NANOG promotes liver cancer cell invasion by inducing epithelial–mesenchymal transition through NODAL/SMAD3 signaling pathway

• High level of Nanog expression is correlated with metastasis and low survival rate in HCC patients.
• Nanog is a novel signaling mediator of HCC metastasis and epithelial–mesenchymal transition.
• Nanog activates Nodal to promote Smad3 phosphorylation and Snail expression.
• The combination of Nanog and P-Smad3 is a potential predictor of poor prognosis of HCC.

NANOG is a major transcription factor essential to the stem cell self-renewal and is associated with tumor malignancy, but the NANOG signaling in cancer metastasis is still elusive. In this study, we determined the expression of NANOG in hepatocellular carcinoma (HCC) and investigated its underlying mechanism in the metastasis of HCC. The expression levels of NANOG were examined in tumor tissues by immunohistochemistry. Functional effect of NANOG was investigated both in vivo and in vitro. Our data shows that high level of NANOG expression correlates with metastasis and low survival rate in HCC. HCC cells overexpressing NANOG are characterized by active epithelial–mesenchymal transition (EMT), and exhibit increased ability of invasion, soft agar colonization, sphere formation and drug resistance, whereas SB-431542, an antagonist of activin receptor-like kinase (ALK) receptors, attenuates EMT and invasion of HCC cells. NANOG activates NODAL and CRIPTO-1 to promote SMAD3 phosphorylation and SNAIL expression. The transcriptional activity of NODAL gene is dependent on two NANOG binding motifs in its promoter region. This study shows a significant correlation between the NANOG expression and the expression of NODAL, P-SMAD3 or SNAIL, and the combination of NANOG and P-SMAD3 is a potential predictor of poor prognosis of HCC. Additionally, cells in the tumor edge area displays higher NANOG expression than cells in the tumor center. These results present novel mechanistic insight into an important role of NANOG in HCC metastasis, and suggest a potential application of NANOG in HCC prognosis and treatment.