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Activating transcription factor 4 (ATF4) is a transcription factor induced under severe hypoxia and a component of the PERK pathway involved in the unfolded protein response (UPR), a process that protects cells from the negative consequences of endoplasmic reticulum (ER) stress. In this study, we have used small interfering RNA (siRNA) and microarray analysis to provide the first whole-genome analysis of genes regulated by ATF4 in cancer cells in response to severe and prolonged hypoxic stress. We show that ATF4 is required for ER stress and hypoxia-induced expansion of autophagy. MAP1LC3B (LC3B) is a key component of the autophagosomal membrane, and in this study we demonstrate that ATF4 facilitates autophagy through direct binding to a cyclic AMP response element binding site in the LC3B promoter, resulting in LC3B upregulation. Previously, we have shown that Bortezomib-induced ATF4 stabilization, which then upregulated LC3B expression and had a critical role in activating autophagy, protecting cells from Bortezomib-induced cell death. We also showed that severe hypoxia stabilizes ATF4. In this study, we demonstrate that severe hypoxia leads to ER stress and induces ATF4-dependent autophagy through LC3 as a survival mechanism. In summary, we show that ATF4 has a key role in the regulation of autophagy in response to ER stress and provide a direct mechanistic link between the UPR and the autophagic machinery.

The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacA s1m1 -induced cell death and apoptosis, and apoptosis inhibitor Z-VAD had no significant effect on autophagy induced by VacA s1m1 . Thus, these results suggested that VacA causes autophagic cell death via ER stress in gastric epithelial cells.

The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1 −/− mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart. Beneficial and detrimental effects have been ascribed to the different Thrombospondin (Thbs) proteins in the adult mammalian heart. Here, the authors show that Thbs1-mediated activation of PERK-eIF2α-ATF4-induced autophagy regulates adult cardiomyocyte size in the stressed heart.

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis. When building healthy tissue, the human body must carefully control the growth of new cells to prevent them from becoming cancerous. A core component of this regulation is the protein mTORC1, which responds to various growth-stimulating factors and nutrients, and activates the chemical reactions cells need to grow. Part of this process involves controlling ‘nutrient-sensing transcription factors’ – proteins that regulate the activity of specific genes based on the availability of different nutrients. One of these nutrient-sensing transcription factors, ATF4, has recently been shown to be involved in some of the processes triggered by mTORC1. The role this factor plays in how cells respond to stress – such as when specific nutrients are depleted, protein folding is disrupted or toxins are present – is well-studied. But how it reacts to the activation of mTORC1 is less clear. To bridge this gap, Torrence et al. studied mouse embryonic cells and human prostate cancer cells grown in the laboratory, to see whether mTORC1 influenced the behavior of ATF4 differently than cellular stress. Cells were treated either with insulin, which activates mTORC1, or an antibiotic that sparks the stress response. The cells were then analyzed using a molecular tool to see which genes were switched on by ATF4 following treatment. This revealed that less than 10% of the genes activated by ATF4 during cellular stress are also activated in response to mTORC1-driven growth. Many of the genes activated in both scenarios were involved in synthesizing and preparing the building blocks that make up proteins. This was consistent with the discovery that ATF4 helps mTORC1 stimulate growth by promoting protein synthesis. Torrence et al. also found that mTORC1’s regulation of ATF4 stimulated the synthesis of glutathione, the most abundant antioxidant in cells. The central role mTORC1 plays in controlling cell growth means it is important to understand how it works and how it can lead to uncontrolled growth in human diseases. mTORC1 is activated in many overgrowth syndromes and the majority of human cancers. These new findings could provide insight into how tumors coordinate their drive for growth while adapting to cellular stress, and reveal new drug targets for cancer treatment.

Activating transcription factor 4 (ATF4), a member of the ATF/cAMP response element-binding (CREB) family, plays a critical role as a stress-induced transcription factor. It orchestrates cellular responses, particularly in the management of endoplasmic reticulum stress, amino acid deprivation, and oxidative challenges. ATF4's primary function lies in regulating gene expression to ensure cell survival during stressful conditions. However, when considering its involvement in ferroptosis, characterized by severe lipid peroxidation and pronounced endoplasmic reticulum stress, the ATF4 pathway can either inhibit or promote ferroptosis. This intricate relationship underscores the complexity of cellular responses to varying stress levels. Understanding the connections between ATF4, ferroptosis, and endoplasmic reticulum stress holds promise for innovative cancer therapies, especially in addressing apoptosis-resistant cells. In this review, we provide an overview of ATF4, including its structure, modifications, and functions, and delve into its dual role in both ferroptosis and cancer.

Activating transcription factor 4 (ATF4) is a critical mediator of metabolic and oxidative homeostasis and cell survival. ATF4 is elevated in response to diverse microenvironmental stresses, including starvation, ER stress damages and exposure to toxic factors. Here we show that ATF4 expression fosters the malignancy of primary brain tumors (WHO grade III and IV gliomas) and increases proliferation and tumor angiogenesis. Hence, ATF4 expression promotes cell migration and anchorage-independent cell growth, whereas siRNA-mediated knockdown of ATF4 attenuates these features of malignancy in human gliomas. Further experiments revealed that ATF4-dependent tumor promoting effects are mediated by transcriptional targeting the glutamate antiporter xCT/SCL7A11 (also known as system Xc - ). Thus, xCT is elevated as a consequence of ATF4 activation. We further found evidence that ATF4-induced proliferation can be attenuated by pharmacological or genetic xCT inhibition and ferroptosis inducers such as sorafenib, erastin and GPx4 inhibitor RSL3. Further, fostered xCT expression promotes cell survival and growth in ATF4 knockdown cells. Moreover, increased xCT levels ameliorate sorafenib and erastin-induced ferroptosis. Conversely, ATF4 knockdown renders cells susceptible for erastin, sorafenib and RSL3-induced ferroptosis. We further identified that ATF4 promotes tumor-mediated neuronal cell death which can be alleviated by xCT inhibition. Moreover, elevated ATF4 expression in gliomas promotes tumor angiogenesis. Noteworthy, ATF4-induced angiogenesis could be diminished by ferroptosis inducers erastin and by GPx4 inhibitor RSL3. Our data provide proof-of-principle evidence that ATF4 fosters proliferation and induces a toxic microenvironmental niche. Furthermore, ATF4 increases tumor angiogenesis and shapes the vascular architecture in a xCT-dependent manner. Thus, inhibition of ATF4 is a valid target for diminishing tumor growth and vasculature via sensitizing tumor cells for ferroptosis.

Understanding the intrinsic mediators that render CD8 + T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8 + T cells. Chop expression is increased in tumor-infiltrating CD8 + T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8 + T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8 + T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8 + T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity. T-cell function impairment is one of the major determinants of tumour immune evasion. Here, the authors show that the hostile conditions in the tumour microenvironment lead to C/EBP homologous-protein upregulation in T cells via ER stress, resulting in repression of T-bet and consequent inhibition of CD8 + T cell function.”

The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis - a specialized form of apoptosis - following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5′ leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR . Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5′ leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo. Translation of ATF4 mRNA is stimulated during the integrated stress response. Here the authors show that two noncanonical translation initiation factors eIF2D and DENR are required for translational induction of ATF4 mRNA in Drosophila and human cells.