinase function is required for transforming growthfactor beta-mediated epithelial to mesenchymal transition and cell migration

We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt sig naling in transforming growth factor beta (TGF beta)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGF beta1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinas e activity against GrSK-3 beta within 30 min. These responses were temporal ly correlated with delocalization of E-cadherin, ZO-1, and integrin beta (1 ) from cell junctions and the acquisition of spindle cell morphology. LY294 002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-neg ative mutant of Akt blocked the delocalization of ZO-1 induced by TGF beta1 , whereas transfection of constitutively active p110 induced loss of ZO-1 f rom tight junctions. In addition, LY294002 blocked TGF beta -mediated C-ter minal phosphorylation of Smad2. Consistent with these data, TGF beta -induc ed p3TP-Lux and p(CAGA)(12). Lux reporter activities were inhibited by LY29 4002 and transiently expressed dominant-negative p85 and Akt mutants in NMu MG and 4T1 cells. Dominant-negative RhoA inhibited TGF beta -induced phosph orylation of Akt at Ser-473, whereas constitutively active RhoA increased t he basal phosphorylation of Akt, suggesting that RhoA in involved in TGF be ta -induced EMT. Finally, LY294002 and neutralizing TGF beta1 antibodies in hibited ligand-independent constitutively active Akt as well as basal and T GF beta -stimulated migration in 4T1 and EMT6 breast tumor cells. Taken tog ether, these data suggest that P13K-Akt signaling is required for TGF beta -induced transcriptional responses, EMT, and cell migration.