Inhibiting translation elongation by reducing eIF5A activity induces feedback inhibition of initiation, limiting tumour cell proliferation

Abstract

Cancer development is associated with dysregulation of the translatome, and targeting canonical eukaryotic initiation and elongation factors can offer treatment avenues for various neoplasms. Emerging evidence indicates that dysregulated mRNA elongation, involving alterations in eEF2 activity and eIF5A expression, also contributes to tumour cell growth. In this study, we investigate whether targeting eIF5A with the inhibitor GC7 is a viable strategy to curtail aberrant cell growth. Our findings demonstrate that inhibiting elongation by reducing eIF5A activity induces feedback inhibition of initiation through eIF2α phosphorylation, decreasing ternary complex formation and shutting down bulk protein synthesis. Employing dynamic SILAC, we identify proteins impacted by reduced eIF5A activity, and show their decreased translation results from feedback inhibition to initiation or other processes downstream of eIF5A. Decreased eIF5A activity impairs mitochondrial function, which activates signalling through HRI to eIF2α phosphorylation, reducing cancer cell proliferation. These effects are reversed by treatment with the integrated stress response inhibitor, implying that the impact of GC7 on cancer cell proliferation is mediated via translation initiation rather than elongation inhibition. These data suggest that eIF5A inhibition could be used to target cancer cells that depend on mitochondrial function for their proliferation and survival.

Fig. 1. Hypusination inhibition leads to sequential elongation and initiation blocks in A549 cells.

a Representative western blots for the indicated targets in A549 cells treated with 10 μM GC7 for the indicated time points. b–f Densitometry from the indicated targets presented in (a). Error bars represent means ± SD (n = 7 independent experiments) and are plotted with individual values. Statistical analysis was carried out using one-way ANOVA with Dunnett’s multiple comparisons test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) relative to the untreated (0 h) sample. g Representative western blot analysis of puromycin incorporation in A549 cells treated with 10 μM GC7 for the indicated time points. h Quantification of puromycin intensity presented in (g) normalized to total protein levels. Error bars represent means ± SD (n = 4 independent experiments) and are plotted with individual values. Statistical analysis was carried out using one-way ANOVA with Dunnett’s multiple comparisons test (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) relative to the untreated sample. i Polysome profiling of A549 cells either untreated or treated with 10 μM GC7 for 0.5, 6 or 24 h. j Percentage of polysomes remaining in A549 cells following 5-min treatment with Harringtonine in either untreated cells or cells pre-treated with 10 μM GC7 for 3 h. Error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values. Statistical analysis was carried out using one-tailed unpaired student’s t test (* = p < 0.05). k Schematic representation of hypusination inhibition and its downstream effects on translation. Source data are provided within the Source Data file.

Fig. 2. Hypusination inhibition causes significant loss of newly synthesized proteins from 6 h.

a The proportion of newly synthesized proteins in GC7 and control treated A549 cells determined by dynamic SILAC. Three independent biological replates were used for each condition and each time point. b Volcano plots showing proteins significantly changing after GC7 treatment at each time point. Differential abundance of newly synthesized proteins was analyzed using DEqMS with the log₂-transformed ratio of heavy to light intensities. Tests were two-sided and p-values were adjusted for multiple testing using the Benjamini–Hochberg False Discovery Rate (FDR) procedure. c Gene Ontology (GO) terms significantly over-represented among proteins showing a significant loss of synthesis at 6 h. Over-representation analysis was performed using goseq and the reported one-sided p-values were adjusted for multiple testing using the FDR procedure. BP Biological Process; CC Cellular Compartment; MF Molecular Function. d Empirical cumulative density plots for the difference in newly synthesized protein between GC7 and control for proteins with known mitochondrial subcellular localizations.

Fig. 3. ISRIB leads to recovery of p-eIF2α specific targets following inhibition of hypusination.

a Representative western blots of A549 cells treated with 10 μM GC7 for either 3 h or 24 h in the presence of ISRIB (200 nM) or normal growing conditions. b–k Densitometry of 24-h GC7 treatments (+/- ISRIB) from (a) for the indicated proteins. Error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values. Statistical analysis was carried out using a two-tailed unpaired t-test (ns = not significant, * = p < 0.05). Source data are provided within the Source Data file.

Fig. 4. Translation initiation inhibition following eIF5A inhibition is driven by PERK and HRI activation and eIF2α phosphorylation.

a Representative western blots for the indicated targets from A549 cells transfected with siRNAs against HRI and treated with 10 μM of GC7 for 24 h. b–d Densitometry of western blots for the indicated targets from (a). e Representative western blots for the indicated targets from A549 cells transfected with siRNAs against PERK and treated with 10 μM of GC7 for 24 h. f–h Densitometry of western blots for the indicated targets from (e). All error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values Statistical analysis was carried out using One-Way ANOVA with Tukey’s multiple comparisons test (ns = not significant, * = p < 0.05, ** = p < 0.01, ***  = p < 0.001, **** = p < 0.0001). i Schematic representation of the proposed regulation of eIF2 following treatment with GC7. Created in BioRender. Harvey, R. (2025) https://BioRender.com/44scex7. Source data are provided within the Source Data file.

Fig. 5. Mitochondrial dysfunction leads to OMA1 and DELE1 dependent HRI activation.

a Confocal microscopy of A549 cells stained with TIMM44 (Green) and treated with 10 μM of GC7 for 3 and 24 h. Nuclei were stained with Hoechst (Blue). Scale bar: 20 μm. b Quantification of mitochondria shape from (a). Error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values. Statistical analysis was carried out using two-way ANOVA with Sidak’s multiple comparisons test (ns = not significant and *** = p < 0.001). c ROS detection assay in A549 cells treated with 10 μM GC7 for the indicated time points. Error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values. Statistical analysis was carried out using one-way ANOVA with Dunnett’s multiple comparisons test (**** = p < 0.0001). A 2-h treatment with 20 μM menadione was used as a positive control for ROS production. d Representative western blots for the indicated targets from A549 cells transfected with siRNAs against OMA1 and treated with 10 μM of GC7 for 24 h. e, f Densitometry of western blots for the indicated targets from (d). Error bars represent means ± SD (n = 3 independent experiments). g Representative western blots for the indicated targets from A549 cells transfected with siRNAs against DELE1 and treated with 10 μM of GC7 for 24 h. h, i Densitometry of western blots for the indicated targets from (g). Error bars represent means ± SD (n = 3 independent experiments) and are plotted with individual values. Statistical analysis (in e–i) was carried out using one-way ANOVA with Tukey’s multiple comparisons test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). j Schematic representation of OMA1 and DELE1 function. Created in BioRender. Harvey, R. (2025) https://BioRender.com/r1ph8ig. Source data are provided within the Source Data file.

Fig. 6. Reduced cell proliferation following eIF5A inhibition is driven by mitochondrial dysfunction and eIF2α phosphorylation.

Representative xCELLigence RTCA impendence measurements of (a) A549 and (b) MCF10A cells treated with 10 μM GC7 (indicated by the dotted line) and continuously monitored in technical duplicate. Cell index is an arbitrary value of impedance generated from proliferating cells. c)Cell index at 48 h 10 μM GC7 treatment in A549 and MCF10A cells. Error bars represent means ± SD (n = 3 independent experiments). Statistical analysis was carried out using two-tailed unpaired student’s t test (ns = not significant and **** = p < 0.0001). d Basal, maximum and reserve capacity of oxygen consumption rates (OCR) were measured in A549 (n = 4 technical replicates) and MCF10A (n = 5 technical replicates) cells. OCR was normalized to total protein and error bars represent means ± SD. e Basal OCR of A549 and MCF10A cells treated with 10 μM GC7 for 6 and 24 h. OCR was normalized to total protein. Data shown represent mean ± SD from technical replicates for untreated (n = 4), 6 h (n = 6) and 24 h (n = 5). f Left, representative xCELLigence RTCA impendence measurements of A549 cells treated with 10 μM GC7 (red), 200 mM ISRIB (grey), GC7 plus ISRIB (blue), or untreated (black). Error bars represent mean ± SD (n = 3 technical replicates) and treatment point is indicated by red line. Right, cell index at 96 h. Error bars represent means ± SD (n = 3 independent experiments). g Cell cycle distribution of A549 cells treated with 10 μM GC7 and 200 mM ISRIB for 72 h. DNA was stained using FxCycle violet dye and quantified using the Dean-Jett-Fox model. Error bars represent means ± SD (n = 3 independent experiments). Cell index from xCELLigence RTCA instrument of (h) MEF WT and MEF S51A cells, or A549 following transfection with siRNAs specific to (i) HRI, (j) PERK, (k) OMA1 and (l) DELE-1, treated with GC7 for 72 h. Error bars represent means ± SD (n = 3 independent experiments). Statistical analysis (in f–l) was carried out using two-way ANOVA with Tukey’s multiple comparisons test (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Source data are provided within the Source Data file.

Fig. 7. A proposed model of the events following inhibition of hypusination and subsequent mitochondrial dysfunction.

Inhibiting eIF5A hypusination with GC7 leads to the inhibition of translation elongation. Subsequently, mitochondrial protein expression is reduced leading to mitochondrial dysfunction, which is sensed by OMA1-DELE-1, and activation of HRI. Additionally, unfolded proteins accumulate in the endoplasmic reticulum, inducing the UPR and activating PERK. HRI and PERK converge to phosphorylate eIF2α which inhibits translation initiation and drives cancer cell cycle arrest. Created in BioRender. Harvey, R. (2025) https://BioRender.com/6lko9fz.