6A). NEDD9 knockdown decreased FoxC1-enhanced cell invasion (Fig. 6B). In vivo metastatic assays confirmed

that 5 mice developed lung metastases in the control group (SMMC7721-FoxC1 plus LV-shcontrol). However, there were only two cases of lung metastasis in the NEDD9-knockdown group (SMMC7721-FoxC1 plus LV-shNEDD9) (Fig. 6C1,C2,C5). The number of metastatic lung nodules was significantly reduced in the NEDD9-knockdown group, compared to the control group (Fig. 6C3). Moreover, the NEDD9-knockdown group had a longer OS time than the control group (Fig. 6C4). IHC assays showed that FoxC1 expression was positively correlated with NEDD9 expression in human HCC tissues Sirolimus (Fig. 6D1,D2). Kaplan-Meier’s analysis showed statistically distinct recurrence and survival patterns among the four subgroups, among which patients with positive coexpression of FoxC1 and NEDD9 had the highest recurrence and lowest OS (Fig. 6E2). Furthermore, NEDD9 expression was higher in metastatic tissues than in primary HCC tissues (Supporting Fig. 2). These results suggested that FoxC1 promoted HCC metastasis by up-regulating NEDD9 expression. ß-catenin has been implicated in promoting HCC progression in several studies.27, 28 In this study, we found that FoxC1

overexpression decreased expression JQ1 in vitro of ß-catenin. To determine whether FoxC1 regulated ß-catenin transcription, a ß-catenin promoter luciferase construct (pGL3-CTNNB1) was cotransfected with pCMV-FoxC1. The luciferase reporter assay showed that FoxC1 had no effect on ß-catenin transcription (Supporting Fig. 6A). These data suggest that FoxC1 did not regulate the ß-catenin promoter in HCC cells. Recent studies reported that MCE公司 the expression level of ß-catenin could be regulated by multiple microRNAs (miRNAs).29, 30 We speculate that FoxC1 may decrease ß-catenin expression through regulating miRNA expression. Expression levels of ß-catenin were also measured in 406 HCC tissues.

Increased ß-catenin accumulation was detected in 220 of 406 (54.2%) HCC tissues, compared to adjacent nontumor tissues. Nuclear ß-catenin staining was detected in 41 cases (41 of 220; 18.6%), with the remaining cases showing staining in the cytoplasm (Supporting Fig. 6B). These results were consistent with those of previous studies.27, 31 However, these data were inconsistent with our findings in HCC cell lines. These differences may be attributed to the existence of other mechanisms that regulate ß-catenin expression (e.g., the Wnt pathway). In the absence of Wnt signaling, ß-catenin is bound to E-cadherin at adherens junctions. N-terminally phosphorylated ß-catenin is targeted for ubiquitination and subsequent proteasomal degradation.32 Deregulation of E-cadherin by FoxC1 may decrease the level of ß-catenin in the membrane and increase ubiquitination of ß-catenin.