Fig 2: Synthetic lethal short hairpin RNA (shRNA) screening shows that ring finger protein 183 (RNF183) confers resistance to trametinib treatment in colorectal cancer (CRC) cells. a HT29 cells were infected with a pooled shRNA library, and the puromycin-selected stable cells were treated with dimethyl sulfoxide (DMSO) or 20 nmol/L trametinib for 8 days. Then, shRNA sequences were amplified by PCR, and next-generation sequencing was conducted to calculate their abundance. b The abundance of RNF183 shRNA decreased in cells treated with trametinib compared with those treated with DMSO. T/D ratio of abundance, the ratio of abundance in the trametinib group to abundance in the DMSO group

Fig 3: NF-κB-IL-8 axis is indispensible for the oncogenic function of RNF183. (aand b) NF-κB inhibitor BAY11-7085 attenuates RNF183 overexpression induced migration (a) and invasion (b) of DLD-1 cells. (c) HCT116 cells were co-transfected with RNF183 plasmid and siRNA-targeting IL-8 as indicated. Recombinant IL-8 was also supplemented in the knockdown group to rescue the phenotype. The pictures of migrated cells (left) and quantification (right) were showed. (d) Luciferase activity of IL-8 promoter was evaluated in HCT116 cells with overexpression of control plasmid, wild-type RNF183 and truncated RNF183 without E3 ubiquitin ligase activity. (e–g) Effects of wild-type and truncated RNF183 on proliferation (e), colony formation (f) and migration (g) of HCT116 cells. Mean±S.D. (n=3). Scale bar: 50 μm. **P<0.01, ***P<0.001

Fig 4: RNF183 promotes metastasis of CRC cells in vivo. (a) Lungs were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Pulmonary histopathology was examined by H&E staining. Mean±S.D. (n=7). (b) Livers were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Hepatic histopathology was examined by H&E staining. Mean±S.D. (n=7). (c) Lungs were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Pulmonary histopathology was examined by H&E staining mean±S.D. (n=8). (d) Livers were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Hepatic histopathology was examined by H&E staining. Mean±S.D. (n=7). Scale bar: 100 μm (× 100 and × 200) or 500 μm (× 40). **P<0.01

Fig 5: RNF183 promotes IL-8 transcription through NF-κB. (a) The effects of RNF183 knockdown on the mRNA abundance of several NF-κB downstream genes in HCT116 cells. (b) Knockdown RNF183 expression significantly reduced IL-8 secretion in HCT116 (left) and DLD-1 (right) cells. (c) Enforced RNF183 expression augments IL-8 transcription (left) and IL-8 secretion (right) in DLD-1 cells. (d) Stable RNF183 overexpression increased IL-8 transcription in xenograft tumors as shown in Figure 2g. (e) Effects of RNF183 on the activity of luciferase reporter with wild-type or NF-κB binding site deleted (△NF-κB) IL-8 promoter in HCT116 cells. (f) Expression of several proteins in NF-κB pathway was examined by western blots with enforced RNF183 expression in HCT116 cells or with RNF183 knockdown in DLD-1 cells. (g) Chromatin immunoprecipitation (ChIP) assays were carried out to determine the binding of P65 on IL-8 promoter with or without RNF183 enforced expression. (h) The expression of P65 and RNF183 were evaluated in forty CRC tissues. The correlation of these two proteins and the significance were also calculated. Mean±S.D. (n=3). Scale bar: 100 μm. ***P<0.001