Conversely, silencing endogenous FoxC1 expression markedly reduced cell migration and invasion in HCCLM3 cells (high initial metastatic potential) Selleckchem VX-809 (Fig. 1D). To further explore the role of FoxC1 in tumor metastasis in vivo, cells were transplanted into livers of nude mice. Representative bioluminescent imaging (BLI) of the different groups is shown in Fig. 1E1. Histological
analysis (Fig. 1E5) further confirmed that the incidence of lung metastasis in the SMMC7721-FoxC1 group was significantly increased, compared to that in the control group (60% versus 10%). In the HCCLM3-shcontrol group, all of the mice developed lung metastases; however, only 5 mice in the HCCLM3-shFoxC1 group developed lung metastases (100% versus 50%; Fig. 1E1,E2). The number of lung metastatic nodules in the SMMC7721-FoxC1 group was
increased, compared to that in the SMMC7721-control group; however, the number of lung metastatic nodules in the HCCLM3-shFoxC1 group was significantly reduced, compared to that in the HCCLM3-shcontrol group (Fig. 1E3). Furthermore, the SMMC7721-FoxC1 group had a shorter OS time than the SMMC7721-control group, whereas the HCCLM3-shFoxC1 Smad inhibitor group had a longer OS time than the HCCLM3-shcontrol group (Fig. 1E4). These data suggested that FoxC1 promoted HCC invasion and metastasis. EMT plays a critical role in metastasis. Specifically, EMT induces tumor-associated epithelial Tau-protein kinase cells to obtain mesenchymal features, which results in reduced cell-cell
contact and increased motility.22 Up-regulation of FoxC1 in SMMC7721 cells resulted in the decreased expression of epithelial markers (E-cadherin and ß-catenin) and increased expression of mesenchymal markers (vimentin and fibronectin), as evidenced by immunofluorescence (IF), western blotting analysis, and real-time PCR. After FoxC1 knockdown in HCCLM3 cells, expression of epithelial markers was significantly increased and expression of mesenchymal markers was markedly decreased (Fig. 2A-C). These findings suggested that FoxC1 induced EMT in HCC cells. Functional loss of E-cadherin is considered a hallmark of EMT.23 A major mechanism of E-cadherin down-regulation is its direct transcriptional repression by repressors, including Snai1, Twist, Slug, Zeb1, and SIP1.24 We determined whether FoxC1 inhibited E-cadherin expression by regulating the expression of these repressors. Real-time PCR analysis showed that FoxC1 markedly increased Snai1 expression, but had no significant effect on mRNA levels of Twist, Slug, Zeb1, or SIP1 (Fig. 3A1). Furthermore, FoxC1 up-regulated Snai1 expression and decreased E-cadherin expression in SMMC7721 cells, whereas the inhibition of Snai1 expression using the lentivirus, LV-shSnai1, significantly attenuated the loss of E-cadherin expression induced by FoxC1.