Figure 7: Inhibition of MAPK14 induces oxaliplatin resistance in CRC cells.

(a) MAPK14 was specifically depleted from HCT116 and SW620 cells by transfection of a pool of MAPK14 targeting siRNAs (siR^MAPK14) 48 h before being treated with 64 μM oxPt for 48 h (or left unexposed; see Supplementary Fig. 9 for knockdown efficiencies). The impact on cell death (64 μM/0 μM) was determined by LDH and are displayed relative to cells transfected with a scrambled siRNA (siR^scr, set to 1). Mean±s.e.m. from at least n=4 experiments with ‘*’ indicating a significant reduction in oxPt-induced cells death compared with siR^scr transfected cells (P≤0.05, t-test). (b) A phospho-specific western blot versus the MAPK14/MAPKAPK2 substrate Ser82-HSPB1 was applied to show increased MAPK14 activity after oxPt treatment and the inhibitory effect of 10 μM SB203580 on this activity. (c) HCT116 and SW620 cells were treated for 1 h with MAPK11/14 inhibitors SB203580 (10 μM, blue) or SB202190 (5 μM, purple), then exposed to 64 μM oxPt (or left unexposed) for 48 h before the increase in cell death (64 μM/0 μM) was determined by LDH. Presented relative to cells not treated with inhibitor (DMSO treated; mean±s.e.m. from at least n=4 experiments with ‘*’ indicating a significant reduction in oxPt-induced death compared with DMSO-treated cells, P≤0.05, t-test). (d) Stable, inducible expression of miR-625-3p was generated using pSBInducer transposition in HCC2998 CRC cells (left). Phospho-specific western blot for MAP2K6 and MAPK14 activity 48 h after DOX induction of HCC2998.ctrl and HCC2998.625 cells (right). (e) HCC2298.ctrl and HCC2998.625 cells DOX-induced for 48 h, then treated (or left untreated) with 64 μM oxPt for 48 h before the increase in cell death (64 μM/0 μM) was determined by LDH. Results are displayed relative to control cells (set to 1; mean±s.e.m. from n=3 experiments; *P≤0.05, t-test). (f,g) HCC2998, Colo205, DLD1, HT29 and LoVo CRC cells were treated for 1 h with MAPK11/14 inhibitors SB203580 (10 μM, blue) or SB202190 (5 μM, purple) then exposed to 64 μM oxPt (or left unexposed) for 48 h before the increase in cell death (64 μM/0 μM) was determined by LDH. Displayed relative to DMSO-treated cells (mean±s.e.m. from n=3–4 experiments with ‘*’ indicating a significant reduction, P≤0.05, t-test). (h) A schematic model showing how miR-625-3p mediated downregulation of MAP2K6 could modulate response to oxPt by abrogating MAPK14 stress-induced signalling. In the canonical model MAP2K6 in complex with MAP2K3 phosphorylates and activates MAPK14, which in turn—directly or indirectly via substrate kinases such as MAPKAPK2—activates a diverse number of target proteins central to stress-induced transcription, translation, cell cycle control and apoptosis.