Lipocalin-2 Negatively Modulates the Epithelial-to-Mesenchymal Transition in Hepatocellular Carcinoma Through the Epidermal Growth Factor (TGF-beta1)/Lcn2/Twist1 Pathway

Lipocalin-2 (Lcn2) is preferentially expressed in hepatocellular carcinoma (HCC). However, the functional role of Lcn2 in HCC progression is still poorly understood, particularly with respect to its involvement in invasion and metastasis. The purpose of this study was to investigate whether Lcn2 is associated with the epithelial-mesenchymal transition (EMT) in HCC and to elucidate the underlying signaling pathway(s). Lcn2 was preferentially expressed in well-differentiated HCC versus liver cirrhosis tissues, and its expression was positively correlated with the stage of HCC. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH-J1 cells, including the down-regulation of N-cadherin, vimentin, alpha-smooth muscle actin, and fibronectin, and the concomitant up-regulation of CK8, CK18, and desmoplakin I/II. Knockdown of Lcn2 by short hairpin RNA (shRNA) in HKK-2 cells expressing high levels of Lcn2 was associated with EMT. Epidermal growth factor (EGF) or transforming growth factor beta1 (TGF-1) treatment resulted in down-regulation of Lcn2, accompanied by an increase in Twist1 expression and EMT in HCC cells. Stable Lcn2 expression in SH-J1 cells reduced Twist1 expression, inhibited cell proliferation and invasion in vitro, and suppressed tumor growth and metastasis in a mouse model. Furthermore, EGF or TGF-1 treatment barely changed EMT marker expression in SH-J1 cells ectopically expressing Lcn2. Ectopic expression of Twist1 induced EMT marker expression even in cells expressing Lcn2, indicating that Lcn2 functions downstream of growth factors and upstream of Twist1. Conclusion: Together, our findings indicate that Lcn2 can negatively modulate the EMT in HCC cells through an EGF (or TGF-1)/Lcn2/Twist1 pathway. Thus, Lcn2 may be a candidate metastasis suppressor and a potential therapeutic target in HCC. (Hepatology 2013;58:1349-1361)