Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells

Abstract

The uncontrolled growth of tumor can lead to the formation of area deprived in nutrients. Due to their high genetic instability, tumor cells can adapt and develop resistance to this pro-apoptotic environment. Among the resistance mechanisms, those involved in the resistance to long-term amino acid restriction are not elucidated. A long-term amino acid restriction is particularly deleterious since nine of them cannot be synthetized by the cells. In order to determine how cancer cells face a long-term amino acid deprivation, we developed a cell model selected for its capacity to resist a long-term amino acid limitation. We exerted a selection pressure on mouse embryonic fibroblast to isolate clones able to survive with low amino acid concentration. The study of several clones revealed an alteration of the eiF2α/ATF4 pathway. Compared to the parental cells, the clones exhibited a decreased expression of the transcription factor ATF4 and its target genes. Likewise, the knock-down of ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover, this association between a low level of ATF4 protein and the resistance to amino acid deprivation was also observed in the cancer cell line BxPC-3. This resistance was abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression may be one important mechanism for cancer cells to survive under prolonged amino acid deprivation.

Keywords: ATF4; amino acids; apoptosis; cancer; resistance.

Figure 1. The AADR clones are resistant to the apoptosis induced by amino acid deprivation

(A) Proliferation assay of parental cells (Parental) and 3 independent clones (Clone 1, 3, 5) cultured for 24, 48 and 120 hours in 2% medium (Concentration of amino acid compared to the control medium). Cell numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h. The graph show means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; *** indicates a significant difference (p<0,001) between clones and parental cells. (B) Early apoptosis detection using Hoechst 33342 labelling in parental cells and AADR clones cultured for 0, 24 and 48 hours in 2% medium. Results are expressed as the percentage of cells presenting high Hoechst staining. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; *** indicates a significant difference (p<0,001) between clones and parental cells. (C) Parental cells and AADR clones were cultured in control medium (Ctl) or in 2% medium (2%) for 24h and 48h. Immunoblot analyses of caspase 3 cleavage were performed. (D) Parental cells (Parental) and AADR clones (Clone 1, 2, 3) were cultured in medium lacking all amino acids for 48 hours and 72 hours. Cell numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; *** indicates a significant difference (p<0,001) between parental cells and clones.

Figure 2. The GCN2 and the mTORC1 pathways are still regulated in the AADR clones

Parental cells (Parental) and AADR clones (Clone 1, 3, 5) were cultured in control medium (Ctl) or in 2% medium (2%) for 24h and 48h (A) Immunoblot analyses of GCN2, eIF2α, S6K1 and their phosphorylated form were performed. (B) (Upper panel) Protein synthesis was measured by SUnSet method, immunoblot analyses of puromycin incorporation into proteins were performed. (Lower panel) Puromycin incorporation was determined by densitometry analysis. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) compared to parental cells in control medium. (C) Parental cells and AADR clones were cultured for 24h in control medium or in 2% medium. When indicated, 20μM of chloroquine (Cq) was added during the last hour of treatment. (Upper panel) Immunoblot analyses of eiF2α and LC3B processing (LC3-I and LC3-II) were performed. (Lower panel) LC3-II level was determined by densitometry analysis and normalized by red ponceau. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; *** indicates a significant difference (p<0,001) between clones and parental cells for a same treatment, # indicates a significant difference (p<0,01) between cells treated or not with chloroquine. (D) Parental cells and AADR clones were cultured in control medium or in 2% medium for 24h. Map1lc3b mRNA level was determined and normalized by the level of β-actin mRNA, results are expressed relative to the value observed in parental cells in control medium. Graph shows means ± S.E.M. of 5 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) compared to parental cells in control medium, * indicates a significant difference (p<0,05) between clones and parental cells.

Figure 3. The expression of ATF4 protein and its target gene is diminished in the AADR clones

Parental cells (Parental) and AADR clones (Clone 1, 2, 3) were cultured in control medium (Ctl) or in 2% medium (2%) for 24h and 48h. (A) (Left panel) Immunoblot analyses of ATF4 and eIF2α were performed. (Right panel) ATF4 level was determined by densitometry analysis. Graph shows means ± S.E.M. of 5 independent experiments. Differences were assessed by 1-way ANOVA. # indicates a significant difference (p<0,001) compared to parental cells in control medium., ** indicates a significant difference (p<0,01) compared to parental cells in 2% medium. (B) Parental cells and AADR clones were cultured in control medium or in 2% medium for 24h. Asns, Atf3, Atg12 and Trb3 mRNA levels were determined and normalized by the level of β-actin mRNA, results are expressed relative to the value observed in parental cells in control medium. Graph show means ± S.E.M. of 5 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) compared to parental cells in control medium, * p<0,05 compared to parental cells in 2% medium.

Figure 4. The knock-down of ATF4 expression in MEFs increases cell survival during amino acid deprivation

MEFs were infected with a lentivirus expressing either a ShRNA control (ShCtl) or a ShRNA targeting ATF4 (ShATF4). (A) ShCtl cells (ShCtl) and ShATF4 cells (ShATF4) were cultured in control medium (Ctl) or in 2% medium (2%) for 24 hours. (Left panel) Immunoblot analyses of ATF4 and eIF2α were performed. (Right panel) ATF4 mRNA level was determined and normalized by the level of β-actin mRNA, results are expressed relative to the value observed in ShCtl cells in control medium. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) compared to control medium *** indicates a significant difference (p<0,001) compared to ShCtl cells in 2% medium. (B) ShCtl cells and ShATF4 cells were cultured in control medium or in 2%medium for 24 and 48 hours. Immunoblot analyses of caspase 3 cleavage were performed. (C) ShCtl cells and ShATF4 cells were cultured in 2% medium for 5 days (D5) and 7 days (D7). Cell numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h (represented by the black line). Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,01) compared to ShCtl cells, ** indicates a significant difference (p<0,01) compared to 0h.

Figure 5. The expression of ATF4 is associated to the resistance to amino acid deprivation in the cancer cell line BxPC-3

(A) HeLa, MIA PaCa-2 and BxPC-3 were cultured in 2% medium (2%) for 72 hours. Cell numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h (represented by the black line). Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) between cell lines, ** indicates a significant difference (p<0,01) compared to 0h. (B) MIA PaCa-2, BxPC-3 and HeLa cells were cultured control medium or 2% medium for 24 hours. Immunoblot analyses of ATF4, eIF2α and its phosphorylated form and the cleaved form of PARP were performed.

Figure 6. The overexpression of ATF4 in BXPC-3 induces cell death and reduces cell proliferation during amino acid deprivation

BxPC-3 cells were infected with adenovirus expressing either GFP (Ad-GFP) or mouse form of ATF4 (Ad-ATF4). (A) Cells infected with either Ad-GFP or Ad-ATF4 were cultured in control medium (Ctl) or in 2% medium (2%) for 24 hours. Immunoblot analyses of ATF4, eIF2α and the cleaved form of PARP were performed. The arrow indicates the mouse form of ATF4. (B) Early apoptosis detection using hoechst 33342 labelling in cells infected with either Ad-GFP or Ad-ATF4 cultured in 2% medium for 24 and 48 hours. Results are expressed as the percentage of cells presenting high hoechst staining. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; ** indicates a significant difference (p<0,01) between compared to 0h. (C) Cells infected with either Ad-GFP or Ad-ATF4 were cultured in 2% medium for 24 and 48 hours. Cell numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h (represented by the black line). Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) between cells infected with Ad-GFP and Ad-ATF4, ** indicates a significant difference (p<0,01) compared to 0h.

Figure 7. Summary diagram of the AADR clones

In the short term, the 2% amino acid medium induces rapidly the apoptosis of mouse embryonic fibroblasts, through notably the induction of ATF4 expression. Nevertheless, this high selective pressure results in the selection of Amino Acid Deprivation Resistant (AADR) clones. These clones present an alteration of the activation of the GCN2/eIF2α/ATF4 pathway, despite the phosphorylation of GCN2 and eIF2α, the level of ATF4 remains low, preventing them from apoptosis.