Figure 7: CERK inhibition increased MMP in Mut KRAS NSCLC cells by limiting VDAC-tubulin interaction and AKT activation.

(A, B) CERK inhibition decreased VDAC-tubulin binding and increased MMP in Mut KRAS NSCLC cells, which was reverted by tubulin-destabilizer, colchicine (Col), treatment. (A) A549/H838 cells (both LUAD-derived) were treated with NVP 231 (0-400 nM) for 24h hours, then mitochondria fractions prepared from the cell samples were subjected to co-IP assay for VDAC1 followed by SDS-PAGE/immunoblotting. (B) A549 cells were treated with vehicle (Veh) or NVP-231 (400 nM) for 24 hours then cells were subjected to VDAC-tubulin colocalization assay. (C, D) A549 cells were treated with Veh or NVP 231 (400 nM) for 24h hours followed by 2-hour treatment with Col (1 μM), then mitochondria fractions prepared from the cell samples were subjected to co-IP assay for VDAC followed by SDS-PAGE/immunoblotting (C) or post-treatment cells were subjected to MMP assay (D). (E) A549 cells treated with Veh, NVP-231 (400 nM) or AKT inhibitor, BEZ235 (AKTi; 50 nM) were subjected to Western immunoblotting. (F, G) AKT inhibition decreased VDAC-tubulin binding and increased MMP in Mut KRAS cells, which was reverted by Col treatment. Cells treated with Veh or AKTi as in (E) were additionally treated with Col (1 μM) for 2 hours before utilized in co-IP assay for VDAC followed by SDS-PAGE/immunoblotting (F) or MMP assay (G). (H-J) A549 cells were transfected with adenovirus control (Ad-Null) or expressing constitutively active AKT2 (Ad-caAKT) for 24 hours followed by 24-hour treatment of Veh or NVP 231 (400 nM); cells were then subjected to SDS-PAGE/immunoblotting (H), MMP assay (I) or clonogenic cell survival assay (J). Data in graphs are means ± SD; n = 12 in (B); n=6-7 in (D), (G), (I), (J) from two independent occasions. *p<0.01, **p<0.005, ***p<0.0005. Unless otherwise noted by an * or depicted p-value, data are not significant (NS) between depicted groups; p>0.05.