Characterization of Novel Canonical WNT Signaling Inhibitors in Prostate and Colorectal Cancers

Abstract

Objectives: The objectives of this study were to (1) characterize the levels of WNT signaling in DLD-1 colorectal cancer cells with mutant APC and DU145 prostate cancer cells with wild type APC, (2) to conduct an investigation of novel tankyrase inhibitors in both colorectal and pancreatic cancer cells. WNT signaling has been shown to play an integral role in the proliferation and metastasis of both cancer types; therefore, it is of interest to evaluate inhibitors of the signaling pathway to assess their potential applications in targeted cancer therapeutics. Methods: To characterize WNT signaling activity in colorectal cancer cells (1), DLD-1 cells transduced with a TCF/LEF luciferase reporter and selected with puromycin were grown out in both a two dimensional culture and as tumorspheres to comparatively examine signaling between the different morphologies, as well as at different plating densities. To probe the mechanism of the tankyrase inhibitors (2), DLD-1 and DU145 pancreatic cancer cells were grown to confluency, treated with 1 uM of each inhibitor, and harvested for lysate preparation after 24 hours. Western blots of β-catenin, AXIN1, and AXIN2 were performed and quantified for comparative analysis. Additional assays were performed using DLD-1 cells to assess effects of the inhibitors on proliferation and migration. Results: In DLD-1 cells, it was observed that all tumorsphere cultures exhibited significantly decreased levels of WNT signaling. In DU145 cells, inhibitor D was demonstrated (western blot) to knock down β- catenin levels, as well as stabilize both AXIN1 and AXIN2 levels. Inhibitor A3 was able to decrease β- catenin levels, as well as stabilize AXIN2 levels. Surprisingly, inhibitor A1 was found (via densitometry) to increase both beta-catenin levels and AXIN2 levels. A2 and IWR were found to only decrease β- catenin protein amounts. In DLD-1 cells, all inhibitors exhibited some decrease in beta-catenin levels; however, only A1, A3, A5, and A6 were found to significantly decrease proliferation (MTT assay), and only A1, A4, and A7 were found to significantly stunt migration (scratch assay). Conclusions: In DU145 cells, tankyrase inhibitors D and A3 display the most promise by being able to modulate both catenin and AXIN protein levels. In DLD-1 cells, growth and proliferation assays demonstrate that some inhibitors result in differential phenotypic effects, suggesting that different gene targets downstream of β-catenin are being effected. Further blots for such targets will prove useful to clarify these results. In addition, similar phenotypic characterizations in DU145 cells are necessary to connect the tankyrase mechanisms to observable effects.