Abstract 601: In vitro and in vivo inhibition of β-catenin by two novel β-catenin RNA antagonists, EZN-3889 and 3892

Background: β-Catenin is a subunit of the cadherin protein complex and a key integral component in the Wnt signaling pathway. Therefore, it not only plays an important role in cell-cell adhesion but also in tumorgenesis. β-catenin is activated in most colon cancers due to either mutations in APC or activating mutations in β-catenin itself. Furthermore, activating β-catenin mutations have been found in a variety of other tumors such as melanomas, hepatocellular carcinomas, breast, and prostate cancer, whereas β-catenin is not activated in most normal tissues. Therefore, inhibition of β-catenin is likely to have therapeutic effects in many cancers. Unfortunately, because β-catenin is devoid of any enzymatic activity, the development of inhibitors of β-catenin has been slow. We present here both in vitro and in vivo evidence that the lack of specificity of small-molecule inhibitors may be overcome by two novel β-catenin LNA-based mRNA antagonists, EZN-3889 and EZN-3892. Material and Methods: In vitro, the ability of the compounds to inhibit mRNA, cell growth, and reporter gene were evaluated by qRT-PCR, MTS, and luciferase assays respectively, in multiple cell lines. In vivo, β-catenin mRNA down-modulation in liver and human tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the compounds. Results: Without lipofection, we found that these two antagonists could specifically down modulate β-catenin mRNA and protein effectively in multiple cell lines with a long-lasting effect. Additionally, these two molecules effectively inhibited β-catenin transcriptional activity in SW480 colorectal and H1581 lung cancer cells stably expressing a TCF/LEF luciferase reporter gene, resulting in growth inhibition. Combination of EZN-3889 or 3892 with sulindac demonstrated improved activities in both growth and reporter assay. Moreover, EZN-3889 and 3892 inhibited the spontaneous growth of spheroids in SW480 culture and convert SW480 spheroids to monolayers. Preliminary data suggest that these spheroids were more tumorgenic than the monolayer cells. In mice, both molecules significantly down modulated β-catenin mRNA in the liver. Conclusions: Without lipofection, β-catenin antagonists potently and specifically inhibited β-catenin mRNA expression both in vitro and in vivo after intravenous injection. Target inhibition correlated with growth inhibition and phenotypical change of SW480 spheroids. Further studies will examine the si