Western blot analyses for read me MAP kinases revealed that the phosphorylation level of JNK was decreased whereas expression and phosphorylation levels of other kinases were not (Figure 2C). As expected from this result and the fact that c-jun is a Wnt target gene (Mann et al, 1999), expression and phosphorylation of c-Jun were also reduced (Figure 2C). A recent finding that RhoA stimulates c-Jun expression through ROCK, which in turn activates JNK (Marinissen et al, 2004), prompted us to examine RhoA activation. As shown in Figure 2D, it was clearly decreased with FZD7_siRNA transfection, suggesting that FZD7 may be a receptor for the non-canonical Wnt/JNK signalling pathway in colon cancer cells.

These data may provide a molecular explanation for the previous findings that inhibition of FZD7 expression decreased the migratory activity of colon cancer cells (Vincan et al, 2007) and hapatocellular carcinoma cells (Merle et al, 2004). We have also demonstrated decreased invasion activity of FZD7-down-regulated HCT-116 cells (Figure 2E). In addition to migratory activity, proteolytic lysis of Matrigel is required for cells to penetrate the membrane in the invasion assay we used. The canonical Wnt pathway may be responsible for this process, because it involves several extracellular proteinases such as urokinase-type plasminogen activator (uPA) (Hiendlmeyer et al, 2004), uPA receptor (Mann et al, 1999), CD44 (Wielenga et al, 1999), matrix metalloproteinase (MMP)-7 (Brabletz et al, 1999) and MT1-MMP/MMP-14 (Takahashi et al, 2002).

It is also known that the ��-catenin/Tcf target gene fra-1 directly induces MMP-1 and MMP-9 promoter activity (Belguise et al, 2005). Our quantitative RT-PCR data showed that the expression levels of CD44v8-9 and MT1-MMP were decreased after FZD7_siRNA transfection into HCT-116 cells (Figure 2F). It remains to be determined how non-canonical Wnt signalling interacts with the canonical Wnt/��-catenin/Tcf signalling in colon cancer cells. It was shown that inhibitors of the canonical signalling, Dickkopf (DKK)-1 and a dominant-negative Tcf construct, did not reduce Wnt3a-dependent motility of CHO-K1 cells (Endo et al, 2005), and that DKK-1 and DKK-2 had no effect on Wnt3a-induced migration of myeloma cells (Weeraratna et al, 2002). In the former cell model system, RhoA was also activated with Wnt3a, whereas the activation of PKC family proteins including PKC��, PKC�� and PKC�� as well as RhoA was found in the latter.

These findings suggest that cell migration stimulated with Wnt may be independent AV-951 of canonical signalling. We have shown that stable transfectants of HCT-116 cells harbouring the FZD7_siRNA have decreased Tcf activity, viability and invasion (Figures 3B�CD), and less in vivo metastatic activity using a liver metastasis model of HCT-116 cells in nude mice (Bouvet et al, 2006).