Fig. 7.

ZMYND8 promotes tumor migration and invasion via interaction with EZH2 in ccRCC cells. (A) 786-O and A498 cells stably infected with lentivirus for sgControl or sgZMYND8 were transfected with empty vector (EV) or EZH2. Cells were harvested for WB analysis of indicated proteins. (B and C) 786-O and A498 stable cell lines were infected with lentivirus as in A and cultured to confluence on six-well plates. The cell layer was scratched with a 200-μL pipette tip. For each sample, at least three scratched fields were photographed immediately (0 h) or 16 h after scratching. Photographs of representative images were taken at 100× magnification and results are shown in B, and the quantified data are presented in C. (Scale bars: 200 μm.) ***P < 0.001; n.s., not significant. (D and E) 786-O and A498 stable cell lines were infected as in A and subjected to Matrigel invasion assays. Representative images of invasion assay are shown in D, and the quantified data are presented in E. (Scale bars: 200 μm.) **P < 0.01; n.s., not significant. (F) sgZMND8 786-O and A498 stable cells were transfected with indicated plasmids, and cells were harvested for WB analysis of indicated proteins. (G and H) 786-O and A498 stable cell lines were transfected as in F and cultured to confluence on six-well plates. The cell layer was scratched with a 200-μL pipette tip. For each sample, at least three scratched fields were photographed immediately (0 h) or 16 h after scratching. Photographs of representative images were taken at 100× magnification and are shown in G, and the quantified data are presented in H. (Scale bars: 200 μm.) *P < 0.05; **P < 0.01; ***P < 0.001. (I and J) 786-O and A498 stable cell lines were transfected as in F and subjected to Matrigel invasion assays. Representative images of invasion assay are shown in I, and the quantified data are presented in J. (Scale bars: 200 μm.) **P < 0.01; ***P < 0.001. (K) Schematic model depicting the function switch of EZH2 mediated by ZMYND8. In hypoxia-exposed or VHL-deficient cells, EZH2 expression is up-regulated, while other components of PRC2, such as SUZ12 and EED, are down-regulated. Under this condition, overexpressed ZMYND8 preferentially binds to T487-phosphorylated EZH2 mediated by CDK1, which not only is critical for the maintenance of EZH2 phosphorylation and inhibition of assembly of the PRC2 complex, but also mediates EZH2 interaction with FOXM1, thereby promoting FOXM1-dependent expression of MMP genes and cancer invasion and progression.