Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention

Abstract

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Keywords: Anticancer drugs; EGR1; Lomustine; Stress granules; Translational control; mRNA translation.

Fig. 1.

Formation of SGs upon lomustine exposure. (A) Survival curve of U2OS cells exposed to lomustine for 24 h, as determined by a CytoTox-Glo™ Cytotoxicity Assay (Promega). Results are mean±s.d., n=3. (B) Quantification of the percentage of U2OS cells with SGs upon lomustine treatment (no drug, 1, 2, 4, 8, 16, 31, 63, 125, 250, 500 and 1000 μM for 24 h). Results are mean±s.d., n=3. P-values are shown (two-tailed paired t-test between no drug control and individual drug concentrations). (C) Fluorescence microscopy image of U2OS cells revealing the presence of SGs activated by 200 μM lomustine for 1 h, using SG markers G3BP1, eIF4G, and eIF3b. The merged image is also shown. (D) Lomustine-induced SGs, contain various SG-associated proteins involved in signaling pathways. (E) Detailed list of proteins associated with lomustine-activated SGs. (F) An impact of N-acetylcysteine (NAC) on the formation of lomustine-activated SG. (G) IF detection of ribosomal protein S6 (RPS6) in the lomustine-induced SGs. Images in C, D, F and G are representative of three repeats.

Fig. 2.

Lomustine inhibits protein synthesis. (A) Analysis of single cell in situ translation activity in U2OS cells in the presence of lomustine at different concentrations (160 and 200 μM). FXR1 protein was used as a SG marker. Puromycin levels reflecting mRNA translation activity are depicted using color gradients corresponding to drug concentration (color scale). (B) Polysome analysis of control, 250 mM NaAsO₂- and 200 mM lomustine-treated cells for 2 h (upper graph) with a statistical polysome/monosome ratio analysis (lower graph). (C) Analysis of translation activity in HAP1-S51A cells, which have a non-phosphorylatable variant of eIF2α, and ΔHRI, ΔGCN2, ΔPKR and ΔPERK HAP1 cells. Translation activity was measured by ribopuromycylation. ATF4 expression was monitored as a control. Results are in arbitrary units (mean±s.d., n=3).

Fig. 3.

Lomustine activates HRI kinase. (A) Effect of lomustine on eIF2α phosphorylation in genetically modified HAP1 cells (S51A, ΔHRI, ΔGCN2, ΔPKR or ΔPERK). Results are in arbitrary units (mean±s.d., n=3). (B) The assessment of the integrity of the eIF4F initiation complex and eIF4E–4E-BP1 association in response to lomustine treatment. Results are in arbitrary units (mean±s.d., n=3). ns, not significant; *P<0.05; **P<0.01 (two-tailed paired t-test). (C) Analysis of the percentage of cells with SGs upon for in S51A, ΔHRI, ΔGCN2, ΔPKR and ΔPERK HAP1 cells. SGs were quantified by counting at least 200 cells. P-values are shown, ns, not significant (two-tailed paired t-test). (D) SG formation in ΔHRI U2OS cells exposed to NaAsO₂ or lomustine. Images in D are representative of three repeats.

Fig. 4.

Transcriptome analysis in cells treated with lomustine. (A) Transcripts showing at least a two-fold increase in expression upon the indicated treatment. Control (PAR) and S51A, ΔHRI, ΔGCN2, ΔPKR, ΔPERK HAP1 cells exposed to lomustine (200 μM, 2 h) or NaAsO₂ (100 μM, 2 h) were analyzed. Results are from three repeats. (B) Expression levels of specific mRNAs in drug-treated and untreated cells. Results are from three repeats. (C) RT-qPCR-based analysis for genes exhibiting the highest expression in response to drug treatments. Results are mean±s.d., n=3.

Fig. 5.

Subcellular localization of EGR1 and DDIT4 mRNAs. FISH was used to visualize EGR1 and DDIT4 transcripts and total polyadenylated RNAs after treatment with lomustine or NaAsO₂. The colocalization was determined by splitting channels, demonstrated as in-image box selections, and quantified using ImageJ software (JACoP plugin). The Spearman's coefficient value (r) was calculated for each analyzed colocalization using the JACoP plugin. Images are representative of three repeats, and r was calculated from >5000 colocalizations (pixels) across three experimental repeats.

Fig. 6.

Analysis of EGR1 protein expression in cells exposed to lomustine and NaAsO2. (A) Analysis of EGR1 protein expression in control S51A, ΔHRI, ΔGCN2, ΔPKR and ΔPERK HAP1 cells treated with lomustine and NaAsO₂. The changes in EGR1 protein levels were quantified using ImageJ. Results are in arbitrary units (mean±s.d., n=3). (B) EGR1 and ATF4 protein expression in MEFs cells (S/S and A/A) after treatment with lomustine and NaAsO_2. The changes in EGR1 and ATF4 protein levels were quantified using ImageJ. Results are in arbitrary units (mean±s.d., n=3). The right panel shows a simultaneous analysis of SG activation in A/A and S/S cells upon treatment with 100 mM NaAsO₂ for 1 h and 200 mM lomustine for 1 h. The results demonstrated on the graph show the percentage of cells with SGs (mean±s.d., n=3).

Fig. 7.

Correlation between SG formation and EGR1 protein expression in U2OS cells. (A) Concentration-dependent SG formation in U2OS cells treated with lomustine. (B) Western blot showing level of EGR1 protein expression in U2OS cells exposed to increasing concentration of lomustine. (C) Parametric evaluation of the percentage of cells with SGs (from A) and percentage of EGR1 production in U2OS cells exposed to lomustine relative to 100% production of this protein in the untreated sample (from B). Results are representative of three repeats.

Fig. 8.

Effects of lomustine on cell viablity in EGR1- and SG-dependent manners. (A) Characterization of EGR1 knockout in HAP1 cells. EGR1 and β-tubulin levels were determined and used for quantification of western blots using the ImageJ software. Results are in arbitrary units (mean±s.d., n=3). (B) Determination of viability of EGR1-negative cells exposed to lomustine for 2 h and after 48 h relief. Results are mean±s.d., n=3. ns, not significant; *P<0.05 (paired two-tailed t-test). (C) A comparison of viability for lomustine-exposed parental (PAR), S51A-modified cells, and ΔEGR1 HAP1 cells. The cells were exposed to lomustine for 2 h and then released for 48 h. Results are mean±s.d., n=3. (D) A putative model for SG influence on cell viability under lomustine treatment. See text for more details.