Fig. 4

Synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer cells. A, Immunoblots of H358 cells expressing scramble control or NOP56-specific shRNAs after treated with rapamycin (1 μM) for 24 h. B-G H358 cells expressing scramble control or shNOP56-specific shRNAs were transfected with control siRNAs or the indicated siRNAs specifically targeting S6, eIF4E, alone and in combination. The cells were then subjected to immunoblots (B, D, F) and viability assay (C, E, G) 72 h post-transfection. Data are presented as mean ± SD (n = 3). H, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with IRE1α-specific or control siRNAs for 48 h, followed by treatment with rapamycin (1 μM) for 24 h before immunoblotting. I, H358 cells expressing control or NOP56-specific shRNAs were transfected with IRE1α-specific or control siRNAs for 24 h, followed by treatment with rapamycin (5 μM) for 72 h before apoptosis assay. Data are presented as mean ± SD (n = 3). *p < 0.05, ***P < 0.001 and ****P < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. J, H358 cells expressing control or NOP56-specific shRNAs were preincubated overnight with vehicle (DMSO) or the JNK inhibitor SP600125, followed by treatment with rapamycin for 72 h before apoptosis assay. Data are presented as mean ± SD (n = 3). **p < 0.01, ***P < 0.001 and ns P>0.05 by two-way ANOVA with Tukey’s multiple comparisons test. K, Proposed model of cellular gauge for IRE1α-regulated UPR. In KRAS-mutant cancer cells, intact NOP56 keeps ROS in check so that IRE1α-regulated UPR is minimal (basal level; left). Intermediate levels of IRE1α-regulated UPR ensue from NOP56 depletion, which activates p38-AKT/mTOR and promotes cell survival (middle). At “dangerous” level of ROS, IRE1α-regulated UPR initiates JNK-dependent apoptosis (right)