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.
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.
This article is featured in Highlights of This Issue, p. 2341
Non–small cell lung cancers (NSCLC) are highly heterogeneous not only in terms of their cellular origins and molecular landscapes, but also in their abilities to adapt to cellular and metabolic stresses (1, 2). Oncogenic mutations can induce intrinsic cellular stress (3). Moreover, lung tumor cells may be exposed to extrinsic cellular stressors in the lung tumor microenvironment, which can include amino acid deprivation, glucose starvation, hypoxia, and acidosis at various stages of cancer progression (4). Malignant cells must therefore co-opt different adaptive responses to mitigate these detrimental effects as they expand and disseminate (5). The current standard of care for NSCLC includes immunotherapy and targeted therapies for tumors with a known oncogenic driver mutation (6). However, most NSCLC patients eventually become refractory to these treatments. Identifying and understanding the adaptive responses of lung cancer cells to metabolic stress may unveil additional therapeutic vulnerabilities of NSCLCs.
One highly conserved stress-induced pathway is the integrated stress response (ISR; ref. 7). The ISR maintains proteostasis by halting global translation when environmental conditions are such that continued translation would be detrimental to cellular fitness. The ISR consists of four kinases which sense unique stressors and phosphorylate eIF2α, attenuating cap-dependent translation (8). The sensor PERK is activated by an accumulation of unfolded protein in the endoplasmic reticulum generated by metabolic stresses such as glucose deprivation and hypoxia, whereas the sensor GCN2 is activated by an accumulation of uncharged transfer RNAs produced as a consequence of amino acid deprivation. Under nutrient stress, attenuation of global translation also allows for selective translation of the transcription factor ATF4 through bypass of an upstream open reading frame (9, 10). ATF4 activates diverse transcriptional programs to either regain homeostasis or, in the setting of chronic stress, trigger cell death (11, 12). The adaptive consequences of ISR activation are highly context dependent and can be influenced by cell type, the nature of the stress, the sensor, and the amplitude and duration of the stress.
Several cancers types have been shown to be regulated by ATF4 and the ISR. For instance, genetic suppression of ATF4 in fibrosarcoma and prostate cancer results in tumor cell apoptosis (13, 14). Conversely, PERK and ATF4 can inhibit tumor progression in melanoma (15). In NSCLC, tumors driven by KRAS mutations can activate ATF4 upon nutrient depletion (16). However, it remains unclear if ATF4 can regulate other molecular subtypes of lung cancer. Importantly, given the context-dependent consequences of ISR activation, there remains a need to determine which of its effector functions are required for the fitness of lung cancer cells at different stages of tumor progression.
Materials and Methods
Cell lines and culture
Cell lines were cultured as recommended by the ATCC and routinely tested for mycoplasma using the Universal mycoplasma detection kit (#30-1012k). Cells were cultured in RPMI 1640 (Thermo Fisher Scientific #11875093) containing 10% FBS (Thermo Fisher Scientific #10437-028), 1% penicillin–streptomycin (Thermo Fisher Scientific #15140122), and 0.2% amphotericin B (Sigma-Aldrich #A2942). Treatment media were prepared by adding back all constituents (Sigma #LAA21-1kt and #G7021), except those indicated, to RPMI 1640 without glucose and amino acids (US Biological #R9010-01). Clonogenic, cell viability, anoikis, bivariate cell-cycle analysis, cleaved caspase-3 staining, CellROX, transwell migration assays, and scratch assays were performed as described in Supplementary Materials and Methods.
shRNA and cDNA expression
Independent shRNAs (Dharmacon) against ATF4 (a and b) or ASNS were subcloned into pINDUCER10 (17). See Supplementary Materials and Methods for sequences. ASNS (#OHS5897-202616233), ATF4 (#OHS5899-202616733), and RFP-expressing control (#OHS5832) constructs (pLOC) were purchased from Dharmacon. For both knockdown and overexpression, lentivirus was generated using standard calcium phosphate transfection protocols, concentrated, titered, and used to infect cell lines using standard methods (MOI = 0.5–4). Infected cells were selected using puromycin (for pINDUCER10) or blasticidin (for pLOC) over 3 or 7 days, respectively. For shRNA induction, cells were incubated with 1 μg/mL doxycycline for 1 to 5 days.
Gene set enrichment analysis and pathway analysis
For gene set enrichment analysis (GSEA; ref. 18), a gene list consisting of 472 ATF4 target genes as defined by Han and colleagues (19) was used. Preranked gene lists from ANOVA analyses for indicated comparisons were generated for either all The Cancer Genome Atlas (TCGA) lung adenocarcinomas (n = 489 tumors; ref. 20), the TCGA Nature Core samples (n = 230 tumors and 45 matched normal tissues which include exome sequencing), or the Director's Challenge Cohort of lung adenocarcinomas (n = 442; ref. 21) where appropriate. DAVID analysis of leading edge genes from the GSEA analysis was performed as previously described (22). Additional details are provided in Supplementary Materials and Methods.
Quantitative real-time PCR
Total RNA was extracted using an RNeasy kit (Qiagen #74106), and 1 μg was used to generate cDNA with an iScript cDNA Synthesis Kit (Bio-Rad #1708890). cDNA was diluted 1:10, mixed with Fast SYBR Green master mix (Thermo Fisher Scientific #4385614), and technical quadruplicates were amplified and measured using a ViiA 7 Real-Time PCR machine (Thermo Fisher Scientific).
Cells were rinsed with PBS and lysed directly in the plate, using RIPA buffer, protease inhibitors (Roche # 11836170001), and phosphatase inhibitors (Sigma #P5726 and #P0044). Cells were incubated on ice for 30 minutes, vortexing every 10 minutes. Lysates were clarified by centrifugation for 15 minutes. Protein was quantified using the DC Protein Assay (Bio-Rad # 500-0112) and analyzed by SDS-PAGE using the Mini-PROTEAN system (Bio-Rad). Protein was transferred to either nitrocellulose or PVDF and membranes blocked using 5% milk in TBST (0.1% Tween20). Blots were incubated with primary antibodies at 4°C overnight, then horseradish peroxidase–secondary antibodies for 1 hour at room temperature. ECL was used to develop blots, and they were imaged using either a KwikQuant imaging system (Kindle Biosciences) or ChemiDoc Imaging System (Bio-Rad).
RNA sequencing and pathway analysis
RNA sequencing (RNA-seq) was performed by the Yale Center for Genome Analysis. Subsequent ANOVA analysis of all genes significantly changed (FDR < 0.05 by Benjamini–Hochberg step-up method) by at least 1.5-fold was performed using Partek Genomics Suite (Partek). All data are deposited in NCBI's Gene Expression Omnibus under accession number GSE126232. Ingenuity Pathway Analysis software (Qiagen) was used to predict changes in upstream regulators and canonical pathways. Additional details are provided in Supplementary Materials and Methods.
Cells were starved of l-methionine for 30 minutes and subsequently incubated with 50 μmol/L homopropargylglycine (HPG; Life Technologies #C10186) for 1 to 4 hours in treatment media. Cells were then trypsinized and a Click-IT kit (Life Technologies #C10269) used to label HPG. Labeled cells were analyzed using an LSR II flow cytometer (BD Biosciences), and data were processed using FlowJo (FlowJo, LLC). Translation rates were determined based on the slope of HPG incorporation over time.
Reverse phase protein arrays
Reverse phase protein array (RPPA) was performed by the MD Anderson Reverse Phase Protein Array Core Facility. Of the antibodies plotted in Fig. 6, FOXO3A antibodies were annotated as “use with caution.” Partek Genomic Suite software (Partek, Inc.) was used for ANOVA analysis (Supplementary Table S4). Proteins whose expression changed by at least 1.5-fold with FDR < 0.05 by Benjamini–Hochberg step-up method were considered significantly changed. A heatmap showing representative proteins that significantly changed was generated using Prism software (GraphPad). Supplementary Table S4 was filtered for targets whose protein levels changed in the RPPA, but whose corresponding mRNA levels from the RNA-seq data remained unchanged. Independently, Spearman correlation of ASNS expression in the TCGA with corresponding RPPA levels was performed for all lung adenocarcinoma samples with available RPPA data (n = 181; ref. 20) in order to determine proteins that clinically correlate with ASNS expression. Targets that changed at the protein but not RNA levels in our experiments were plotted against the corresponding correlation between protein levels and ASNS expression from the TCGA. Additional details are available in Supplementary Materials and Methods.
All work was done in accordance with Yale Institutional Animal Care and Use Committee policies (protocol #201611338). Isonitrogenous, amino acid–defined chow containing (#TD01084) or lacking (#TD160365) asparagine was purchased from Envigo. For experiments that involved knockdown, the mouse chow also contained 625 ppm doxycycline (#TD160774 and #TD160775). Trans-ISRIB (Tocris #5284) was sterilely dissolved using the following formulation: 5:2:20:73 v/v DMSO:Tween80:PEG400:D5W. Cells were suspended in sterile PBS and mixed 1:1 with growth factor–reduced matrigel (Corning #356231), and 100 μL, containing 5e4 cells, was injected s.c. in the flanks of 4- to 5-week-old male athymic nu/nu mice (Charles River Laboratories #088). Tumor volume was measured using calipers and calculated using the formula: tumor volume = (major axis)(minor axis2) × 0.52.
Data were presented as mean ± SEM or SD, as indicated, with P values calculated by two-tailed Student t test for in vitro work and Mann–Whitney test for in vivo work. FDR (Benjamini–Hochberg step-up method) was used for multiple test correction where appropriate. Statistics were performed using Prism software (GraphPad) or Partek Genomics Suite (Partek).
ATF4-regulated amino acid stress response is activated in advanced stage lung adenocarcinoma and can be inhibited by ISRIB
To understand the biological and mechanistic context of ISR regulation in human lung cancer, we compared the activation status of this pathway across different stages and subtypes of primary human lung adenocarcinoma (20), which is the most frequently diagnosed type of NSCLC. We inferred ISR pathway activation by performing GSEA using conserved transcriptional targets of ATF4 (18, 19). We did not observe a bias in ATF4 target gene activation when comparing lung adenocarcinomas expressing several major driver mutations or as a function of mutation burden, although we note that KRAS-mutant tumors had higher ATF4 target gene expression when compared with EGFR-mutant tumors specifically (Fig. 1A). ATF4 targets genes were significantly enriched in late stage (III/IV) versus early stage (I/II) lung adenocarcinomas (Fig. 1A). ATF4 genes were also preferentially activated in transcriptional subtypes of lung adenocarcinoma with poor prognosis and a high probability for relapse (e.g., proximal inflammatory and distal alveolar stem cell–like tumors; refs. 20, 23; Fig. 1A; Supplementary Fig. S1A).
Increased levels of phosphorylated eIF2α (p-eIF2α) have been observed in human lung cancer tissue (24). Moreover, in late stage lung adenocarcinomas, activated ATF4 targets were enriched for genes involved in amino acid metabolism and, in particular, serine and asparagine biosynthesis (Supplementary Fig. S1B; Supplementary Table S1). These findings suggest that adaptive responses to amino acid supply may be a major determinant of ISR activation throughout tumor progression and across oncogenic subtypes of lung adenocarcinoma. Accordingly, we tested the role of the ISR in the H2030 and PC9 lung adenocarcinoma cell lines which are dependent on prototypical oncogenic mutant KRAS or mutant EGFR, respectively, for their viability. Reducing ATF4 using two independent shRNAs (a and b; Supplementary Fig. S1C) robustly decreased the expression of ASNS, which drives asparagine biosynthesis, and more modestly reduced PSAT1, a gene involved in serine biosynthesis (Fig. 1B). ATF4 knockdown did not affect the outgrowth of these cell lines under complete amino acid conditions (+Asn or +Ser), but concurrent removal of asparagine or serine from the media (−Asn or −Ser) significantly reduced their clonogenicity (Fig. 1C and D; Supplementary Fig. S1D and S1E), as has been shown in other types of cancers (13).
To ascertain the dependency of lung adenocarcinoma cells on the ATF4 regulator eIF2α, we tested the effects of the integrated stress response inhibitor (ISRIB) on cell outgrowth. ISRIB is a small molecule that targets eIF2α's guanine exchange factor, eIF2B, stabilizing it in an activate state (25, 26). ISRIB can maintain global translation, thus blocking ATF4 translation, in the presence of cellular stress (27). We confirmed that asparagine starvation increases eIF2α phosphorylation and ATF4 protein levels and that the latter can be blocked by ISRIB in many, but not all, cell lines (Supplementary Fig. S1F; Fig. 1E). We next evaluated the effects of ISRIB on the outgrowth of a panel of lung adenocarcinoma cell lines, including tumor cells derived from a treatment-refractory lung adenocarcinoma patient (YLR086). Cells were cultured under ISRIB treatment in the presence or absence of asparagine. Whereas ISRIB treatment alone (+Asn ISRIB; Fig. 1F, left column) had no significant effect, several lines demonstrated modest decreases in outgrowth upon asparagine deprivation alone (-Asn Vehicle; Fig. 1F, middle column). Combining asparagine limitation with ISRIB treatment (-Asn ISRIB; dual treatment) inhibited the outgrowth of most of the lines tested (8 of 12), with some lines responding additively or selectively (e.g., PC9, HCC827, and H441) to the dual treatment (Fig. 1F, right column).
ISRIB did not further sensitize PC9 cells to the deprivation of serine, glutamine, or glucose (Supplementary Fig. S1G–S1I), indicating that lung adenocarcinoma cells can be preferentially dependent on the ISR when extracellular asparagine is limiting. A smaller proportion of lung adenocarcinoma cell lines were partially or fully resistant to ISRIB under asparagine deprivation (e.g., H2030, H1975, H23, and HCC4006; Fig. 1F). For the H2030 cell line, although its clonogenicity required ATF4 induction under asparagine starvation (Fig. 1C), ATF4 levels were not reduced by ISRIB (Fig. 1E), suggesting that the pharmacologic activity of ISRIB or inhibition of its target may be abated in this line. Recent studies suggest that asparagine is an essential amino acid for some tumor cells specifically as extracellular glutamine levels become limiting (28, 29). Hence, we also tested ISRIB on two other resistant lines, HCC4006 and H1975, when deprived of both asparagine and glutamine. In this setting, ISRIB had an additive effect on the growth inhibition of both cell lines (Supplementary Fig. S1J). In summary, despite variable adaptive responses to amino acid stress, most lung adenocarcinoma cells can be sensitized to asparagine limitation by targeting eIF2α and ATF4.
Pharmacologic inhibition of the ISR arrests the proliferation and motility of lung adenocarcinoma cells during amino acid deprivation
Inhibition of the ISR causes a variety of cell biological effects in a context-dependent manner. By using the well-characterized PC9 lung adenocarcinoma cell line, dual treatment was shown to significantly decrease viability in an ISRIB dose-dependent manner (Fig. 2A and B). Bivariate cell-cycle analysis demonstrated that dual treatment caused an arrest in G2–M phases of the cell cycle by 72 hours (Fig. 2C). Apoptosis, as measured by cleaved caspase-3 staining, increased slightly, but only after 9 days of treatment (Fig. 2D). These data suggest that the proximal effects of dual treatment are cytostatic. Moreover, growth inhibition was only partially reversed by either halting ISRIB treatment (ISRIB removed) or replenishing the cells with extracellular asparagine (Asn added; Fig. 2E). Clonogenic outgrowth under dual treatment could also be rescued by ectopic expression of ATF4 (Supplementary Fig. S2A and S2B).
Given that ATF4 activity may be increased in lung adenocarcinoma subtypes with a higher probability of metastatic relapse (Fig. 1A), we also evaluated the effects of dual treatment on anchorage-independent growth and cell motility, two cellular phenotypes that are regulated by amino acid accessibility or metabolic stress (30, 31) and are required for tumor cell dissemination. When PC9 cells were cultured in suspension, their growth was significantly inhibited by dual treatment (Fig. 2F). Moreover, a significant decrease in the migratory ability of PC9 cells was observed for dual treated cells (Fig. 2G and H; Supplementary Fig. S2C and S2D). We conclude that ISRIB also blocks adaptive anchorage-independent tumor cell growth and motility in response to amino acid stress.
Direct and indirect molecular effects of ATF4 inhibition and ISRIB during asparagine starvation
Because eIF2α can regulate other stress response proteins in addition to ATF4 (7), we next comprehensively analyzed the molecular effects of ISRIB treatment. Based on the kinetics of ATF4 activation in PC9 cells starved of asparagine, we analyzed by RNA-seq the acute (6 hours) and long-term (24 hours) effects of ISRIB treatment, amino acid deprivation, or dual treatment. ISRIB alone did not affect the transcriptome of PC9 cells, consistent with its minimal biological effects in the absence of stress (Supplementary Table S2). Following 6 hours of asparagine deprivation, 42 genes were downregulated, whereas 284 genes were upregulated by at least 1.5-fold (FDR < 0.05) of which 48 are known ATF4 targets (Fig. 3A). Twenty-four hours following asparagine starvation alone, the expression of known ATF4 targets returned to baseline levels, with ASNS being the only target to remain relatively elevated. Of the acutely induced ATF4 targets, 42 were blocked by ISRIB (Fig. 3A). Pathway analysis identified ATF4 as well as the eIF2α kinases PERK and GCN2 as potential regulators of these targets (Fig. 3B; Supplementary Table S3), confirming that the ISR pathway is directly inhibited by ISRIB in lung adenocarcinoma cells. Genes that are acutely regulated by asparagine starvation and blocked by ISRIB include regulators of amino acid biosynthesis (ASNS), autophagy (MAP1LC3B), and amino acid transport (SLC7A11; Fig. 3C). ASNS was also confirmed to be inhibited by ATF4 knockdown under similar conditions (Supplementary Fig. S3A).
Long-term dual treatment increased the number of differentially expressed genes (604 upregulated and 396 downregulated; Fig. 3A) in lung adenocarcinoma cells and was predicted to inhibit several signaling pathways, the most significant of these being the NRF2-mediated oxidative stress response (Fig. 3D; Supplementary Table S3). Previous studies suggest that NRF2 functions upstream or in conjunction with ATF4 to activate genes that control reactive oxygen species (ROS) and amino acid metabolism (32, 33). Given the decrease in NRF2 targets by dual treatment, we evaluated mRNA and protein levels of NFE2L2/NRF2 itself. Although NFE2L2 was induced by asparagine limitation, this induction was not affected by ISRIB or ATF4 knockdown (Supplementary Fig. S3B). However, under stress, NRF2 protein levels were decreased by ISRIB or ATF4 knockdown (Fig. 3E), and this reduction correlated with decreased expression of the NRF2 target gene HMOX1 (Supplementary Fig. S3C). These results indicate that, upon amino acid starvation, NRF2 protein levels and activity can be indirectly regulated downstream of ATF4.
Oxidative stress and decreased autophagy are two potential consequences of perturbed ATF4 and NRF2 activity (34, 35). Indeed, in PC9 cells, asparagine deprivation combined with genetic or ISRIB-mediated reduction of ATF4 led to increases in intracellular ROS (Fig. 3F). However, although n-acetylcysteine (NAC) treatment reduced ROS back to baseline levels, it was not sufficient to restore cell proliferation (Supplementary Fig. S3D; Fig. 3G). We also measured autophagic flux by monitoring the accumulation of microtubule-associated proteins 1A/1B light chain 3B (LC3-II) in the presence of lysosomal protease inhibitors. Neither ISRIB treatment nor ATF4 knockdown consistently affected LC3-II accumulation under the conditions tested (Supplementary Fig. S3E and S3F). As such, the regulation of ROS may be indirectly affected by long-term eIF2α or ATF4 inhibition, but neither ROS accumulation nor autophagy is required for the inhibition of lung adenocarcinoma cell growth during asparagine starvation.
ASNS-mediated protein translation is necessary and sufficient for adaptive cell proliferation and migration
Another hallmark of the ISR is attenuation of global protein translation, through phosphorylation of eIF2α (7). We thus ascertained the effects of asparagine starvation and ISRIB on translation in lung adenocarcinoma cells. Protein translation rates were decreased following 6 hours of asparagine deprivation in the PC9 cell line, but were not rescued by ISRIB (Fig. 4A). This is in contrast to the ability of ISRIB to restore translation in cells undergoing ER stress via thapsigargin treatment (Fig. 4A). By 24 hours, translation rates returned to baseline following asparagine starvation alone, whereas translation rates remained inhibited in dual treated cells (Fig. 4B). Knockdown of ATF4 with asparagine deprivation also led to more pronounced decreases in translation (Fig. 4C). Finally, similar findings were observed in other cell lines, where ATF4 was reduced pharmacologically using ISRIB (H1650 and YLR086) or by gene knockdown (H2030; Supplementary Fig. S4A–S4C). This suggests that sustained inhibition of translation in lung adenocarcinoma cells is a broader consequence of ISR inhibition downstream of eIF2α and ATF4.
Mammalian cells do not catabolize asparagine, but instead use asparagine primarily for protein translation (28, 36). We therefore tested the dependence of lung adenocarcinoma cells on the sole asparagine biosynthetic enzyme ASNS, which is a target of ATF4 and regulated by stress (37). Knockdown of ASNS in the PC9 cells similarly decreased their translation rates during asparagine deprivation (Fig. 4C), phenocopied the decreased clonogenicity caused by ATF4 knockdown, and recapitulated the increase in intracellular ROS (Supplementary Fig. S4D–S4F). Importantly, forced expression of ASNS (Supplementary Fig. S4G and S4H) could restore protein translation when ATF4 was reduced by ISRIB treatment or ATF4 knockdown in the PC9 cells (Fig. 4D and E). Rescue of protein translation by ASNS expression was also sufficient to restore the clonogenicity (Fig. 5A–D) and motility (Fig. 5E and F) of PC9 cells under asparagine deprivation. Finally, ASNS was also required by the H2030 cell line during asparagine starvation, and ASNS expression downstream of ATF4 was also necessary and sufficient for adaptive protein translation and proliferation in this model system (Supplementary Fig. S5A–S5E). We conclude that asparagine bioavailability and protein translation are the primary ATF4 effector functions which mediate adaptive cell migration and proliferation by lung adenocarcinoma cells in response to amino acid stress.
ASNS mediates the synthesis of cell-cycle regulators
To identify steady-state changes in protein and phosphoprotein levels resulting from dual treatment, we performed RPPA. No significant changes were observed in either the ISRIB or asparagine-deprived conditions alone. However, upon dual treatment, subsets of 26 or 54 proteins (of 239) and 10 or 18 phosphoproteins (of 65) were increased or decreased by 1.5-fold (FDR P < 0.05) at 24 or 72 hours, respectively (Supplementary Table S4).
Several of these proteins are involved in cell-cycle regulation, cell adhesion/migration, and signal transduction. Specifically, activation of AKT and its target FOXO3A was increased upon dual treatment (Fig. 6A; red). Western blotting confirmed increased phosphorylation of AKT under acute asparagine deprivation that returned to baseline by 24 hours, but was maintained in the presence of dual treatment (Supplementary Fig. S6A). Although no changes in mTOR phosphorylation or its downstream target 4E-BP1 were observed, phosphorylation of p70S6K, which phosphorylates S6 downstream of mTORC1, was sustained under dual treatment (Supplementary Fig. S6A). These changes correlated with inhibition of TRIB3 and DDIT4 (Supplementary Fig. S6B), two ATF4 targets which are known to inhibit AKT and mTORC1, respectively, during cellular stress (38, 39). Nevertheless, alleviation of these ATF4 feedback mechanisms and activation of the PI3K/mTOR pathway did not restore protein translation under dual treatment (Fig. 4B).
To further identify proteins whose translation and/or synthesis are regulated by the ISR, we integrated RPPA and RNA-seq data, focusing on proteins that were significantly altered by dual treatment independently of changes in their mRNA levels. This included key regulators of cell-cycle progression (CDC25C and cyclin B1; ref. 40) as well as cell adhesion and migration [Rab25 (41), E-Cadherin, fibronectin (FN), and integrin beta 1/CD29 (42); Fig. 6A]. To determine if these proteomic alterations correlated with ASNS expression in human lung adenocarcinoma, we identified proteins that were significantly altered under dual treatment and most positively associated with ASNS mRNA levels in human lung adenocarcinoma from TCGA. Among these, phosphorylation of AKT was negatively correlated with ASNS, whereas cyclin B1 and FN levels were positively correlated with ASNS in human lung adenocarcinomas (Fig. 6B and C). As CDC25C and cyclin B1 are required for progression through the G2–M phase of the cell cycle and cyclin B1 expression in human lung cancers correlates with poor patient outcome (43), Western blotting was used to confirm their expression in the PC9 cells. Asparagine deprivation and/or inhibition of ATF4 alone had mild effects on CDC25C and cyclin B1 levels (Fig. 6D and E). When combined with asparagine deprivation, ISRIB treatment, ATF4 knockdown, or ASNS knockdown more significantly decreased both proteins (Fig. 6D and E; Supplementary Fig. S6C). mRNA levels of CCNB1 remained unchanged following ATF4 knockdown (Supplementary Fig. S6D). In addition, the rates of cyclin B1 turnover did not correlate with the reduction in steady-state levels caused by dual treatment (Supplementary Fig. S6E). Finally, under asparagine-deprived conditions with ISRIB or ATF4 knockdown, ectopic reexpression of ASNS was sufficient to restore levels of CDC25C and cyclin B1 (Fig. 6F and G). Our results demonstrate that the ISR maintains the levels of several key mediators of tumor cell proliferation via adaptive protein biosynthesis.
Dietary asparagine limitation combined with ATF4 inhibition delays lung adenocarcinoma growth in vivo
Because combining ISR inhibition with amino acid deprivation limits the fitness of lung adenocarcinoma cells in vitro, we tested the efficacy of this approach in vivo. First, PC9 cells expressing a control shRNA or shATF4 were injected into the flanks of mice that were placed on an isonitrogenous amino acid–defined diet either containing or lacking asparagine. Analysis of tumor tissue by LC-MS/MS confirmed a reduction in asparagine levels for mice on diets lacking asparagine (Supplementary Fig. S7A). Although ATF4 knockdown alone caused a minor reduction in tumor growth, this did not reach statistical significance (Fig. 7A and B). However, as compared with the control group, mice injected with cells expressing shATF4 had significantly delayed tumorigenic progression and decreased tumor volume when also fed a diet lacking asparagine (Fig. 7A and B). ASNS levels in vivo were effectively reduced by 80% in these residual tumor tissues (Supplementary Fig. S7B).
To test if inhibition of the ISR in combination with dietary asparagine limitation reduces the growth of established tumors, lung adenocarcinoma cells injected into mice were allowed to grow for 10 days at which point they generated palpable tumors. Tumor-bearing mice were then fed diets containing or lacking asparagine and treated with DOX to induce control shRNA, shATF4, or shATF4 with ectopic ASNS re-expression. Similarly, tumor-bearing mice were treated with 5 mg/kg ISRIB or vehicle ISRIB treatment. All treatments were well tolerated in mice over the duration of the experiment, as illustrated by endpoint mouse weights (Supplementary Fig. S7C). Knockdown of ATF4 in combination with asparagine limitation significantly reduced the progression of established tumors (Fig. 7C and D, solid pink circles). ISRIB treatment in combination with asparagine limitation also initially delayed tumor growth, but this effect was not maintained over time (Supplementary Fig. S7D; Fig. 7D, open pink circles). ISRIB treatment did not significantly reduce ASNS expression (Supplementary Fig. S7E), suggesting that the pharmacokinetics of ISRIB and/or targeting of eIF2α may be less efficacious in vivo under these conditions. Importantly, ectopic expression of ASNS was able to rescue the outgrowth of tumors with deficient ISR caused by ATF4 knockdown (Fig. 7C and D; light purple), confirming that this enzyme is a major effector of lung adenocarcinoma tumorigenesis under stress in vivo.
Activation of the ISR has been observed in several types of cancers, but the underlying mechanisms and biological consequences of this are often unclear (11). In this study, we used genetic, pharmacologic, and metabolic perturbations to understand the dependency of lung adenocarcinoma cancer cells on several effectors of the ISR, including eIF2α and ATF4. Moreover, we performed comprehensive transcriptomic as well as proteomic analysis to uncover novel regulatory layers of the ISR in lung adenocarcinoma cancer cells.
Regulation of amino acid stress response by the ISR is a common feature of lung adenocarcinomas
Our biological experiments confirm that a major role of the ISR in lung adenocarcinoma cells is to co-opt amino acid metabolism. We further demonstrate that pharmacologic inhibition of eIF2α in multiple lung adenocarcinoma cell lines can be achieved in vitro using ISRIB, which sensitizes cancer cells to exogenous amino acid availability. Lung cancer cell lines expressing different oncogenic mutations require eIF2α and ATF4 to maintain their fitness in response to amino acid stress, particularly when extracellular asparagine levels are limited. Thus, although this phenomenon may be influenced by genetic alterations in lung adenocarcinoma, it is not necessarily restricted to a particular oncogenic driver.
Inhibition of eIF2α and ATF4 affects not only lung adenocarcinoma cell proliferation, but also anchorage-independent growth and cell motility, suggesting that the ISR may be required for multiple steps of lung adenocarcinoma progression. Accordingly, although knockdown of ATF4 alone did not significantly affect tumorigenesis, it was effective at blocking the growth of PC9 cells in vivo when combined with dietary asparagine limitation. This effect could be rescued by ectopic ASNS re-expression demonstrating that this ATF4 target is rate limiting for lung adenocarcinoma progression in vivo. Asparaginase-mediated depletion of systemic asparagine can be used to inhibit tumorigenesis in childhood leukemia and preclinical models of breast cancer and NSCLC (16, 30, 44). Notably, ASNS overexpression confers asparaginase resistance in leukemia (45). Recent studies indicate that ISRIB can be used as a monotherapy in prostate cancer models (14). In our study, ISRIB initially delayed the growth of lung adenocarcinoma tumors in combination with asparagine limitation, but continuous treatment was not effective, likely due to insufficient reduction of ASNS under the conditions tested herein. Nevertheless, we conclude that ATF4 inhibition paired with exacerbation of cellular stress is a viable therapeutic paradigm for the treatment of lung adenocarcinoma and that pharmacologic optimizations of ISRIB formulation and/or localized delivery are warranted for further preclinical testing in this cancer type.
Molecular link between ATF4-mediated transcriptional response and protein translation
Inhibition of eIF2α can affect global as well as selective protein translation. Our results suggest that the impact of ISRIB during amino acid deprivation of lung adenocarcinoma cells occurs mainly through ATF4 and its target ASNS. ATF4 can activate inhibitors of the AKT and mTOR pathways to suppress translation during nutrient deprivation (38, 39, 46). Consistent with signaling cross-talk, both AKT and mTOR activity were increased by dual treatment. Nevertheless, the net rates of translation in lung adenocarcinoma cells remained decreased upon ISRIB treatment or ATF4 knockdown because of the suppression of ASNS. In mammalian cells, asparagine is primarily used for protein biosynthesis, either through direct protein translation or indirectly as a cotransporter for amino acid uptake (28, 36). ASNS can be activated downstream of the PI3K/mTOR pathways to enhance protein translation in KRAS-mutant NSCLC (16). By performing epistasis experiments, we demonstrated that ASNS expression is also regulated independently of mTORC1 activation and is required for protein translation downstream of eIF2α and ATF4 in lung adenocarcinoma cell lines expressing various oncogenic mutations.
ASNS-mediated protein translation drives adaptive cell proliferation and migration
NRF2 can either independently activate ATF4 transcription or cooperate with ATF4 to induce target genes involved in antioxidant responses (16, 32). Our data reveal an additional molecular link between proteostasis and the control of ROS. Specifically, NRF2 can be regulated downstream of ATF4, via ASNS-mediated protein synthesis. Interestingly, although inhibition of ATF4 causes a decrease in NRF2 protein and accumulation of intracellular ROS, this level of oxidative stress was not responsible for the phenotypes that were incurred under dual treatment. Also, although ATF4 can upregulate autophagy genes (35), this did not correlate with the stress-induced growth arrest of lung adenocarcinoma cells tested here. Alternatively, inhibition of eIF2α, ATF4, or ASNS under asparagine deprivation reduces the expression of a subset of proteins, several of which are important mediators of cell-cycle progression and cell motility. In breast cancer, asparagine bioavailability can also regulate proteins involved in epithelial-to-mesenchymal transition (30). In lung adenocarcinoma cells, ASNS can regulate the G2–M mediator cyclin B1 and cell adhesion/extracellular matrix molecules such as FN. Importantly, ASNS expression is necessary and sufficient for adaptive lung cancer cell proliferation and migration in independent cell lines.
Cyclin B1 expression is associated with poor prognosis in human NSCLC (43), and we found cyclin B1 protein levels, but not CCNB1 mRNA, to correlate with ASNS in human lung adenocarcinomas. As such, the control of cell-cycle progression via cyclin B1 translation is one likely mechanism by which the ISR and ASNS maintain human lung adenocarcinoma progression. Cyclin B1 is not preferentially enriched for asparagine residues, suggesting that the abundance of this amino acid does not necessarily determine which proteins are influenced by ASNS-mediated translation. Alternatively, proteins that have short half-lives may be more sensitive to the limitation of asparagine and ISRIB treatment. Finally, stress granules have been shown to sequester CCNB1 mRNA (47) and could also inhibit protein translation of particular transcripts in response to unresolved amino acid stress and a maladaptive ISR. This potential selective control of proteostasis may provide novel therapeutic opportunities to target lung cancer cells as they attempt to overcome metabolic barriers over the course of treatment and tumor progression.
Disclosure of Potential Conflicts of Interest
D.X. Nguyen reports receiving commercial research grants from AstraZeneca and Leidos. No potential conflicts of interest were disclosed by the other authors.
Conception and design: A.E. Albert, P.D. Cao, D.X. Nguyen
Development of methodology: A.E. Albert, G.W. Cline, Z. Liu
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.E. Albert, S.J. Adua, W.L. Cai, A. Arnal-Estapé, G.W. Cline, M. Zhao
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.E. Albert, W.L. Cai, A. Arnal-Estapé, G.W. Cline, Z. Liu, M. Mariappan, D.X. Nguyen
Writing, review, and/or revision of the manuscript: A.E. Albert, W.L. Cai, S.J. Adua, A. Arnal-Estapé, G.W. Cline, M. Mariappan, D.X. Nguyen
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.E. Albert, M. Zhao, D.X. Nguyen
Study supervision: D.X. Nguyen
This study was funded by grants from the NIH/NCI to D.X. Nguyen (R01CA166376 and R01CA191489) and M. Mariappan (R01GM11738601). A.E. Albert was supported by Training Grants from the NIH (T32GM007205) and the Yale Cancer Center. The MD Anderson Reverse Phase Protein Array Core is funded by NCI #CA16672. We thank Dr. Katerina Politi for comments on the article and for providing the patient-derived NSCLC cell line YLR086. We thank Kevin B. Huang for his work related to this project.
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