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. 2019 Dec;17(12):2343-2355.
doi: 10.1158/1541-7786.MCR-19-0245. Epub 2019 Sep 24.

Adaptive Protein Translation by the Integrated Stress Response Maintains the Proliferative and Migratory Capacity of Lung Adenocarcinoma Cells

Alexandra E Albert  1 , Sally J Adua #  2 , Wesley L Cai #  2 , Anna Arnal-Estapé  2   3 , Gary W Cline  4 , Zongzhi Liu  2 , Minghui Zhao  2 , Paul D Cao  2 , Malaiyalam Mariappan  1 , Don X Nguyen  5   3   6
Affiliations

Adaptive Protein Translation by the Integrated Stress Response Maintains the Proliferative and Migratory Capacity of Lung Adenocarcinoma Cells

Alexandra E Albert et al. Mol Cancer Res. 2019 Dec.

Abstract

The integrated stress response (ISR) is a conserved pathway that is activated by cells that are exposed to stress. In lung adenocarcinoma, activation of the ATF4 branch of the ISR by certain oncogenic mutations has been linked to the regulation of amino acid metabolism. In the present study, we provide evidence for ATF4 activation across multiple stages and molecular subtypes of human lung adenocarcinoma. In response to extracellular amino acid limitation, lung adenocarcinoma cells with diverse genotypes commonly induce ATF4 in an eIF2α-dependent manner, which can be blocked pharmacologically using an ISR inhibitor. Although suppressing eIF2α or ATF4 can trigger different biological consequences, adaptive cell-cycle progression and cell migration are particularly sensitive to inhibition of the ISR. These phenotypes require the ATF4 target gene asparagine synthetase (ASNS), which maintains protein translation independently of the mTOR/PI3K pathway. Moreover, NRF2 protein levels and oxidative stress can be modulated by the ISR downstream of ASNS. Finally, we demonstrate that ASNS controls the biosynthesis of select proteins, including the cell-cycle regulator cyclin B1, which are associated with poor lung adenocarcinoma patient outcome. Our findings uncover new regulatory layers of the ISR pathway and its control of proteostasis in lung cancer cells. IMPLICATIONS: We reveal novel regulatory mechanisms by which the ISR controls selective protein translation and is required for cell-cycle progression and migration of lung cancer cells.

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Conflict of interest statement

Conflict of interest statement: D.X.N. has received research funding from AstraZeneca, Inc and Leidos.

Figures

Figure 1.
Figure 1.. The ISR is activated in diverse molecular subtypes and stages of LUAD and can be inhibited by ISRIB.
A) Gene set enrichment analysis (GSEA) of ATF4 targets in LUAD samples from the TCGA (either n = 489 or n = 275 with paired RNA seq and whole exome sequencing data. Comparisons are stage III-IV (late) versus I-II (early) disease, proximal inflammatory (PI) versus terminal respiratory unit (TRU) tumors, proximal proliferative (PP) versus TRU tumors, DASC-like versus alveolar-like tumors, non-silent mutation high versus low tumors, KRAS mutant versus wildtype tumors, EGFR mutant versus wildtype tumors, and KRAS versus EGFR mutant tumors. NES (normalized enrichment score) and -log10(FDR) are listed for each analysis. Dashed line is FDR < 0.05. B) qRT-PCR of ATF4 target genes involved in amino acid biosynthesis in the H2030 and PC9 cell lines with doxycycline-induced control shRNA or shRNAs against ATF4. Data for two independent shRNAs (a and b) are shown. Relative expression normalized to GUSB with upper and lower limits of a representative experiment shown; n = 2. ns = not significant. C) Representative images of clonogenic assay with the indicated cells plated in +Asn or -Asn media. D) Quantification of C) showing mean and SEM. E) Representative western blots for ATF4 and GAPDH (loading control) from PC9, H1650, YLR086, and H2030 cells following incubation in -Asn media with vehicle (DMSO) or 200 nM ISRIB for the indicated times. F) Heat map depicting quantification of clonogenic assay of a panel of LUAD cell lines treated as indicated. +Asn ISRIB: ISRIB treatment alone. -Asn Vehicle: asparagine deprivation alone. -Asn ISRIB: asparagine deprivation with ISRIB treatment (dual treatment). Asterisks indicate FDR < 0.05 for indicated condition versus (*) +Asn Vehicle or (**) -Asn Vehicle. Unless otherwise noted n = 3 and p-values by Unpaired t-test.
Figure 2.
Figure 2.. ISRIB arrests the adaptive proliferation and motility of LUAD cells under amino acid stress.
A) PC9 cell viability under the indicated treatments was measured over time by resazurin. ****p = 5.6e-6, ***p = 1.9e-4. B) Drug dose curve showing cell viability over a range of ISRIB concentrations in +Asn or -Asn media on day 9. C) Bivariate cell cycle analysis using BrdU and 7-AAD as analyzed by flow cytometry following 3 days of indicated treatment. Data was plotted as % of total tumor cells in a particular phase of the cell cycle. D) Staining for cleaved caspase-3 in PC9 cells treated as indicated with percent positive cells of total cells per sample shown. E) Clonogenic assays; top wells were treated for 9 days as indicated and harvested. Bottom wells were first treated for 9 days with –Asn/ISRIB and then either ISRIB was removed (ISRIB removed) or asparagine replenished (Asn added) for an additional 9 days before harvest. Representative images shown. F) Cells were cultured for 3 days under anchorage-independent growth conditions and indicated treatments. Cell viability was performed as in A); n = 2. G) Trans-well migration assay with cells plated in the indicated treatment media containing 0.2% FBS and using the corresponding treatment media containing 10% serum as a chemoattractant. After 24 hr, migrating cells were stained for DAPI, immunofluorescent images captured, and quantified. H) Scratch assay to measure cell migration over time under the indicated conditions. Values indicate remaining wound area as a percentage of initial wound area. p-value shown for 6 hr treatment. Unless indicated otherwise, n = 3, error bars show SEM, and p-values by Unpaired t-test.
Figure 3.
Figure 3.. Molecular effects of ATF4 inhibition and ISRIB during asparagine starvation.
A) RNAseq of PC9 cells treated as indicated for either 6 hr (light green) or 24 hr (dark green). The heat map depicts the z-scores for genes that were significantly changed (FDR < 0.05 and fold change < −1.5 or > 1.5) in any treatment comparisons (6 hr or 24 hr). The burgundy box highlights acute changes activated by the ISR at 6 hr, while the aqua box highlights changes associated with chronic asparagine starvation at 24 hr. Scale narrowed to −2 to +2 for increased contrast. B) Volcano plot of IPA upstream regulators analysis for -Asn ISRIB versus -Asn Vehicle at 6 hr. C) qRT-PCR of representative ATF4 targets in PC9 cells treated as indicated for 6 hr; veh = vehicle. Relative expression normalized to GUSB with upper and lower limits shown as representative experiment. D) Volcano plot of pathway analysis for -Asn ISRIB versus +Asn Vehicle at 24 hr. E) Western blotting for NRF2 and GAPDH (loading control) in PC9 cells treated as indicated. F) Flow cytometry analysis of ROS generation using CellROX probe in PC9 cells treated as indicated for 48 hr. Fold changes between controls (dark blue) and dual treatments (light pink) are indicated. Representative experiment depicted; n = 2. G) Cell viability of cells treated as indicated for 6 days with the addition of increasing concentrations of NAC. AFU is arbitrary fluorescence units. Error bars show SEM; n = 2. Unless otherwise noted, n = 3 and p-values by Unpaired t-test.
Figure 4.
Figure 4.. ASNS mediates protein translation during asparagine deprivation.
A-E) Translation rates as measured by incorporation of the methionine analog homopropargylglycine (HPG) over time and evaluated by flow cytometry. Each data point represents the slope of HPG incorporation for at least four time points using median fluorescence intensity (MFI) from an independent experiment. A) PC9 cells following 6 hr treatment as indicated. Thaps = 100 nM thapsigargin. CHX = 100 μg/mL cycloheximide, used as a control to completely block de novo protein translation. B) PC9 cells following 24 hr treatment as indicated. C) PC9 cells expressing the indicated shRNAs in +Asn or -Asn conditions for 24 hr. D) PC9 cells expressing control shRNA (Ctrl) or ectopic ASNS (ASNS) treated as indicated for 24 hr. E) PC9 cells expressing the indicated shRNAs with control or ectopic ASNS expression cultured in -Asn for 24 hr. n = 3 for all experiments, error bars show SD, and p-values by Unpaired t-test.
Figure 5.
Figure 5.. ASNS-mediated protein translation is sufficient for adaptive cell proliferation and migration.
A) Representative images of clonogenic assay of the indicated PC9 cell lines treated as shown. B) Quantification of A). Error bars show SEM. C) Representative images of clonogenic assay of the indicated PC9 cells in + Asn or -Asn conditions. D) Quantification of C). Error bars show SEM. E) Trans-well migration assay of PC9 cells expressing empty vector control (Ctrl) or ASNS in treatment media with 0.2% FBS and using treatment media with 10% FBS as a chemoattractant for 24 hr. Migrating cells stained for DAPI and quantified using ImageJ. F) Trans-well migration assay of the indicated PC9 cell lines treated as indicated for 24 hr and quantified as in E). n = 3 for all experiments, error bars show SD, and p-values by Unpaired t-test.
Figure 6.
Figure 6.. Cyclin B1 protein synthesis is regulated by ASNS in LUAD cells under stress.
A) Heat map of reverse phase protein array (RPPA) for PC9 cells treated as indicated for 24 hr (indicated by asterisk) or 72 hr. Representative proteins that were significantly increased or decreased by dual treatment (FDR < 0.05 and 1.5 fold change) are plotted as z-scores. Functional categories indicated as cell cycle (green), cell adhesion/migration (purple), and signaling (red). B) Volcano plot of Spearman correlation between ASNS expression and protein levels as evaluated by RPPA, using LUADs from the TCGA with matching RPPA and RNA seq data (n = 181). C) Proteins that were differentially expressed in -Asn ISRIB versus +Asn Vehicle groups at 24 hr, but whose corresponding mRNA levels remained unchanged were identified. Fold change for these proteins were plotted against corresponding Spearman correlations between TCGA RPPA and ASNS from B). Green data points have a Spearman correlation with FDR < 0.05. D-G) Representative western blots for CDC25C, cyclin B1, and GAPDH (loading control) in PC9 cells lines modified and treated as indicated for D) 24, 48, or 72 hr, E) 72 hr, or F-G) 24 hr. Representative westerns shown, n = 3 for all experiments.
Figure 7.
Figure 7.. Dietary asparagine limitation combined with ISR inhibition delays LUAD growth in vivo.
A) Mice were placed on an amino acid-defined diet containing doxycycline (DOX) with or without asparagine. After 7 days, 5e4 PC9 cells expressing the indicated shRNAs were injected into the flanks of each mouse and tumor volume measured over time; n = 5 tumors. Error bars show SEM. B) Tumor volume as measured by area under the curve (AUC) until endpoint from A). C) 5e4 PC9 cells expressing the indicated shRNAs and vector control (no label) or ectopic ASNS were injected into the flanks of mice. Once tumors were palpable (10 days), mice were placed on an amino acid-defined diet containing DOX with or without asparagine and tumor volume was measured over time; n = 10 tumors. Error bars show SEM. D) Tumor volume as measured by area under the curve (AUC) until endpoint from C) and Supplementary Fig. S7D. Unless otherwise indicated error bars show SD and p-values by Mann-Whitney test. ns = not significant.
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