Figure 6.

β-catenin–LEF-1 is sufficient to promote EMT without upstream TGF-β or Snail/Slug signaling. (A) Due to the APC mutation in DLD1 cells, exposure to an adenovirus containing an LEF-1 expression plasmid was sufficient to induce EMT via β-catenin–LEF-1 complexes alone, as show by immunocytochemistry. (B) LEF-1 transcriptional activity was confirmed with the pTOPFLASH-Lux reporter plasmid. Data represent mean±SD; n=3; *p<0.01 compared with control. (C) Immunoblotting showing that Snail protein expression was greatly reduced in the presence of Snail siRNA. Exogenous LEF-1 had no effect on Snail expression with or without the presence of Snail siRNA. (D) Similar results were found for Slug expression in the presence of Slug siRNA. (E) EMT was observed by assessing protein expression of E-cadherin and vimentin. Exposure to adenoviral LEF-1 caused complete inhibition E-cadherin expression, while increasing levels of vimentin and fibronectin. Presence of Snail siRNA, Slug siRNA, AS TCF-4, or a neutralizing antibody against TGF-β1, TGF-β2, and TGF-β3 (TGF-β1, 2, 3 Ab) had no effect, but AS β-catenin prevented LEF-1-induced EMT. (F) Similar results were observed for LEF-1-induced cell invasion/migration.