Another interesting approach targeting active Wnt/P-catenin signaling is the use of recombinant viruses, which either carry a "death-inducing gene" under the control of a Tcf-dependent promoter or the replication of which is dependent on active P-catenin/Tcf signaling. A recombinant adenovirus containing the apoptosis inducing gene FADD (Fas-associated death domain-containing protein) under the control of a Tcf-dependent promoter has been generated (180). Colorectal cancer cells with activated P-catenin/Tcf signaling were selectively and efficiently killed by the virus, supporting the idea that aberrantly activated P-catenin can be used to selectively target colon cancer cells. In order to maximize the tumor-killing effect in colorectal cancer cells with deregulated Wnt signaling a recombinant adenovirus was generated which carries a herpes simplex virus thymidine kinase gene (HSV-TK) under the control of a Tcf-responsive promoter (181). Treatment of nude mice xenografted with human DLD-1 colon cancer cells with the recombinant adenovirus and ganciclovir significantly suppressed the growth of the tumor cells. Control mice xenografted with a human hepatoma cell line did not respond to this treatment, demonstrating that this approach selectively targets tumor cells with aberrant activation of P-catenin.
Oncolytic viruses selectively lyse malignant cells by cytopathic effects (182). The major advantage of this strategy is the amplification of the virus at the site of the tumor. Brunori and coworkers engineered an oncolytic adenovirus that selectively replicates in cells with aberrantly high P-catenin expression (183). This adenovirus expresses the viral E1B and E2 genes from promoters controlled by b-catenin/Tcf. The Tcf-E1B and Tcf-E2 promoters were found to be active in many cell lines with activated Wnt signaling. Viruses with Tcf-dependent regulation of E2 expression replicated efficiently in SW480 colon cancer cells, but showed a significantly reduced replication in H1299 lung cancer cells and WI38 normal fibroblasts. As a proof of concept, the authors introduced a stable P-catenin mutant into normal WI38 fibroblasts, which rendered these cells permissive for virus replication. Another oncolytic virus with Tcf-binding sites integrated into the promoters of the E1A, E1B, E2, and E4 genes exhibited a strong selectivity for cells with deregulated Wnt signaling (184). This adenovirus preferentially replicated in cells with activated Wnt signaling and resulted in a dramatic increased efficacy in cytopathic assays. Based on a similar idea, Toth and coworkers generated an adenovirus with a synthetic promoter containing five Tcf-consensus binding sites replacing the wild-type E4 promoter (185). The virus preferentially replicated in cells with activated Wnt signaling. In a xenograft model the virus effectively suppressed the growth of SW480 colorectal cancer cells but not of control cells without deregulated Wnt signaling. Instead of using adenoviral vectors Malerba et al. (186) modified the minute parvovirus of mice to contain Tcf-binding sites within the P4 promoter. Replication and cytopathic effects of this recombinant virus were also strongly dependent on P-catenin/Tcf activity.
So far, two screens for small molecular compounds targeting active Wnt/P-catenin signaling in cancer have been published. In search for small molecule inhibitors of Wnt signaling, Lepourcelet and coworkers performed a systematic screen of libraries of natural compounds that specifically disrupt the
P-catenin/Tcf-4 complex (187). Among 7000 compounds screened, 8 showed dose-dependent inhibition of the P-catenin/Tcf-4 complex. Each of the compounds was able to inhibit P-catenin/Tcf reporter gene activity and transcription of known P-catenin/Tcf target genes. Subsequent analyses revealed that three of the compounds abrogated axis duplication in Xenopus embryos, which was induced by injection of P-catenin mRNA. Finally, two compounds of fungal origin were identified as specific inhibitors of the P-catenin/Tcf complex in vitro and in vivo. However, the compounds were also found to inhibit P-catenin binding to APC, which might affect P-catenin phosphorylation by inhibition of its binding to the multiprotein phosphorylation complex. Another screen of 5000 compounds for small-molecule inhibitors of P-catenin-mediated signaling identified a compound, ICG-001, which specifically inhibits the cyclic AMP response element-binding protein, a p300-related coactivator of P-catenin-mediated transcription (188). Treatment of cells with ICG-001 inhibited transcription of a reporter gene and the transcription of P-catenin/Tcf target genes was downregulated. ICG-001 inhibited the proliferation of colorectal cancer cells in vitro and of tumor xenografts in nude mice. These two studies are encouraging as they demonstrate the feasibility of identifying small molecule inhibitors that target protein-protein interactions. However, more studies are needed to exclude toxic side effects and negative effects on tissue homeostasis and regeneration as a compound effective against cancer cells would be expected to also target normal cells that depend on the same pathway, i.e., intestinal epithelium.
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