Figure 4.

WWC3 activates the Hippo pathway via LATS1, suppressing the EMT process.

Notes: (A) YAP transfection in H1299 upregulates Snail and Slug expression. GAPDH was used as a loading control. (B) WT WWC3 upregulated Hippo-related key molecules, the phosphorylation levels of LATS1, and YAP, whereas mutant could not. WT or mutant plasmid was transfected into H1299 cells, and subsequently, the proteins were analyzed by immunoblotting. Total LATS1 and YAP were used as loading controls. (C and D) LATS1 or YAP knockdown reversed the changes of EMT caused by WWC3 silencing. A549 cells were transfected with shCTL, shWWC3, and siRNA-LATS1/siRNA-YAP as indicated, lysed, and analyzed by immunoblotting. (E) VP reversed the changes of EMT caused by WWC3 silencing. A549 cells were transfected with shRNA-WWC3 or control vector, and 36 h later, the YAP–TEAD interaction inhibitor VP was treated for 12 h. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (F) YAP silencing reversed the enhanced invasive ability of lung cancer cells caused by WWC3 knockdown. Boyden chamber transwell assay of cellular invasion and the mean number of cells in five fields per membrane (A549, shWWC3 vs control, number: 213±14 vs 583±17, P<0.001; siRNA-YAP vs shWWC3+siRNA-YAP, number: 95±10 vs 85±5, P>0.05). The magnification is 400×.

Abbreviations: WWC3, WW and C2 domain containing protein-3; LATS1, large tumor suppressor-1; EMT, epithelial–mesenchymal transition; YAP, yes-associated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; shCTL, short-hairpin RNA control; VP, verteporfin; TEAD, TEA domain transcription factors; EV, empty vector.