Interestingly, FoxC1 is reported

to induce EMT. FoxC1 induces EMT through the inhibition of E-cadherin expression in mammary epithelial cells and promotes their migration and invasion. Additionally, FoxC1 overexpression is strongly correlated with poor survival in breast cancer patients.18, 19 Several recent studies also reported that FoxC1 increases the migration and invasion of breast cancer cells, and that FoxC1 overexpression predicts poor overall survival (OS) in patients with FG-4592 price breast cancer.20, 21 These studies indicate that FoxC1 might promote tumor metastasis and malignant progression by inducing EMT. To date, no studies have reported on the clinicopathologic significance of FoxC1 in HCC. In this study, we present the first evidence that FoxC1 promotes HCC invasion and metastasis by not only inducing selleck kinase inhibitor EMT, but also by up-regulating NEDD9 expression. FoxC1 overexpression predicts poor prognosis in HCC patients after curative resection. The molecular mechanism of these effects involves

the transactivation of Snai1 and NEDD9 expression by FoxC1 through direct binding to their promoters. BLI, bioluminescent imaging; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; EGF, epidermal growth factor; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-related kinase; FoxC1, forkhead box C1; HBV, hepatitis B virus; HBx, hepatitis B virus x; HCC, hepatocellular carcinoma; IF, immunofluorescence; IHC, immunohistochemical; miRNAs, microRNAs; mRNA, messenger selleck chemicals RNA; NEDD9, neural precursor cell expressed, developmentally down-regulated 9; OS, overall survival; PCR, polymerase chain reaction; siRNA, short interfering RNA; TNM, tumor-node-metastasis; VEGF, vascular endothelial growth factor. Plasmids were constructed according to the standard procedures in our previous study.12 All of the primers are shown in Supporting Table 2. The Snai1 promoter construct (−1511/+140)snail was generated from human genomic DNA corresponding to the sequence from −1511 to +140 (relative to the transcriptional start site) of the 5′-flanking region of the human

Snai1 gene. This construct was generated with the forward and reverse primers incorporating KpnI and HindIII sites at the 5′- and 3′-ends, respectively. The polymerase chain reaction (PCR) product was cloned into the KpnI and HindIII sites of the pGL3-Basic vector (Promega, Madison, WI). The 5′-flanking deletion constructs of the FoxC1 promoter ([−922/+140]Snail, [−694/+140]Snail, and [−354/+140]Snail) were similarly generated with the (−1511/+140)Snail construct as a template. Other promoter constructs ([−2056/+121]NEDD9, [−1762/+121]NEDD9, [−1324/+121]NEDD9, [−1007/+121]NEDD9, [−478/+121]NEDD9, and pGL3-E-cadherin) were cloned in the same manner. FoxC1-binding sites in the Snai1 and NEDD9 promoters were mutated with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA).