Figure 5

Effects of dnhWnt-2 expression in NSCLC cell lines. (A) Colony formation was determined in NSCLC cell lines A549 and A427. Expression of dnhWnt-2 construct and endogenous Wnt-2 was detected using semi-quantitative RT-PCR in these cell lines. (B) TCF transcriptional activity was measured in A549 and A427 cells in the present of dnhWnt-2 or vector control. Triplicate measurements were made and the level of expression was shown as relative fold activation (TOP/FOP) (mean ± standard deviation). (C) Models of Wnt-2 signaling regulated in NSCLC cell line A549. The Wnt canonical pathway is activated when endogenous Wnt-2 ligand (large triangle) binds to Frizzled-8 receptor (Fzd-8), which recruits the intracellular protein disheveled (Dvl) to plasma membrane. Activation of Wnt canonical pathway prevents the phosphorylation of β-catenin, resulting in the stabilization and translocation of β-catenin in the nucleus, where it activates target genes through binding to TCF transcription factors. In the presence (+) of dnhWnt-2 construct (small triangle), endogenous Wnt-2 is prevented from binding to Frizzled-8 receptor. The activation of Wnt canonical pathway is inhibited, resulting in the degradation of β-catenin and blockage of TCF transcriptional activity. (D) A model demonstrates Wnt-2 signaling regulation in NSCLC cell line A427, which harbors a β-catenin mutant (star). In A427 cells, the dnhWnt-2 construct competes the binding of Frizzled-8 receptor with endogenous Wnt-2 and inhibits the activation of Wnt canonical pathway. Instead of being phosphorylated and degraded, the β-catenin mutant is constitutively expressed and activates downstream Wnt signaling regardless of the presence of Wnt-2- ligand.