Using the pSBT-PGK-Puro plasmid as a template the SB right inverted repeat (SB-RIR) and the mouse polyadenylation segment (PGApA) cloning fragments were PCR-amplified with primer pairs MreI-SB-RIR and HindIII-XcaI-c (SB-RIR), and HindIII-PacI-PGKpA and MreI-c (PGKpA), respectively (the oligos are shown in Supplementary Data 3). a promising predictive marker. Herein, we show that functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome profiles confirm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of was reported to be positively associated with lack of response to first-line oxaliplatin (oxPt)-based treatment in two independent cohorts of patients with metastatic CRC (mCRC)5. While that study suggested high expression of to be a novel predictive marker for oxPt-resistance in a subset of mCRC patients, a possible functional relationship between and cellular drug sensitivity was not examined. Here, we have constructed a transposon-based doxycycline (DOX) inducible vector to investigate the role of in Rabbit Polyclonal to TCEAL1 modulating oxPt sensitivity in CRC cells increases cell viability by decreasing apoptosis. Furthermore, we have identified direct and indirect targets of dysregulation in these cells and in mCRC patients treated with first-line oxPt. We show that directly targets and inhibits the mitogen activated protein kinase (MAPK) kinase MAP2K6 (also known as MKK6). As a consequence, we find that promotes oxPt resistance We constructed a (SB) transposon vector (pSBInducer), which allows for stable expression of small interfering RNAs (siRNAs) and miRNAs in a DOX-inducible manner (Supplementary Fig. 1), and consequently, robust downregulation of targeted genes in mammalian cells (Supplementary Fig. 2). We used pSBInducer to introduce expression (or control shRNA designed not to target any human transcripts) in the microsatellite stable and microsatellite instable CRC cell lines SW620 and HCT116, respectively (Supplementary Fig. 1). Forty-eight hours of DOX induction raised the level of approximately three-fold in HCT116.625 cells, which is comparable to the previously reported difference in expression between responder and non-responder patients (Supplementary Fig. 3)5. In SW620.625 cells, DOX treatment induced by more than 400 fold (Supplementary Fig. 3). Ectopic expression of had no significant effect on cell growth in SW620 cells, whereas in HCT116 cells, a slight (28%) increased viability was observed (Fig. 1a). Open in a separate window Figure 1 Ectopic expression of is associated with increased viability in oxPt medium.(a) Cell proliferation upon DOX induction of in the CRC cell lines HCT116.625, SW620.625 and control cells expressing a scrambled shRNA was determined by an MTT assay after 72?h of growth. Displayed as means.e.m. (induction increased oxPt resistance RI-1 over a range of concentrations (Fig. 1b), which translated into an increase in the half maximum inhibitory concentration IC50 (causing 50% inhibition of viability) from 1.6?M in HCT116.ctrl to 28.8?M in HCT116.625, and from 1.3?M in SW620.ctrl control cells to 6.1?M in SW620.625 cells (Fig. 1c). There was no difference in IC50 between vector control cells and their parental wild-type counterparts (Fig. 1c). This indicates that functionally is associated with increased resistance to oxPt in CRC cells. Increased expression reduces oxPt-induced cell death To determine whether inhibition of cell death was a contributing factor to the observed oxPt resistance in HCT116.625 and SW620.625 cells, we performed a lactate RI-1 dehydrogenase activity (LDH) assay. Induction of in HCT116.625 cells inhibited drug-induced cell death when exposed to oxPt (Fig. 2a). A small decrease in cell death was also observed for RI-1 2 and 8?M oxPt in overexpressing SW620.625 cells although this was only borderline significant (Fig. 2a). Open in a separate window Figure 2 inhibits oxPt-induced cell death in CRC cell lines.(a) DOX-induced HCT116.625 and SW620.625 together with control cells were treated for 48?h with oxPt. Cell death was determined with the LDH assay as 100%*(LDHmedium/(LDHmedium+LDHlysate)). Displayed as means.e.m. (induction, we used a flow cytometry-based Annexin-V/propidium iodide (PI) cell death assay on three randomly selected, independent HCT116.625 single cell clones (these are biological replicates since mediated transposition is near-random and individual low-passage cell clones harbour unique pSBInducer integrations6). In agreement with the LDH assay, the Annexin-V/PI assay demonstrated that indeed reduced oxPt-induced cell death (Fig. 2b). The percentage of apoptotic cells in non-treated cells was similar in control and cell clones, while the death rate upon exposure to oxPt was reduced from 81% in control cells to below 50% in the HCT116.625 cell clones. The same experiment was also performed with a single cell-derived SW620 clone, which revealed a similar effect (reduction in death.