Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil

Abstract

Ribosome quality control (RQC) resolves collided ribosomes, thus preventing their cytotoxic effects. The chemotherapeutic agent 5-Fluorouracil (5FU) is best known for its misincorporation into DNA and inhibition of thymidylate synthase. However, while a major determinant of 5FU's anticancer activity is its misincorporation into RNAs, the mechanisms by which cancer cells overcome the RNA-dependent 5FU toxicity remain ill-defined. Here, we report a role for RQC in mitigating the cytotoxic effects of 5FU. We show that 5FU treatment results in rapid induction of the mTOR signalling pathway, enhanced rate of mRNA translation initiation, and increased ribosome collisions. Consistently, a defective RQC exacerbates the 5FU-induced cell death, which is mitigated by blocking mTOR pathway or mRNA translation initiation. Furthermore, 5FU treatment enhances the expression of the key RQC factors ZNF598 and GIGYF2 via an mTOR-dependent post-translational mechanism. This adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment.

Graphical Abstract

Figure 1.

5FU treatment enhances global mRNA translation. (A) Left: HCT116 Cells were treated with 5FU (2.5 μM) for 0.5, 4 and 24 h for immunofluorescence analysis of 5FU incorporation. Cells were stained for 5-FU (green) using anti-BrdU antibody and counterstained with the nucleic acid dye DAPI (blue). An untreated control (0 h) was included, which displays low level of background. Right: Graph showing 5FU fluorescence per cell (29 cells for the 0 h and 50 cells were quantified in the 0.5, 4 and 24 h groups) relative to 0 h control. **** P< 0.0001, one way ANOVA with Dunnett's multiple comparisons test. Scale bar = 45 μm. (B) Quantification of new protein synthesis by Surface Sensing of Translation (SUnSET) assay in HCT116 cells treated with 5FU (2.5 μM) for the indicated times. Left: Representatives immunoblot analysis of lysates probed with the indicated antibodies. Right: The bar graph represents the relative change in puromycin/β-actin signal density in each group, measured by ImageJ. Data are presented as mean ± SD; n = 4 independent replicates; **P< 0.01, two-tailed Student's t-test. (C) Quantification of new protein synthesis by pulse labelling with the methionine analogue L-homopropargylglycine (HPG). HCT116 cells were treated with 5FU (2.5 μM) for the indicated times. The signal was normalised against the unstained samples for each group. Data are presented as mean ± SD; n = 3 independent replicates; *P< 0.05, two-tailed Student's t-test. (D) Analysis of general mRNA–ribosome association in HCT116 cells. Left. Cells were treated with 5FU (2.5 μM) for the indicated times before harvesting and analysis using polysome profiling assay. A shift from the sub-polysome to polysomes fractions indicates enhanced mRNAs-ribosomes association. Right. Quantification of the polysome/sub-polysome ratio in control and 5FU treated cells. Data are presented as mean ± SD; n = 3 independent replicates; **P< 0.01, two-tailed Student's t-test. (E) Western blot analysis of effects of 5FU treatment (2.5 μM) for the indicated times on the signalling pathways regulating mRNA translation machinery in HCT116 cells. To avoid excessive non-specific signals due to repeated re-probing, identical samples were run on separate gels. β-Actin was used as loading control for each membrane. Quantification of the pS6 and p4E-BP1 expression is presented in Supplementary Figure S1E and F. Also see the relatedSupplementary Figure S1.

Figure 2.

ZNF598 resolves 5FU induced collided ribosomes. (A) Western blot analysis of mono-ubiquitination of eS10 upon 5FU treatment (2.5 μM) for the indicated time in HCT116 cells. β-actin was used as a loading control. Treatment with Anisomycin (5 μg/ml) for 1 h was used as a positive control. (B) Left. Western blot analysis of mono-ubiquitination of eS10 in parental and ZNF598-KO HCT116 cells upon 5FU treatment (2.5 μM) for 1 h. β-actin was used as loading control. Right: Densitometric quantitation of the mono-ubiquitinated eS10/total eS10. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 4 independent replicates; *P< 0.05, two-tailed Student's t-test. (C) Left: Polysome profiling analysis of general mRNA–ribosome association in in ZNF598-KO HCT116 cells treated with 5FU (2.5 μM) for 6 h or vehicle. Right: Quantification of the polysome/sub-polysome ratio in vehicle and 5FU treated cells. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 4 independent replicates; *P< 0.05, two-tailed Student's t-test. (D) Quantification of new protein synthesis by pulse labelling with the methionine analogue l-homopropargylglycine in parental and ZNF598-KO HCT116 cells treated with 5FU (2.5 μM) for 4 h. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; **P< 0.01, two-tailed Student's t-test. (E) Left: Assessment of ribosome collisions in parental and ZNF598-KO HCT116 cells by polysome profiling following micrococcal nuclease (MNase) digestion of cell lysates. Right: Quantification of the disome/monosome ratio in each condition. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; ns = non-significant; two-tailed Student's t-test. (F) Left: MNase digestion in parental and ZNF598-KO HCT116 cells treated with 5FU (2.5 μM) for 12 h. Right: Quantification of the disome/monosome ratio in each condition. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; **P< 0.01, two-tailed Student's t-test. Also see the relatedSupplementary Figure S2 .

Figure 3.

ZNF598 deficiency enhances the sensitivity of cancer cells to 5FU. (A) Cell growth assay with parental and ZNF598-KO HCT116 cells after the indicated times. Data are shown as mean ± SD; n = 3 independent replicates; *P< 0.05, two-tailed Student's t-test. (B) Dose-response assay for measurement of sensitivity of parental and ZNF598-KO HCT116 cells to 5FU. Cell viability was measured 72 h post-treatment using CellTiter-Glo® luminescent cell viability assay. (C) Left: Colony formation assay with parental and ZNF598-KO HCT116 cells 12 days post-seeding. Cells were treated with the indicated concentration of 5FU. Right: Quantification of number of colonies with a diameter >200 μm in each well. Data are presented as mean ± SD; n = 3 independent replicates; *P< 0.05; **P< 0.01; ***P< 0.001, two-tailed Student's t-test. (D) Western blot analysis of expression of cleaved PARP (c-PARP) using lysates derived from parental and ZNF598-KO HCT116 cells treated with the indicated doses of 5FU for 72 h. β-actin was used as loading control. (E–G) Dose-response assay for measurement of sensitivity of parental and ZNF598-KO HCT116 cells to (E) FUDR, the precursor of the DNA-incorporating 5FU metabolite, (F) RNA-incorporating 5FU metabolite FUTP, and (G) Gemcitabine. 72 h post-treatment, cell viability was measured using CellTiter-Glo® luminescent cell viability assay. Also see the relatedSupplementary Figure S3.

Figure 4.

Inhibition of translation initiation reduces the 5FU-induced ribosome collisions and cell death in ZNF598-deficient cells. (A) Top: Western blot analysis of mono-ubiquitination of eS10 in lysates derived from HCT116 cells pre-treated with Torin-1 (15 nM) or vehicle for 24 h, followed by treatment with 5FU (2.5 μM) for 1 h. β-actin was used as loading control. Bottom: Densitometric quantitation of the mono-ubiquitinated eS10/total eS10. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; *P< 0.05, two-tailed Student's t-test. (B) Left: MNase digestion in ZNF598-KO HCT116 cells pre-treated with Torin-1 (15 nM) or vehicle for 24 h, followed by treatment with 5FU (2.5 μM) for 12 h. Right: Quantitation of the disome/monosome ratios. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; **P< 0.01, two-tailed Student's t-test. (C) Dose-response assay for measurement of 5FU sensitivity of parental and ZNF598-KO HCT116 cells in the presence of Torin-1(15 nM). 72 h post-treatment cell viability was measured using CellTiter-Glo® luminescent cell viability assay. (D) Left. MNase digestion in ZNF598-KO HCT116 cells pre-treated with 4EGI-1 (25 μM) or vehicle for 24 h, followed by treatment with 5FU (2.5 μM) for 12 h. Right. Quantification of the disome/monosome ratio. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; *P< 0.05, two-tailed Student's t-test. (E) Dose-response assay for measurement of 5FU sensitivity of parental and ZNF598-KO HCT116 cells in the presence of 4EGI-1 (25 μM). 72 h post-treatment cell viability was measured using CellTiter-Glo® luminescent cell viability assay.

Figure 5.

mTOR-dependent upregulation of expression of ZNF598 and GIGYF2 by 5FU-derived metabolites and UTP. (A) Western blot analysis of expression of indicated proteins in lysates derived from HCT116 cells treated with 5FU (2.5 μM) for the indicated times. Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S4A and B. (B) Western blot analysis of expression of the indicated proteins in lysates derived from HCT116 cells treated with 5FU (2.5 μM) for the indicated times. Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S4D and E. (C) Quantitative RT-PCR analysis of expression of ZNF598 and GIGYF2 mRNAs in HCT116 cells upon 5FU treatment (2.5 μM) for the indicated times. Data are presented as mean ± SD; n = 3 independent replicates; ns = non-significant; two-tailed Student's t-test. (D) Western blot analysis of expression of the indicated proteins in lysates derived from HCT116 cells pre-treated with 5FU (2.5 μM) for 1 h followed by incubation for the indicated times in the presence or absence of 100 μg/ml Cycloheximide (CHX). Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S4H and I. (E) Western blot analysis of expression of the indicated proteins in lysates derived from HCT116 cells pre-treated with Torin-1 (15 nM) or vehicle for 24 h, followed by treatment with 5FU (2.5 μM) for the indicated times. Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S5C and D. (F, G) Western blot analysis of lysates derived from HCT116 cells treated with (F) FUTP (1 μM) or (G) UTP (1 μM) for the indicated times. Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S5E and F (FUTP) and Supplementary Figure S5I and J (UTP). (H) Western blot analysis of lysates derived from HCT116 cells treated with the small molecule mTOR activator MHY1485 (200 nM) for the indicated times. Quantification of the ZNF598 and GIGYF2 protein expression is presented in Supplementary Figure S5K and L. (I) Top. Western blot analysis of lysates derived from HCT116 cells treated with the small molecule mTOR activator MHY1485 (200 nM) for the indicated times. Bottom. Densitometric quantitation of the mono-ubiquitinated eS10 / total eS10. Data are normalised against the respective control for each replicate and presented as mean ± SD; n = 3 independent replicates; ns = non-significant; **P< 0.01, two-tailed Student's t-test. Treatment with Anisomycin (5 μg/ml for 1 h) was used as a positive control. Also see the relatedSupplementary Figures S4 and S5.

Figure 6.

mTOR couples the elevated mRNA translation initiation and increased rate of ribosome collisions with enhanced expression of RQC factors in 5FU-treated cells. (A) Proposed model for mechanism of 5FU-induced enhanced ribosome collisions and modulation of RQC activity. 5FU rapidly enhances mTOR activity by an unknown mechanism, leading to increased translation initiation via phosphorylation of factors such as 4E-BPs. Increased translation initiation elevates the risk of ribosome collisions. Fluorinated Uridines may also increase the risk of ribosome stalling through direct incorporation into the mRNA ORF or non-coding RNAs such as rRNAs, thereby further increasing the chances of ribosome collision. In parallel, activation of mTOR by 5FU also leads to stabilisation of ZNF598 and GIGYF2, which further bolsters RQC mechanism and contributes to mitigating the cytotoxic consequences of 5FU-induced ribosome stalling and collisions. Image was generated with BioRender.com. (B-D) Protein expression levels of (B) ZNF598, (C) GIGYF2, and (D) ASCC3 in normal colon and colon adenocarcinoma samples. Data are extracted from The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) (52) using UALCAN data analysis platform. Z-values (number of standard deviations away from the mean) as well as Pvalues are annotated for each protein.