Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

Abstract

Background: The mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer (CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells, using a synthetic lethal short hairpin RNA (shRNA) screening approach.

Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183 (RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA (siRNA) was used to knock down RNF183 transiently. Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the protein abundance. MTT assay, colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.

Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8 (IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.

Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.

Keywords: Colorectal cancer; Mitogen-activated extracellular signal-regulated kinase 1/2; Ring finger protein 183; Synthetic lethal; Trametinib.

Fig. 1

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. 2

RNF183 expression was increased after trametinib treatment, and its knockdown synergistically repressed the proliferation of CRC cells in combination with trametinib. RNF183 mRNA abundance was examined using quantitative real-time PCR (qPCR), and its protein level was detected using Western blotting in HT29 (a) and HCT116 cells (b) with or without 20 nmol/L trametinib for 48 h. Arrows indicate the band of RNF183, which was obviously induced by trametinib. Effects of trametinib treatment in combination with RNF183 overexpression or knockdown on the proliferation of HT29 (c) and HCT116 cells (d) measured using MTT assay. RNF183 conferred resistance to trametinib-induced inhibition on proliferation in both cell lines. Effects of trametinib treatment in combination with RNF183 overexpression or knockdown on the apoptosis of HT29 (e) and HCT116 cells (f). Effects of trametinib treatment in combination with RNF183 overexpression or knockdown on lactate dehydrogenase (LDH) release of HT29 (g) and HCT116 cells (h). Experiments were repeated three times. Data are presented as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001. Tra trametinib, Ctrl stable cell lines transfected with pcDNA4-myc/his, RNF183 stable cell lines transfected with pcDNA4-myc/his-RNF183, shNC stable cell lines transfected with lentiviral vectors of negative control shRNA, shRNF183 stable cell lines transfected with lentiviral vectors of RNF183-targeted shRNA

Fig. 3

Induction of interleukin-8 (IL-8) transcription by RNF183 promoted cell proliferation and conferred resistance to trametinib. a mRNA levels of nuclear factor-kappa B (NF-κB) downstream genes in HCT116 cells transfected with small interfering RNAs (siRNAs) of RNF183 (siRNF183) or non-specific control (siNC) were detected by qPCR. IL-8 mRNA level is down-regulated in HCT116 cells transfected with siRNF183. b IL-8 mRNA levels are decreased in HT29 and HCT116 cells treated with 20 nmol/L trametinib for 48 h. c IL-8 protein levels are down-regulated in HT29 and HCT116 cells treated with 20 nmol/L trametinib for 48 h. Effects of trametinib treatment in combination with RNF183 overexpression or knockdown on IL-8 mRNA (d) and protein (e) levels in HT29 cells. f Effects of RNF183 overexpression with or without IL-8 knockdown on HT29 cell colony formation. g HT29 and HCT116 cells were transfected with siNC or IL-8-targeted siRNA (siIL-8) for 24 h; then the cells were treated with 20 nmol/L trametinib for an additional 72 h, and the cell viability was measured with MTT assay. All experiments were repeated at least three times, and data are presented as mean ± SD. SELE selectin E, PLAU plasminogen activator urokinase, ICAM1 intercellular adhesion molecule 1, CXCR4 C-X-C motif chemokine receptor 4, IL-6 interleukin-6, MMP9 matrix metallopeptidase 9. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Synergistic antitumor effects of RNF183 knockdown and trametinib treatment on xenograft tumor growth of HCT116 cells. a A representative image of tumors collected from mice inoculated with HCT116 cells that were stably transfected with shRNF183 or shNC and treated with or without trametinib. When tumor reached 100 mm³, 6 mice respectively from the shRNF183 or shNC groups were given 300 μg/kg trametinib every day for 23 days. The remaining 6 mice in each group were treated with vehicle as control. b RNF183 knockdown and trametinib synergistically reduced tumor volumes (i) and tumor weights (ii). c qPCR assay results show that the IL-8 levels in xenograft tumors were synergistically repressed by RNF183 knockdown and trametinib treatment. d Images exhibiting the hematoxylin–eosin (H&E), RNF183, and Ki-67 staining for xenograft tumors, which indicate decreased intensity of Ki-67 in RNF183-knockdown, trametinib treatment, and combination treatment groups. RNF183 is located in the cytoplasm and nucleus and Ki-67 is located in the nucleus. ***P < 0.001