Phosphatidylinositol 3-Kinase Function Is Required for Transforming Growth Factor β-mediated Epithelial to Mesenchymal Transition and Cell Migration

TLDR

The study investigates the role of PI3K‑Akt signaling in TGFβ‑mediated epithelial‑to‑mesenchymal transition. TGFβ1 rapidly activates PI3K‑Akt signaling, which is required for junction protein delocalization, transcriptional activation, EMT morphology, and cell migration, as PI3K or Akt inhibition blocks these events and RhoA modulates Akt phosphorylation.

Abstract

We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor β (TGFβ)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGFβ1 induced phosphorylation of Akt at Ser-473 and Akt <i>in vitro</i> kinase activity against GSK-3β within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin β₁ from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGFβ1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGFβ-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGFβ-induced p3TP-Lux and p(CAGA)₁₂-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGFβ-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGFβ-induced EMT. Finally, LY294002 and neutralizing TGFβ1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGFβ-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGFβ-induced transcriptional responses, EMT, and cell migration.