Lipocalin-2-mediated downregulation of Twist1 reverses the epithelial-mesenchymal transition in hepatocellular carcinoma

Elevated lipocalin-2 (Lcn2) has been observed in multiple human cancers, but its biological roles remain unclear. Our purpose in this study was to investigate whether Lcn2 is involved in hepatocellular carcinoma (HCC) development via its involvement in invasion and metastasis. Using unsupervised hierarchical clustering analysis of tissue samples, we identified preferential expression of Lcn2 (>2-fold change) in the tumor group (differentiated group) versus the non-tumor group (liver cirrhosis group). LCN2 immunoreactivity was positively correlated with TNM stage (r = 0.194, P = 0.020), but not with differentiation or recurrence of HCC. Over-expression of Lcn2 was observed in HLK-2, HKK-2, and HLK-5 HCC cells. Knock-down of Lcn2 by shRNA in HKK-2 cells was correlated with the down-regulation of E-cadherin, CK-8, CK-18, and desmoplakin I/II (DesI/II) and the up-regulation of N-cadherin, vimentin (VIM), and fibronectin (FN), which are markers of the epithelial-mesenchymal transition (EMT) associated with tumor progression. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH-J1 cells. EGF or TGF-β treatment resulted in downregulation of Lcn2 with a concomitant change in EMT. Stable Lcn2 expression in HCC cells reduced the expression of the transcription factor Twist1, resulting in the inhibition of cell proliferation, migration, and invasion in vitro, and suppressed tumor growth and metastatic ability in a mouse model. These findings suggest that Lcn2 can reverse the EMT in HCC through transcriptional suppression of Twist 1. Thus, Lcn2 is a candidate metastasis suppressor and a potential therapeutic target for HCC. Unsupervised hierarchical clustering separated the samples into two main groups: a non-tumor group (NT, n = 40) and a HCC group (HCC, n = 40), and into two subgroups: a liver cirrhosis group (LC, n = 20) and a differentiated HCC group (difHCC, n = 20). Lcn2 was one of the unique genes with a two-fold or greater difference in expression from the mean with P < 0.01 based on the t-test for hierarchical clustering analysis.