Figure 2
PTENWT melanoma cells exhibit reduced motility and metastasis in response to WNT/β-catenin signaling. (a) Fold-change in cell migration over 24 h in PTENWT and PTENMut control and WNT3A overexpression cells. Cells were grown to confluency before the monolayer was wounded and scratch size measured before, and after 24 h. (b) Representative images of 3D cell growth following 48h stimulation with carrier or 50 ng/ml rWNT3A. Arrows highlight connected cell clusters, which are indicative of invasive phenotypes in melanoma cells. Scale bar, 200 μm. (c) Fold-change in cell invasion after 48 h compared with control cells. Parental lines were stimulated with carrier or 50 ng/ml rWNT3A. For panels a and c, mean shown±s.d. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. (d) × 0.75 magnification images of mouse lungs 4 weeks after tail vein injections of A375 control or WNT3A overexpression cells. Star highlights metastasis-free lung lobe. Example images shown. (e) Quantitative scoring of S100 staining on mouse lung sections. Mean shown±s.d. ****P<0.0001. (f) Fold-change in cell migration over 24 h in control and PTEN overexpressing A2058 and M229 cells, following 48 h stimulation with carrier or 50 ng/ml rWNT3A. Cells were seeded to confluency before the monolayer was wounded and scratch size measured before, and after 24 h. Mean shown±s.d. *P<0.05, **P<0.01. (g) 2 μM of LY294002 inhibits AKT activity in PTENMut A2058 cell, after 48 h of stimulation. In total, 15 μg of whole-cell lysate was blotted for AKT and P-AKT, following 48 h stimulation with carrier or 50 ng/ml rWNT3A. Ku80 served as the loading control. (h) Fold-change in A2058 cell invasion after 48 h of treatment with 50 ng/ml rWNT3A/carrier with/without 2 μM LY294002, as indicated. LY294002 was kept on the cells throughout the experiment. (i) Percentage of metastatic tumors in cutaneous melanoma patients (TCGA data set) with high AXIN2 expression (n=209). *P<0.05 or **P<0.01. Distant Mets, distant metastasis; Nodes, nodal metastasis.
