The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation
2010; Springer Nature; Volume: 29; Issue: 12 Linguagem: Inglês
10.1038/emboj.2010.81
ISSN1460-2075
AutoresJiangbin Ye, Monika Kumanova, Lori S. Hart, Kelly L. Sloane, Haiyan Zhang, Diego N De Panis, Ekaterina Bobrovnikova-Marjon, J. Alan Diehl, David Ron, Constantinos Koumenis,
Tópico(s)DNA Repair Mechanisms
ResumoArticle14 May 2010free access The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation Jiangbin Ye Jiangbin Ye Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Monika Kumanova Monika Kumanova Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Lori S Hart Lori S Hart Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Kelly Sloane Kelly Sloane Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Haiyan Zhang Haiyan Zhang Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Diego N De Panis Diego N De Panis Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ekaterina Bobrovnikova-Marjon Ekaterina Bobrovnikova-Marjon Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author J Alan Diehl J Alan Diehl Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author David Ron David Ron Department of Medicine, Skirball Institute of Biomolecular Medicine, New York School of Medicine, New York, NY, USA Search for more papers by this author Constantinos Koumenis Corresponding Author Constantinos Koumenis Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Jiangbin Ye Jiangbin Ye Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Monika Kumanova Monika Kumanova Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Lori S Hart Lori S Hart Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Kelly Sloane Kelly Sloane Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Haiyan Zhang Haiyan Zhang Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Diego N De Panis Diego N De Panis Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ekaterina Bobrovnikova-Marjon Ekaterina Bobrovnikova-Marjon Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author J Alan Diehl J Alan Diehl Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author David Ron David Ron Department of Medicine, Skirball Institute of Biomolecular Medicine, New York School of Medicine, New York, NY, USA Search for more papers by this author Constantinos Koumenis Corresponding Author Constantinos Koumenis Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Author Information Jiangbin Ye1, Monika Kumanova1, Lori S Hart1, Kelly Sloane1, Haiyan Zhang1, Diego N De Panis1, Ekaterina Bobrovnikova-Marjon2, J Alan Diehl2, David Ron3 and Constantinos Koumenis 1 1Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 2Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 3Department of Medicine, Skirball Institute of Biomolecular Medicine, New York School of Medicine, New York, NY, USA *Corresponding author. Department of Radiation Oncology, University of Pennsylvania School of Medicine, John Morgan Building, Rm 185, 3620 Hamilton Walk, Philadelphia, PA 19104-6072, USA. Tel.: +1 215 898 0076; Fax: +1 215 898 0090; E-mail: [email protected] The EMBO Journal (2010)29:2082-2096https://doi.org/10.1038/emboj.2010.81 There is a Have you seen ...? (June 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2α (eIF2α) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho-eIF2α were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2-eIF2α-ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti-tumour approaches. Introduction Earlier studies in mouse embryonic fibroblasts (MEFs) showed that the basic leucine zipper transcription factor ATF4 is a critical regulator of genes involved in redox balance and maintenance of amino acid metabolism. Consequently, ATF4−/− MEFs require non-essential amino acids (NEAAs) and antioxidants to survive and proliferate (Harding et al, 2003). ATF4 also appears to have multiple functions during development: ATF4 knockout mice exhibit abnormal lens formation, growth retardation, anemia and delayed bone development (Tanaka et al, 1998; Masuoka and Townes, 2002; Yang et al, 2004). Although ATF4 can be transcriptionally regulated (Siu et al, 2002), it is the translational upregulation of ATF4 that has received the most attention, because of the unusual mode of translational regulation of its mRNA in response to stress through phosphorylation of the eukaryotic initiation factor 2α (eIF2α) (Harding et al, 2000; Lu et al, 2004; Vattem and Wek, 2004). The endoplasmic reticulum kinase PERK (activated by misfolded/unfolded proteins in the ER) or the cytoplasmic kinase GCN2 (activated by amino acid deprivation) phosphorylate eIF2α at Ser51, thereby downregulating global translation. Paradoxically, a group of stress-responsive mRNAs that include ATF4 are translated more efficiently when eIF2α is phosphorylated. In the case of ATF4, this is due to the presence of the two upstream open reading frames located in the 5′UTR of the mRNA. These two elements repress ATF4 translation under unstressed conditions but enable its translation under stressed conditions (eIF2α phosphorylation). This translational regulation model was first characterized in yeast and later found to also exist in mammalian cells (Hinnebusch, 1984; Mueller and Hinnebusch, 1986; Harding et al, 2000; Vattem and Wek, 2004). GCN2 is a high molecular weight protein kinase activated by uncharged tRNA (Wek et al, 1990, 1995; Ramirez et al, 1992). Activated GCN2 phosphorylates eIF2α to translationally upregulate ATF4, which in turn increases amino acid biosynthetic and transport pathways (Harding et al, 2000, 2003). GCN2 knockout mice are viable and fertile and display no gross phenotypic abnormalities unless fed diets lacking a single amino acid (Zhang et al, 2002; Anthony et al, 2004). Other than maintaining amino acid homeostasis, GCN2 also regulates synaptic plasticity and memory (Costa-Mattioli et al, 2005), feeding behaviour (Hao et al, 2005; Maurin et al, 2005), as well as lipid metabolism (Guo and Cavener, 2007). GCN2 is also activated by UV radiation and mediates NFκB signalling (Deng et al, 2002; Jiang and Wek, 2005). In the tumour microenvironment, the abnormal development of vasculature results in insufficient blood supply, which is the major reason for the development of acute and chronic hypoxia and has been associated with deprivation of glucose and other nutrients. Earlier, we showed that PERK activation and the resulting eIF2α phosphorylation increase the ability of transformed cells to survive under hypoxia in vitro and in vivo and promote tumour growth (Bi et al, 2005). In the same study, we reported that as a downstream target of PERK and phospho-eIF2α, ATF4 also contributes to hypoxia resistance in MEFs. We and others reported that ATF4 overexpression is elevated in primary tumour tissues and co-localizes with hypoxic regions (Ameri et al, 2004; Bi et al, 2005). However, the precise function of ATF4 in tumour cell survival and proliferation has not been elucidated. In this study, we report that ATF4 is necessary for tumour cells to maintain homeostasis of amino acid metabolism and that activation of GCN2-ATF4-asparagine synthetase (ASNS) pathway promotes tumour cell survival under nutrient (amino acid or glucose) deprivation. GCN2-eIF2α pathway is activated in various human and mouse tumour tissues. Deficiency of ATF4 or GCN2 severely inhibits tumour growth in vivo. Together, these results suggest that GCN2-ATF4-ASNS pathway is a promising target for tumour therapy. Results ATF4 expression is required for survival and proliferation of fibrosarcoma and colorectal adenocarcinoma cells in the absence of non-essential amino acid supplementation To investigate the function of ATF4 in tumour cell proliferation and survival, plasmids expressing ATF4-specific shRNA (pSM2-shATF4) or non-targeting shRNA (pLKO-shNT) were transfected into HT1080 (human fibrosarcoma) or DLD1 (human colorectal adenocarcinoma) cells. Two established HT1080 shATF4 clones (shATF4.cl3 and shATF4.cl4) and one DLD1 shATF4 clone showed 60–70% reduction of ATF4 mRNA levels compared with corresponding shNT clones (Supplementary Figure S1A). As the basal ATF4 protein levels are low in unstressed cells, we treated cells with the ER stress-inducing agent thapsigargin to upregulate ATF4. Consistent with mRNA levels, both HT1080 and DLD1 shATF4 clones showed no ATF4 induction after treatment with thapsigargin (Figure 1A). Figure 1.Knockdown of ATF4 inhibits tumour cell survival and proliferation. (A) ATF4 protein levels from the nuclear fractions of thapsigargin-treated (Tg, 1 μM, 4 h) or DMSO-treated cells. Lamin A/C was used as a loading control. (B) Survival of HT1080 and DLD1 cells cultured in the presence or absence of NEAA (100 μM) measured by MTT assay 48 h after plating (Data represent mean±s.e.m., n=3, *P<0.05). Cell survival was normalized to control (shNT cells without NEAA). (C) Cell proliferation assay. HT1080 cells were plated in DMEM with/without NEAA for 24 h. (Left panel) Fluorescent staining for nuclei (Hoechst, blue) and proliferating cells with EdU (red). (Right panel) Three randomly selected photographs were selected and numbers of EdU positive and total nuclei were counted. Percentage proliferation index was calculated by dividing the number of proliferating nuclei by the total number of nuclei. (Data represent mean±s.e.m., n=3, *P<0.05). Download figure Download PowerPoint It was reported earlier that ATF4−/− MEFs require the presence of NEAAs and antioxidant such as β-mercaptoethanol (β-ME) to survive (Harding et al, 2003) (Supplementary Figure S2). Similar to SV40-immortalized ATF4−/− MEFs, tumour cells expressing ATF4 shRNA showed significantly reduced survival in the absence of NEAA (Figure 1B). A long-term growth assay suggested that shATF4 clones have defects in cell survival and proliferation rates (Supplementary Figure S1B). In contrast, cell survival of ATF4 knockdown cells was not affected by adding β-ME at concentrations from 25 μM to 0.2 mM (data not shown). Transiently knocking down ATF4 also reduced cell survival, indicating that this effect was not due to clonal effects during selection (Supplementary Figure S1C). Reduced cell survival could result from decreased cell proliferation and/or increased cell death. By analysing the levels of fluorescent EdU incorporation in exponentially growing cells, we found that HT1080.shATF4 cells showed a 35% reduction in cell proliferation compared with shNT cells (Figure 1C). Similar findings were seen in DLD1 cells (Supplementary Figure S3A). Adding NEAA to the medium led to full recovery of cell proliferation in both knockdown cell lines (Supplementary Figure S3A). Cell-cycle analysis also showed that shATF4 cells had an increased G1 population compared with shNT cells (Supplementary Figure S3B), indicating that ATF4 knockdown caused G1/S arrest in tumour cells. Addition of NEAA partially reversed the G1 arrest. These data suggest that ATF4 deficiency induces amino acid starvation, which causes G1/S cell-cycle arrest and reduced proliferation. Knockdown of ATF4 in transformed cells induces apoptosis When the ATF4 knockdown cells were cultured in the absence of NEAA, morphological features of apoptosis such as membrane blebbing and cell shrinkage were observed. These apoptotic phenotypes were diminished by the addition of NEAA (Figure 2A). shATF4 cells also had higher levels of cleaved PARP, an apoptosis marker, which was similarly reduced in the presence of NEAA (Figure 2B). To further analyse the levels of apoptosis in shATF4 cells cultured without NEAA, we measured caspase3/7 activities. shATF4 cells exhibited an over 13-fold increase in basal caspase3/7 activity compared with shNT cells; similarly, supplementation with NEAA significantly reduced the caspase activities (Figure 2C). Figure 2.Knockdown of ATF4 induces apoptosis in tumour cells. (A) Phase-contrast images of HT1080 shNT and shATF4 cells growing in DMEM with or without NEAA for 24 h. Images shown were taken at × 400 magnification. (B) Immunoblot for cleaved PARP in HT1080 shNT and shATF4 cells. β-Actin was used as a loading control. HT1080 cells were incubated in DMEM with/without NEAA for 24 h. (C) Caspase3/7 activities normalized to total number of cells. HT1080 cells were incubated in DMEM with/without NEAA for 24 h. (D) Phase-contrast images of HT1080 shNT and shATF4 cells infected with the control (Av-Cre) or adenovirus expressing mouse ATF4 (AV-mATF4) indicated at × 400 magnification. After infection, cells were incubated in DMEM without NEAA. Images were taken 24 h after infection. (E) Relative cell survival in infected cells using an MTT assay. Equal numbers of cells were plated in DMEM without NEAA 24 h after infection. MTT assay was performed after 48 h. Cells infected under the same condition were also collected at 24 h after infection for ATF4 immunoblot (bottom panel). (Data represent mean±s.e.m., n=3, *P<0.05). Download figure Download PowerPoint To exclude the possibility that the defects of shATF4 cells were due to off-target effects of the shRNA or selection-induced mutations, we overexpressed full-length mouse ATF4 that was not targeted by the shRNA against human ATF4 using an adenoviral vector. Overexpression of mATF4 significantly increased cell survival and blocked the apoptotic phenotype of shATF4 cells (Figures 2D and E). These findings further support a pro-survival function of ATF4 in these tumour cells. Knockdown of ATF4 induces a pro-survival autophagic response Autophagy is a lysosomal-dependent intracellular degradation process that is activated by certain stresses, primarily by nutrient starvation. As shATF4 cells rely on NEAA to survive, we hypothesized that autophagy might be induced in the HT1080.shATF4 cells as an initial pro-survival response in the absence of NEAA. This hypothesis was supported by the fact that in the absence of NEAA, shATF4 cells had a smaller size compared with shNT cells and cytoplasmic vacuoles (a sign of autophagosome formation) were observed (data not shown). shATF4 cells had elevated levels of the autophagy marker microtubule-associated protein light chain (LC) 3-II compared with shNT cells (Figure 3A), which were reduced to basal levels by adding NEAA, indicating that autophagy was induced in shATF4 cell because of NEAA shortage. Figure 3.Knockdown of ATF4 induces protective autophagy in tumour cells. (A) Immunoblot for the autophagy marker cleaved-LC3 from whole cell lysates in HT1080 shNT and shATF4 cells incubated in DMEM with/without NEAA for 24 h. β-Actin was used as a loading control. (B) Electron microscopy imaging. Arrows point to double-membrane-containing autophagosomes. HT1080 cells were incubated in DMEM with/without NEAA for 24 h. (C) Top: HT1080 shNT and shATF4 cells were transfected with pCMV-GFP-LC3 and incubated in DMEM. After 48 h, cells were stained with Hoechst before imaging. Bottom: Quantitation of cells with autophagic (punctate) morphology. Percentage autophagic cells were calculated after normalization to the total number of cells with GFP signal. Data represent mean (±s.e.m., n=3). (D) HT1080 cells were transfected with 100 nM of non-targeting siRNA (siNT) or siRNA targeting Atg7 (siAtg7). Atg7, LC3 and cleaved-PARP levels were analysed by immunoblotting 24 h after transfection. α-Tubulin was used as a loading control. (E) HT1080 cells were transfected with 100 nM non-targeting siRNA (siNT) or siRNA targeting ATG7 (siATG7). After 72 h, cell survival of HT1080 and DLD1 cells was measured by MTT assay. Experiment was carried out in triplicate. (Data represent mean±s.e.m., n=3, *P<0.05). Download figure Download PowerPoint Under electron microscopy, the HT1080.shNT cells exhibited typical fibroblast morphology with intact ER and mitochondria, whereas the shATF4 cells were smaller, rounded and contained double-membrane autophagosomes, further confirming extensive induction of autophagy in shATF4 cells (Figure 3B, arrows pointing to autophagosomes). Similar to LC3 processing, addition of NEAA reversed the autophagic phenotype. The induction of autophagy was also confirmed by expressing GFP-LC3 in HT1080 cells. In shNT cells, the GFP signal was distributed evenly throughout the cytoplasm. However, in shATF4 cells, the signal was concentrated in green dots or ring-shaped structures, indicating the formation of autophagosomes (Figure 3C). To test whether the autophagy induced in shATF4 cells was a cytoprotective stress response, an siRNA targeting Atg7 (an E1-like ubiquitin conjugating enzyme required for autophagosomes maturation) was used to inhibit autophagy. When Atg7 levels were reduced and autophagy was blocked, the shATF4 cells showed increased apoptosis compared with shNT cells (Figures 3D and E). These results indicate that the autophagy induced by loss of ATF4 in the HT1080 cells promotes survival. The combination of losing ATF4 and the inhibition of autophagy results in a cooperative enhancement of cell death. Addition of Asn in trans or expression of ASNS, rescues the survival of shATF4 cells The mixture of NEAAs used in the experiments described above was comprised of seven amino acids: Ala, Asp, Asn, Glu, Gly, Pro and Ser. Each amino acid was added to a final concentration of 100 μM in DMEM. Regular DMEM contains 400 μM Gly and Ser but not the other five amino acids (Supplementary Table S1). To determine which amino acid(s) was/were responsible for mediating the pro-survival effects of NEAA, individual amino acids were added into DMEM at a 100 μM final concentration in cultured shATF4 cells. The results indicated that Asn, but not any other individual amino acid, could rescue the survival of shATF4 cells (Figure 4A). An even more substantial pro-survival effect of Asn was observed in a long-term clonogenic survival assay (Figure 4B). Figure 4.Supplementation with Asn or overexpression of ASNS rescues survival of shATF4 cells. (A) Survival of HT1080 (left) or DLD1 (right) cells grown in DMEM with indicated amino acids supplemented at a concentration of 100 μM for 48 h. (B) Clonogenic survival of HT1080 shNT/shATF4 cells. Left: Picture of a representative experiment. Right: Clonogenic survival of HT1080 shNT/shATF4 cells. (Data represent mean±s.e.m., n=4, *P<0.05.) (C) ASNS mRNA levels in HT1080 shNT/shATF4 cells measured using real-time RT–PCR. (Data represent mean±s.e.m., n=3, *P<0.05.) (D) ASNS expression and cell survival in transfected HT1080 cells. (Left panel) Top bands represent HA-tagged ASNS, lower bands represent endogenous ASNS. (Right panel) Survival of HT1080 shNT and shATF4 cells after indicated treatments. After 24 h, transfected cells were harvested for immunoblot or plated for MTT assay (48 h). (Data represent mean±s.e.m., n=3, *P 70% in shATF4 cells compared with shNT cells (Figure 4C). Moreover, adding Asn to HT1080.shATF4 cells rescued NEAA deprivation-induced G1 arrest (Supplementary Figure S3B), a finding that is consistent with reports that ASNS deficiency can induce a G1 arrest (Greco et al, 1987; Gong and Basilico, 1990). Furthermore, overexpression of ASNS in shATF4 cells partially rescued cell survival (Figure 4D) and adding Asn repressed both apoptosis and autophagy in shATF4 cells (Figure 6A). This result suggests that ASNS is an important enzyme for maintaining intracellular asparagine levels, which are crucial for tumour cell survival and cell-cycle progression. The amino acid glutamine (Gln) serves not only as a substrate for nucleotide and protein synthesis, a precursor for the synthesis of Asn, but also as an important energy source for tumour cells (Reitzer et al, 1979; DeBerardinis et al, 2007). To produce Asn, ASNS transfers the amino group from Gln to Asp (Figure 5A). As ASNS requires Gln to synthesize Asn, we wanted to test whether shATF4 cells were more sensitive to Gln deprivation than shNT cells. To test this, shNT and shATF4 cells were cultured in DMEM with/without 4 mM glutamine. MTT assays showed that shATF4 cells showed about 50% reduction in survival in the absence of Gln, whereas the shNT cells exhibited only a 25% reduction after 48 h incubation (Figure 5B). Interestingly, adding Asn (100 μM final concentration) to Gln-deprived cells could partially rescue cell survival (Figure 5C), suggesting that producing Asn may also be an important function of Gln, at least in this tumour cell line. In summary, ATF4 deficiency severely inhibits tumour cell survival in vitro, which is primarily due to Asn deprivation. Figure 5.Knockdown of ATF4 increases sensitivity to glutamine deprivation. (A) The biosynthetic reaction catalysed by ASNS. (B) Survival of HT1080 (left panel) or DLD1 (right panel) cells grown in DMEM with/without 4 mM L-glutamine for 48 h. (C) Survival of HT1080 cells in DMEM with/without Gln or Asn for 48 h. Gln: 4 mM, Asn: 100 μM. (Data represent mean±s.e.m., n=3, *P<0.05). Download figure Download PowerPoint Activation of the GCN2-eIF2α pathway under amino acid deprivation promotes cell survival, upregulates p21 (cip1/waf1) and activates autophagy We hypothesized that if shATF4 cells are deficient in the biosynthesis of NEAAs, this should lead to the activation of the upstream kinase GCN2, completing an autoregulatory feedback loop. Indeed, we found that GCN2 was phosphorylated in HT1080.shATF4 cells and adding Asn or NEAA repressed this phosphorylation (Figure 6A), suggesting that knocking down ATF4 reduces ASNS expression, causing an Asn deficiency, which activated GCN2. eIF2α, the substrate of GCN2, was also phosphorylated in shATF4 cells in response to NEAA and similar to GCN2, its phosphorylation was repressed by addition of Asn or NEAA in trans. The CDK inhibitors p21 and p27 have a critical function in G1/S cell-cycle arrest in response to stress, and it had been reported that they can be induced by amino acid deprivation (Leung-Pineda et al, 2004). shATF4 cells constitutively expressed high levels of p21, which were substantially reduced by adding NEAA or Asn; however, p27 levels were unaffected (Figure 6A). This is consistent to an earlier report that ATF4-null primary mouse bone marrow stromal cells have increased p21 but not p27 expression (Zhang et al, 2008). The induction of p21 is likely responsible for the G1/S cell-cycle arrest in shATF4 cells. Figure 6.Activation of GCN2-eIF2α pathway under amino acid deprivation promotes cell survival, upregulates ATF4 and p21, and activates autophagy. (A) HT1080 shNT and shATF4 cells were incubated in the media indicated for 24 h. Whole cell lysates were harvested for immunoblot (IB) or immunoprecipitation (IP) with the indicated antibodies. (B) GCN2+/+ and GCN2−/− MEFs were incubated with/without 4 mM Gln for 24 h and immunoblotting was performed. (C) eIF2α wt or eIF2α S51A mutant MEFs were incubated with/without 4 mM Gln for 24 h and immunoblotting was performed with indicated antibodies. Numbers below the blots of p-eIF2a and ASNS indicate fold change in levels normalized to those of α-tubulin. Analysis was performed using the Scion Image version of the NIH Image shareware image analysis program. (D) GCN2+/+ and GCN2−/− MEFs were incubated with or without Met or Gln for 48 h. Cell survival was analysed using MTT assay. (Data represent mean±s.e.m., n=3, *P<0.05.) (E) Wild-type, GCN2−/− and eIF2α S51A mutant MEFs were cultured without 4 mM Gln for 1 h or 3 h, cell lysates were subjected to immunoblotting. (F) HT1080 cells stably transfected with shNT or shGCN2 plasmid were cultured without Gln for 1 or 3 h, cell lysates were subjected to immunoblotting with indicated antibodies. Download figure Download PowerPoint As GCN2 is the molecular sensor of amino acid deprivation that induces translational upregulation of ATF4, we tested whether GCN2 activation promotes tumour cell survival when a single amino acid is removed from the culture media. SV40 immortalized, Ras-transformed GCN2+/+ and GCN2−/− MEFs were cultured in DMEM with or without Gln. Under Gln deprivation, GCN2+/+ cells showed enhanced eIF2α phosphorylation and upregulation of ATF4, ASNS and p21, whereas the GCN2−/− cells failed to activate this pathway and had increased levels of cleaved caspase3 (Figure 6B). These results showed that the induction of p21 under amino acid starvation depends on GCN2 activation. As eIF2α is currently the sole known substrate of GCN2, we wanted to further investigate whether the induction of ATF4 and p21 was dependent on eIF2α phosphorylation. To test this, eIF2α wild-type or eIF2α S51A mutant MEFs (a Ser-Ala mutation blocks eIF2α phosphorylation) were incubated in DMEM with or without Gln. Similar to the GCN2−/− cells, eIF2α S51A mutant cells were unable to induce ATF4, ASNS or p21 in the absence of Gln, but had increased levels of apoptosis (Figure 6C). In full DMEM (i.e. +Gln, +Met), GCN2−/− cells showed 25% reduction in cell survival compared with wild-type cells after 48 h incubation, whereas Met or Gln deprivation further reduced the cell survival of GCN2−/− cells to around 50% or 4%, respectively (Figure 6D). In summary, the activation of GCN2-eIF2α-ATF4 pathway is necessary for tumour cell survival under amino acids starvation. It was reported earlier that GCN2 activation and eIF2α phosphorylation induce autophagy in yeast (Talloczy et al, 2002). We also observed a correlation between GCN2 activation with LC3 cleavage in HT1080.shATF4 cells (Figure 6A), suggesting that GCN2 could be the molecular switch that senses amino acid shortage and induces autophagy in mammalian cells. To test this, wild-type, GCN2−/− and eIF2α S51A mutant MEFs were incubated in Gln-free media. GCN2−/− cells had significant lower LC3 processing compared with wild-type cells in response to Gln starvation. eIF2α S51A mutant cells could not induce LC3 processing at all (Figure 6E). To confirm the function of GCN2 in autophagy induction in human tumour cells, HT1080 cells stably transfected with shNT or shGCN2 plasmid, were incubated in Gln-free media. Autophagy was analysed by blotting for an autophagy marker p62/SQSTM1, a long-lived protein that is rapidly degraded during autophagy progression (Klionsky et al, 2008). p62 was degraded in shNT cells on Gln deprivation but stabilized in shGCN2 cells (Figure 6F). In conclusion, a functional GCN2-eIF2α pathway is required for amino acid starvation-activated autophagy in transformed cells. Activation of phospho-eIF2α-ATF4 pathway under glucose deprivation depends on GCN2 Induction of ATF4-ASNS pathway by glucose deprivation has been o
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