Thiazolidinediones (TZDs) downregulate Wnt/   β   -catenin signaling via multiple mechanisms in breast cancer cells Free

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Thiazolidinediones (TZDs), a class of medication used clinically to treat diabetes mellitus, have been demonstrated in vitro and in vivo to possess anti-tumor effects. Though the mechanisms are not well established, effects on Wnt/β-catenin signaling, a key developmental pathway that is dysregulated in many cancers including breast cancer, may be important. Here we show that TZDs downregulate Wnt/β-catenin signaling and decrease proliferation of breast cancer cells. Microarray analysis showed that 100μM rosiglitazone (RG) downregulates mRNA expression of the Wnt co-receptors Frizzled-1 (FZD1) and low-density lipoprotein-related protein 6 (LRP6) as early as 4 h after treatment in MDA-MB-231 cells. These results were confirmed using troglitazone (TG) treatment in MDA-MB-231 and MCF7 cells where FZD1 mRNA levels decreased 4 h after TG treatment in both cell lines; however, LRP6 mRNA and protein levels were only downregulated in MDA-MB-231 cells, but not in MCF7 cells. We then investigated the effects of TG on phosphorylation, transactivation activity, and localization of β-catenin, as well as on cell proliferation. TG downregulated p-β-catenin (S33/S37/T41) but upregulated p-β-catenin (S45) and p-GSK3β (S9). In contrast, we found that p-GSK3β (S9) was decreased instead of increased in response to TG treatment plus Akti-1/2, a specific inhibitor of Akt1 and Akt2. Also, p-β-catenin (S33/S37/T41) increased in response to TG treatment in the presence of Akti-1/2, indicating that the decrease of p-β-catenin (S33/S37/T41) was GSK3β-dependent. Moreover, using okadaic acid to inhibit PP2A activity, p-β-catenin (S33/S37/T41) still decreased in response to TG treatment, indicating that the decrease of p-β-catenin (S33/S37/T41) was PP2A-independent. Furthermore, TG upregulated nuclear β-catenin localization, but downregulated TCF4 protein expression, thereby decreasing β-catenin/TCF4 complex formation. Finally, we found that Akti-1/2 augmented TG-mediated downregulation of the protein levels of LRP6, β-catenin, and TCF4. These results suggest that TZD-mediated anti-tumor effects may be mediated by downregulation of the Wnt/β-catenin signaling pathway and the combination of TZDs and a specific Akt inhibitor may serve as a new option for treatment.