Activating Transcription Factor 4 Activity Research Articles

Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high metastasis and therapeutic resistance. Activating transcription factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to survive. Prior research and data reported provide evidence that high ATF4 expression correlates with worse overall survival in PDAC. Tomatidine, a natural steroidal alkaloid, is associated with inhibition of ATF4 signaling in multiple diseases. Here, we discovered that invitro and invivo tomatidine treatment of PDAC cells inhibits tumor growth. Tomatidine inhibited nuclear translocation of ATF4 and reduced the transcriptional binding of ATF4 with downstream promoters. Tomatidine enhanced gemcitabine chemosensitivity in 3D ECM-hydrogels and invivo. Tomatidine treatment was associated with induction of ferroptosis signaling validated by increased lipid peroxidation, mitochondrial biogenesis, and decreased GPX4 expression in PDAC cells. This study highlights a possible therapeutic approach utilizing a plant-derived metabolite, tomatidine, to target ATF4 activity in PDAC.

TGF-β Promotes Metabolic Reprogramming in Lung Fibroblasts via mTORC1-dependent ATF4 Activation.

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by the TGF-β (transforming growth factor-β)-dependent differentiation of lung fibroblasts into myofibroblasts, which leads to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-β upregulates the expression of the enzymes of the de novo serine-glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here, we demonstrate that TGF-β promotes accumulation of ATF4 (activating transcription factor 4), which is required for increased expression of the serine-glycine synthesis pathway enzymes in response to TGF-β. We found that induction of the integrated stress response (ISR) contributes to TGF-β-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-β is activation of mTORC1 (mTOR Complex 1). TGF-β activates the PI3K-Akt-mTOR pathway, and inhibition of PI3K prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1, had no effect on TGF-β-mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1, completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-β. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.

IL-24 Promotes Apoptosis through cAMP-Dependent PKA Pathways in Human Breast Cancer Cells.

Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24.

Abstract 2035: Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress

Abstract Background. Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer which is characterized by a poor survival rate and high incidence of metastases. The integrated stress response (ISR) is activated under stress conditions (hypoxia, nutrient deprivation, or endoplasmic reticulum stress) and reduces the protein synthesis through phospho-eIF2α/ATF4 (activating transcription factor 4) to regulate cell fate. ATF4, which is overexpressed in breast cancer including TNBC, regulates tumor growth, autophagy, drug resistance, and metastasis during ISR through PERK and GCN2 pathways. We have reported enhanced ATF4 expression in unstressed MCF10A cells treated with TGFβ1. Here, we investigate the potential TGFβ-mediated stress-independent control of ATF4 activity and its impact on the TNBC-associated metastasis. Methods. SUM159PT and BT549 TNBC cell lines were treated with human recombinant TGFβ1 (10 ng/ml) and the TGFBRI kinase inhibitor LY2157299 (5µM) for 72h. Effects of TGFβ on ATF4 expression were assessed by silencing SMAD2/3 and SMAD4 with TGFβ1 for 24h in SUM159PT and BT549 cells. Changes in ATF4 expression by IRS were assessed after PERK, GCN2, PKR, HRI, and eIF2α inhibition with TGFβ1 for 72h in SUM159PT, BT549 and MDA-MB-231 cell lines. ATF4 expression levels were determined by RT-PCR and/or western blot. Effects on TGFβ-induced metastasis were analyzed by ATF4 knockdown for 48h by using two different siRNA sequences (#1 and #2) following treatment with TGFβ1 for 24h. Migration and invasion were performed by wound healing and transwell assays, respectively. Results were compared to a scrambled siRNA as negative control (SCR). Results. Our results show that ATF4 expression (at RNA and protein levels) was abrogated by LY2157299 upon TGFβ activation, suggesting that ATF4 is active downstream of TGFBRI. This result was supported by further SMAD2/3 and SMAD4 knockdown following treatment with TGFβ1, which was correlated with decreased ATF4 expression. Additionally, we assessed whether ATF4 inhibition can reduce the TGFβ-induced metastatic properties of TNCB cell lines. We found that ATF4 depletion inhibited migration and invasiveness in the three cell lines tested. ATF4 is known to exert a pro-metastatic role downstream of ISR through PERK and GCN2 pathways. Our results demonstrate that, upon treatment with TGFβ1, knockdown of the IRS mediators PERK, PKR, GCN2, HRI, and eIF2α did not correlate with a consistent decrease of ATF4 levels in the three cell lines. Conclusion. In conclusion, our results show for the first time that ATF4 is a downstream target of the canonical TGFβ/SMAD pathway in a ISR-independent fashion, and its depletion correlates with an inhibition of the TGFβ1-mediated migration and invasion of TNBC cell lines. Therefore, ATF4 may represent a therapeutic target in TNBC patients with active TGFβ signaling pathway. Citation Format: Jenny C. Chang, Alberto Ramirez, Maria P. Molina, Francisca E. Cara, Wei Qian, Wen Chen, Anthony J. Kozielski, Roberto R. Rosato, Juan A. Marchal, Jose A. Lorente, Pedro Sanchez-Rovira, Sergio Granados-Principal. Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2035.

Identification and Small Molecule Inhibition of an Activating Transcription Factor 4 (ATF4)-dependent Pathway to Age-related Skeletal Muscle Weakness and Atrophy

Aging reduces skeletal muscle mass and strength, but the underlying molecular mechanisms remain elusive. Here, we used mouse models to investigate molecular mechanisms of age-related skeletal muscle weakness and atrophy as well as new potential interventions for these conditions. We identified two small molecules that significantly reduce age-related deficits in skeletal muscle strength, quality, and mass: ursolic acid (a pentacyclic triterpenoid found in apples) and tomatidine (a steroidal alkaloid derived from green tomatoes). Because small molecule inhibitors can sometimes provide mechanistic insight into disease processes, we used ursolic acid and tomatidine to investigate the pathogenesis of age-related muscle weakness and atrophy. We found that ursolic acid and tomatidine generate hundreds of small positive and negative changes in mRNA levels in aged skeletal muscle, and the mRNA expression signatures of the two compounds are remarkably similar. Interestingly, a subset of the mRNAs repressed by ursolic acid and tomatidine in aged muscle are positively regulated by activating transcription factor 4 (ATF4). Based on this finding, we investigated ATF4 as a potential mediator of age-related muscle weakness and atrophy. We found that a targeted reduction in skeletal muscle ATF4 expression reduces age-related deficits in skeletal muscle strength, quality, and mass, similar to ursolic acid and tomatidine. These results elucidate ATF4 as a critical mediator of age-related muscle weakness and atrophy. In addition, these results identify ursolic acid and tomatidine as potential agents and/or lead compounds for reducing ATF4 activity, weakness, and atrophy in aged skeletal muscle.

The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness

Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4−/− littermates at 8 and 20weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4−/− mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4−/− mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness.

Loss Of Tumor Suppressor BTG1 Enhances ATF4 Function and Promotes Cell Survival

We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth.We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells. [Display omitted] As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2). [Display omitted] Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells.Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones.Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean.A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation. Disclosures:No relevant conflicts of interest to declare.

Abstract 1730: RET tyrosine kinase receptor represses Noxa transcription and prevents genotoxic or endoplasmic reticulum stress-induced apoptosis.

Abstract One of the hallmarks of human cancers is the intrinsic or acquired resistance to apoptosis. Apoptosis resistance contributes to carcinogenesis, tumor progression, and treatment resistance. Patients with medullary thyroid carcinoma (MTC) have been shown to be resistant to standard chemotherapy and conventional radiotherapy. Dominant activating mutations in the RET protooncogene are the cause of hereditary MTC and are found in more than 40% of sporadic cases. Despite an ever-growing wealth of information about RET's role in cell survival/proliferation, a detailed understanding of how this tyrosine kinase receptor acts to prevent cell death in tumorigenesis remains elusive. We hypothesized that activated RET prevents apoptosis by repressing the transcription of the proapoptotic gene Noxa in the nucleus in response to genotoxic or endoplasmic reticulum (ER) stress. Here we report that activated RET translocates to the nucleus in cultured MTC cells as well as MTC primary tumor tissues. RET is recruited to the Noxa promoter and is able to repress its transcription. The formation of the RET-Noxa promoter complex depends on RET expression and requires the tyrosine kinase activity of RET. Additionally, stable depletion of RET in MTC cells, using lentiviral shRNA, sensitizes MTC cells to genotoxic or endoplasmic reticulum stress. We specifically show that RET is recruited to the Activating Transcription Factor 4 (ATF4) binding site on the Noxa promoter. RET physically interacts with ATF4 and phosphorylates ATF4 leading to the blockage of its transcriptional activity. Moreover, RET knockdown is associated with increased ATF4-dependent expression of Noxa. Inhibition of RET kinase activity by sunitinib results in ATF4 activation, induction of Noxa expression and apoptosis. Conversely, silencing of ATF4 in MTC cells decreased sunitinib-mediated Noxa induction and apoptosis. ATF4 protein levels markedly decreased and/or localized in the cytoplasm of 60% of primary MTC (n=40) and associated with poor overall survival. Our data suggest that RET activating mutations confers resistance to chemotherapeutic agents by preventing apoptosis through inhibition of ATF4 activity and repression of proapoptotic gene Noxa. These findings reveal the importance of nuclear localization of RET, as newly discovered modulator of nuclear gene expression. This newly identified interaction between RET kinase and ATF4 has important implication for MTC and other RET/ATF4-mediated cancer types. Since ATF4 transcription is induced by endoplasmic reticulum stress, and activation of ATF4 induces cell cycle arrest and apoptosis, the concept of promoting endoplasmic reticulum stress in combination with tyrosine kinase inhibitors could be considered as a therapeutic strategy for cancer. Citation Format: Rozita Bagheri-Yarmand, Anupama E Gururaj, Michelle Williams, Zamal Ahmed, Jean E Ladbury, Oliver Bogler, Sue-chen Huang, Gilbert G Cote, Robert F Gagel. RET tyrosine kinase receptor represses Noxa transcription and prevents genotoxic or endoplasmic reticulum stress-induced apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2013-1730

Activating transcription factor 4 mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes

There is convincing evidence that endoplasmic reticulum (ER) stress is implicated in the pathogenesis of diabetes and its complications; however, the mechanisms are not fully understood. This study aimed to dissect the role and signalling pathways of activating transcription factor 4 (ATF4) in ER-stress-associated endothelial inflammation and diabetic retinopathy. ER stress and ATF4 activity were manipulated by complementary pharmacological and genetic approaches in cultured retinal endothelial (TR-iBRB) cells. Diabetes was induced by streptozotocin in heterozygous Atf4 knockout and wild-type mice. ER stress markers, inflammatory cytokines and adhesion molecules, activation of the signal transducer and activator of transcription 3 (STAT3) pathway, and retinal vascular permeability were measured. High-glucose treatment resulted in rapid induction of ER stress, activation of ATF4, and increased production of inflammatory factors in TR-iBRB cells. Suppressing ER stress or inhibiting ATF4 activity markedly attenuated high-glucose-induced production of intercellular adhesion molecule 1, TNF-α and vascular endothelial growth factor. Conversely, enhancing ER stress or overexpressing Atf4 was sufficient to induce endothelial inflammation, which was, at least in part, through activation of the STAT3 pathway. Furthermore, knockdown of the Stat3 gene or inhibiting STAT3 activity restored ER homeostasis in cells exposed to high glucose and prevented ATF4 activation, suggesting that STAT3 is required for high-glucose-induced ER stress. Finally, we showed that downregulation of Atf4 significantly ameliorated retinal inflammation, STAT3 activation and vascular leakage in a mouse model of type 1 diabetes. Taken together, our data reveal a pivotal role of ER stress and the ATF4/STAT3 pathway in retinal endothelial inflammation in diabetic retinopathy.

Activation of Endoplasmic Reticulum Stress by Hyperglycemia Is Essential for Müller Cell–Derived Inflammatory Cytokine Production in Diabetes

Inflammation plays an important role in diabetes-induced retinal vascular leakage. The purpose of this study is to examine the role of endoplasmic reticulum (ER) stress and the signaling pathway of ER stress–induced activating transcription factor 4 (ATF4) in the regulation of Müller cell–derived inflammatory mediators in diabetic retinopathy. In diabetic animals, elevated ER stress markers, ATF4, and vascular endothelial growth factor (VEGF) expression were partially localized to Müller cells in the retina. In cultured Müller cells, high glucose induced a time-dependent increase of ER stress, ATF4 expression, and inflammatory factor production. Inducing ER stress or overexpressing ATF4 resulted in elevated intracellular adhesion molecule 1 and VEGF proteins in Müller cells. In contrast, alleviation of ER stress or blockade of ATF4 activity attenuated inflammatory gene expression induced by high glucose or hypoxia. Furthermore, we found that ATF4 regulated the c-Jun NH2-terminal kinase pathway resulting in VEGF upregulation. ATF4 was also required for ER stress–induced and hypoxia-inducible factor-1α activation. Finally, we showed that administration of chemical chaperone 4-phenylbutyrate or genetic inhibition of ATF4 successfully attenuated retinal VEGF expression and reduced vascular leakage in mice with STZ-induced diabetes. Taken together, our data indicate that ER stress and ATF4 play a critical role in retinal inflammatory signaling and Müller cell–derived inflammatory cytokine production in diabetes.

Parathyroid Hormone Increases Activating Transcription Factor 4 Expression and Activity in Osteoblasts: Requirement for Osteocalcin Gene Expression

PTH is an important peptide hormone regulator of calcium homeostasis and osteoblast function. However, its mechanism of action in osteoblasts is poorly understood. Our previous study demonstrated that PTH activates mouse osteocalcin (Ocn) gene 2 promoter through the osteoblast-specific element 1 site, a recently identified activating transcription factor-4 (ATF4) -binding element. In the present study, we examined effects of PTH on ATF4 expression and activity as well as the requirement for ATF4 in the regulation of Ocn by PTH. Results show that PTH elevated levels of ATF4 mRNA and protein in a dose- and time-dependent manner. This PTH regulation requires transcriptional activity but not de novo protein synthesis. PTH also increased binding of nuclear extracts to osteoblast-specific element 1 DNA. PTH stimulated ATF4-dependent transcriptional activity mainly through protein kinase A with a lesser requirement for protein kinase C and MAPK/ERK pathways. Lastly, PTH stimulation of Ocn expression was lost by small interfering RNA down-regulation of ATF4 in MC-4 cells and Atf4(-/-) bone marrow stromal cells. Collectively, these studies for the first time demonstrate that PTH increases ATF4 expression and activity and that ATF4 is required for PTH induction of Ocn expression in osteoblasts.