Cancer metastasis is strongly correlated with epithelial-mesenchymal transition (EMT), in which transforming growth factor-beta (TGF-beta) signaling plays a central role. CD44 has emerged as a cancer stem cell (CSC) marker that strongly induces EMT together with TGF-beta 1. This study aimed to investigate the link between high CD44 and TGF-beta 1 levels during EMT in HCC cell lines. FACS analysis showed high expression of CD44 in TGF-beta 1-positive SNU-368 cells and TGF-beta 1-negative SNU-354 cells. SNU-368 CD44(+) cells showed EMT through up-regulation of the AKT/GSK-30/13-catenin pathway. By comparison, SNU-354 CD44(+) cells showed only increased N-cadherin expression, which was not accompanied by a decrease in E-cadherin expression, and also down-regulated the AKT/GSK-3 beta/beta-catenin pathway. However, TGF-beta 1-stimulated SNU-354 cells (CD44/TGF-beta 1(+)) exhibited lower E-cadherin and higher N-cadherin expression with increased AKT/GSK-3 beta/beta-catenin pathway activity. CD44/TGF-beta 1(+) SNU-354 cells also showed enhanced migration and formed larger spheres, while the TGF-beta 1-induced stem cell properties returned to their original state with the TGF-beta 1 inhibitor SB431542. SB431542-treated SNU368 (CD44/TGF-beta 1 cells also showed diminished N-cadherin and AKT/GSK-3 beta/beta-catenin pathway activity and further decreased cell motility in a wound healing assay. However, CD44 knockdown in SNU354 cells did not induce EMT even after treatment with TGF-beta 1. Finally, double inhibition of both CD44 and TGF-beta 1 further decreased migration and sphere formation more strongly than a single inhibition in SNU-368 cells. In conclusion, the current study demonstrated the synergistic interactions between CD44 and TGF-beta 1 in EMT induction and CSC properties through the AKT/GSK-3 beta/beta-catenin pathway in HCC cells. (C) 2016 Elsevier Inc. All rights reserved.