Activating Transcription Factor 4 Silencing Research Articles

Activation of PERK/eIF2α/ATF4/CHOP branch of endoplasmic reticulum stress response and cooperation between HIF-1α and ATF4 promotes Daprodustat-induced vascular calcification.

Introduction: Vascular calcification is accelerated in patients with chronic kidney disease (CKD) and increases the risk of cardiovascular events. CKD is frequently associated with anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor for the treatment of CKD-associated anemia that enhances erythropoiesis through the activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Studies showed that DPD promotes osteogenic differentiation of human aortic smooth muscle cells (HAoSMCs) and increases aorta calcification in mice with CKD. HIF-1 activation has been linked with endoplasmic reticulum (ER) stress; therefore, here we investigated the potential contribution of ER stress, particularly activating transcription factor 4 (ATF4), to the pro-calcification effect of DPD. Methods: Here, we used an adenine-induced CKD mouse model and HAoSMCs as an in vitro vascular calcification model to study the effect of DPD. Results: DPD treatment (15mg/kg/day) corrects anemia but increases the expression of hypoxia (Glut1, VEGFA), ER stress (ATF4, CHOP, and GRP78), and osteo-/chondrogenic (Runx2, Sox9, BMP2, and Msx2) markers and accelerates aorta and kidney calcification in CKD mice. DPD activates the PERK/eIF2α/ATF4/CHOP pathway and promotes high phosphate-induced osteo-/chondrogenic differentiation of HAoSMCs. Inhibition of ER stress with 4-PBA or silencing of ATF4 attenuates HAoSMC calcification. DPD-induced ATF4 expression is abolished in the absence of HIF-1α; however, knockdown of ATF4 does not affect HIF-1α expression. Conclusion: We concluded that DPD induces ER stress in vitro and in vivo, in which ATF4 serves as a downstream effector of HIF-1 activation. Targeting ATF4 could be a potential therapeutic approach to attenuate the pro-calcific effect of DPD.

Glucose promotes cell growth and casein synthesis via ATF4/Nrf2-Sestrin2- AMPK-mTORC1 pathway in dairy cow mammary epithelial cells

In the dairy industry, glucose (Glu) is used as bioactive substance to increase milk yield. However, the molecular regulation underneath needs further clarification. Here, the regulation and its molecular mechanism of Glu on cell growth and casein synthesis of dairy cow mammary epithelial cells (DCMECs) were investigated. When Glu was added from DCMECs, both cell growth, β-casein expression and the mechanistic target of rapamycin complex 1 (mTORC1) pathway were increased. Overexpression and silencing of mTOR revealed that Glu promoted cell growth and β-casein expression through the mTORC1 pathway. When Glu was added from DCMECs, both Adenosine 5'-monophosphate-activated protein kinase α (AMPKα) and Sestrin2 (SESN2) expression were decreased. Overexpression and silencing of AMPKα or SESN2 uncovered that AMPKα suppressed cell growth and β-casein synthesis through inhibiting mTORC1 pathway, and SESN2 suppressed cell growth and β-casein synthesis through activating AMPK pathway. When Glu was depleted from DCMECs, both activating transcription factor 4 (ATF4) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression were increased. Overexpression or silencing of ATF4 or Nrf2 demonstrated that Glu depletion promoted SESN2 expression through ATF4 and Nrf2. Together, these results indicate that in DCMECs, Glu promoted cell growth and casein synthesis via ATF4/Nrf2-SESN2-AMPK-mTORC1 pathway.

Activating transcription factor 4 drives the progression of diabetic cardiac fibrosis.

Diabetic cardiomyopathy (DC) is one of serious complications of diabetic patients. This study investigated the biological function of activating transcription factor 4 (ATF4) in DC. Streptozotocin-treated mice and high glucose (HG)-exposed HL-1 cells were used as the in vivo and in vitro models of DC. Myocardial infarction (MI) was induced by left coronary artery ligation in mice. Cardiac functional parameters were detected by echocardiography. Target molecule expression was determined by real time quantitative PCR and western blotting. Cardiac fibrosis was observed by haematoxylin and eosin and Masson's staining. Cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling. Activities of superoxide dismutase, glutathione peroxidase, and levels of malonic dialdehyde and reactive oxygen species were used to assess oxidative stress damage. Molecular mechanisms were evaluated by chromatin immunoprecipitation, dual luciferase assay, and co-immunoprecipitation. ATF4 was up-regulated in the DC and MI mice (P<0.01). Down-regulation of ATF4 improved cardiac function as evidenced by changes in cardiac functional parameters (P<0.01), inhibited myocardial collagen I (P<0.001) and collagen III (P<0.001) expression, apoptosis (P<0.001), and oxidative stress (P<0.001) in diabetic mice. Collagen I (P<0.01) and collagen III (P<0.01) expression was increased in MI mice, which was reversed by ATF4 silencing (P<0.05). ATF4 depletion enhanced viability (P<0.01), repressed apoptosis (P<0.001), oxidative damage (P<0.001), and collagen I (P<0.001), and collagen III (P<0.001) expression of HG-stimulated HL-1 cells. ATF4 transcriptionally activated Smad ubiquitin regulatory factor 2 (Smurf2, P<0.001) to promote ubiquitination and degradation of homeodomain interacting protein kinase-2 (P<0.001) and subsequently caused inactivation of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway (P<0.001). The inhibitory effects of ATF4 silencing on HG-induced apoptosis (P<0.01), oxidative injury (P<0.01), collagen I (P<0.001), and collagen III (P<0.001) expression were reversed by Smurf2 overexpression. ATF4 facilitates diabetic cardiac fibrosis and oxidative stress by promoting Smurf2-mediated ubiquitination and degradation of homeodomain interacting protein kinase-2 and then inactivation of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway, suggesting ATF4 as a treatment target for DC.

ATF4 Protects the Heart From Failure by Antagonizing Oxidative Stress.

Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes' whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

High ATF4 Expression Is Associated With Poor Prognosis, Amino Acid Metabolism, and Autophagy in Gastric Cancer.

BackgroundThe role of activating transcription factor 4 (ATF4) underlying gastric cancer (GC) remains unclear. The purpose of this study was to investigate the expression levels and biological functions of ATF4 in GC.MethodsExpression of ATF4 was detected by quantitative PCR (qPCR), Western blotting, and immunohistochemistry. Cox regression was used for survival analysis and the construction of the nomogram. Immunofluorescence was used to identify the intracellular localization of ATF4. Knockdown and overexpression of ATF4 in GC cells followed by wound healing and Transwell assays, EdU and Calcein-AM/propidium iodide (PI) staining, and cell cycle detection were performed to examine its function in vitro. Transmission electron microscopy was performed to assess the autophagy levels upon ATF4 silencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) were used to determine gene enrichment. SPSS 22.0 software, GraphPad Prism 7.0, and R version 3.6.1 were used for statistical analysis.ResultsATF4 expression was upregulated in GC cells and tissues compared with corresponding normal tissues. Survival analysis suggested that a high ATF4 expression was strongly associated with worse overall survival (OS) of GC patients (p < 0.001). The nomogram and the receiver operating characteristic (ROC) curves demonstrated that ATF4 was a highly sensitive and specific prognostic marker of GC [C-index = 0.797, area under the ROC curve (AUC) of 3-year OS = 0.855, and AUC of 5-year OS = 0.863]. In addition, ATF4 knockdown inhibited the cell proliferation, migration, invasion, and cell cycle progression of GC cells in vitro, while overexpression of ATF4 exerted the opposite effects. Bioinformatics analysis showed that ATF4 could promote GC progression possibly by regulating asparagine (Asn) metabolism and autophagy pathways. Further experiments indicated that ATF4 expression was significantly positively correlated with ASNS expression. The inhibition of cell clone formation in Asn-deprived conditions was more significant in the shATF4 group. Finally, we found that ATF4 promoted autophagy through regulating the mTORC1 pathway in GC cells.ConclusionThese findings suggested that ATF4 can significantly promote GC development and serve as an independent prognostic factor for GC.

Downregulation of activating transcription factor 4 attenuates lysophosphatidycholine-induced inflammation via the NF-κB pathway

Lysophosphatidycholine (LPC) is the main active component in oxidized low-density lipoprotein (ox-LDL). The pathological function of ox-LDL has been broadly studied in atherosclerosis. However, the specific relationship between LPC-induced unfolded protein response (UPR) and inflammation in human umbilical vein endothelial cells (HUVECs) remains elusive. In this study, we found elevated serum levels of LPC in atherosclerotic patients. LPC stimulation resulted in elevated secretion of interleukin (IL)-6 and IL-8 in HUVECs, accompanied with the activation of ER stress and NF-κB pathway. Additionally, suppression of ER stress by 4-phenylbutric acid (4-PBA), an ER stress inhibitor, alleviated the activation of the NF-κB pathway and secretion of inflammatory factors. Moreover, activating transcription factor 4 (ATF4) silencing inhibited the transcription and secretion of IL-6 and IL-8, and suppressed the adhesion of THP-1 cells to HUVECs. Activation of the NF-κB pathway and expression of its upstream factors, including Toll like receptor 4 and cellular inhibitor of apoptosis, were also inhibited by ATF4 silencing. The present findings suggest that suppression of UPR alleviates LPC-induced HUVECs inflammation by inhibition of NF-κB pathway, and indicate ATF4 as a potential target for the treatment of atherosclerosis.

SY8-1. Maternal diet and gut microbiota: effects on gut blood pressure and metabolism

Lysophosphatidycholine (LPC) is the main active component in oxidized low-density lipoprotein (ox-LDL). The pathological function of ox-LDL has been broadly studied in atherosclerosis. However, the specific relationship between LPC-induced unfolded protein response (UPR) and inflammation in human umbilical vein endothelial cells (HUVECs) remains elusive. In this study, we found elevated serum levels of LPC in atherosclerotic patients. LPC stimulation resulted in elevated secretion of interleukin (IL)-6 and IL-8 in HUVECs, accompanied with the activation of ER stress and NF-κB pathway. Additionally, suppression of ER stress by 4-phenylbutric acid (4-PBA), an ER stress inhibitor, alleviated the activation of the NF-κB pathway and secretion of inflammatory factors. Moreover, activating transcription factor 4 (ATF4) silencing inhibited the transcription and secretion of IL-6 and IL-8, and suppressed the adhesion of THP-1 cells to HUVECs. Activation of the NF-κB pathway and expression of its upstream factors, including Toll like receptor 4 and cellular inhibitor of apoptosis, were also inhibited by ATF4 silencing. The present findings suggest that suppression of UPR alleviates LPC-induced HUVECs inflammation by inhibition of NF-κB pathway, and indicate ATF4 as a potential target for the treatment of atherosclerosis.

Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells.

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

Hydrogen peroxide induces nucleus pulposus cell apoptosis by ATF4/CHOP signaling pathway.

Oxidative stress induces excessive apoptosis resulting in the reduction of intervertebral disc cells, the consequent reduction of extracellular matrix (ECM) synthesis, and compositional changes, which is the pathological basis for intervertebral disc degeneration (IVDD). The present study explored the activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway in the H2O2-induced nucleus pulposus (NP) cell apoptosis. Human degenerated intervertebral discs were collected from Lumbar disc surgery. NP cells isolated from the tissues were cultured with H2O2 to induce apoptosis in vitro. Malondialdehyde (MDA) analysis was performed to determine the reactive oxygen species (ROS) of the tissue. Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed to analyze collagen II, ATF4, CHOP, and caspase-9 gene expression. Flow cytometry was used to determine the apoptotic ratio of NP cells. siRNA was also used to silence ATF4 and CHOP gene expression. NP tissues in higher degenerated degree underwent much more MDA, expressed less collagen II, more ATF4, CHOP, and caspase-9 compared with the mildly degenerated tissues. H2O2 induced NP cell apoptosis by upregulating expression of ATF4, CHOP and caspase-9. The silencing of ATF4 or CHOP alleviated NP cell apoptosis by suppressing caspase-9 expression. Inhibiting caspase-9 did not affect ATF4 and CHOP expression but protected NP cells from apoptosis. In this study, we found H2O2 could promote NP cell apoptosis by activating the ATF4/CHOP signaling pathway resulting in the upregulation of caspase-9. Interdict of ATF4, CHOP, or caspase-9 contributed to the reduction of apoptosis caused by H2O2.

Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors.

Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies.

Sestrin2 Negatively Regulates Casein Synthesis through the SH3BP4-mTORC1 Pathway in Response to AA Depletion or Supplementation in Cow Mammary Epithelial Cells.

Sestrin2 (SESN2) negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) pathway and casein synthesis in response to amino acid (AA) depletion in cow mammary epithelial cells (CMECs); however, the underlying mechanism is unclear. In the current study, the regulation of SESN2 on AA-mediated β-casein (CSN2) synthesis in CMECs and its mechanism were investigated. Overexpression and silencing of SESN2 demonstrated that SESN2 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway. Co-immunoprecipitation analysis showed that SESN2 interacted with SH3 domain-binding protein 4 (SH3BP4). Overexpression and silencing of SH3BP4 demonstrated that SH3BP4 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway and that SESN2 negatively regulated expression of CSN2 and mTORC1 pathway through the SH3BP4 in the presence and absence of AA. The absence or presence of AA demonstrated that AA negatively regulated expression and nuclear localization of activating transcription factor 4 (ATF4). Overexpression and silencing of ATF4 demonstrated that AA negatively regulated SESN2 expression through ATF4. Together, these results indicate that SESN2 negatively regulates the mTORC1 pathway and subsequent CSN2 synthesis through the SH3BP4 in response to AA absence or presence in CMECs.

ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma

Rationale: Activating transcription factor 4 (ATF4) is a central regulator of the cellular stress response and reduces tumor burden by controlling the expression of target genes implicated in the induction of apoptosis. Evidence shows ATF4 activation is responsible for proteasome inhibitor bortezomib (BTZ)-induced osteosarcoma (OS) cell death. However, it remains unclear how such suppressive function is impaired during prolonged therapeutic interventions.Methods: Stable cells and in vivo xenograft models were generated to reveal the essential role of ATF4 in cell apoptosis and tumor growth. Fluorescence in situ hybridization (FISH) and immunohistochemistry were employed to detect the expression and significance of ATF4 in the specimens from osteosarcoma patients. Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by proliferation, apoptosis, real-time PCR, immunoblotting and immunofluorescence. Promoter activity was analysed using the luciferase reporter assay. Immunoprecipitation was used to explore the interaction of proteins with other proteins or DNAs.Results: ATF4 significantly inhibited OS tumorigenesis, whereas knockdown of ATF4 prevented the antitumor effects of BTZ. Normal osteoblasts are supposed to preferentially express ATF4, but ATF4 silencing was detected in both OS clinical samples and BTZ-resistant sublines (OS/BTZ). We found that ATF4 downregulation was tightly linked to the aberrant expression of RET, primarily due to RET stabilization in OS/BTZ cells. Loss of RET upregulated ATF4 and potentiated the apoptotic response to BTZ. ATF4 recognized the TK domain of RET by recruiting its transactivated E3 ligase Cbl-c to accelerate RET proteasomal turnover, which in turn prevented BTZ resistance. In contrast, the chaperone GRP78 bound to RET and interfered with ATF4/RET interactions, promoted RET stabilization. Intriguingly, ATF4 repressed GRP78 transcription in OS/BTZ cells via the first ERSE, instead of transactivating GRP78 in wild-type OS via classical CRE element, revealing a dual targeting of RET and GRP78 to overcome chemoresistance.Conclusion: The results uncover a crucial role for ATF4 in blocking the progression and resistance response in RET/GRP78-positive human osteosarcoma.

Histone deacetylase 6 reduction promotes aortic valve calcification via an endoplasmic reticulum stress-mediated osteogenic pathway

ObjectiveAortic valve (AoV) calcification occurs via a pathophysiologic process that includes osteoblastic differentiation of valvular interstitial cells (VICs). Histone deacetylases (HDACs) have been shown to be involved in the pathogenesis of vascular diseases. Here, we investigated the role of HDAC6 in AoV calcification. MethodsAoV cusps from patients with aortic stenosis (n = 7) and normal controls (n = 7) were subjected to determination of calcified nodules and HDAC6 expression. Human VICs were cultured in osteogenic media and treated with 10 uM tubacin or HDAC6 small interfering RNA silencing to inhibit HDAC6. Treatment with 100 uM tauroursodeoxycholic acid was used to suppress endoplasmic reticulum stress. Activating transcription factor 4 (ATF4) small interfering RNA was used to knock down ATF4. Alizarin red staining was used to evaluate calcified nodules formation of VICs cultured with osteogenic media for 14 days. ResultsHDAC6 expression was significantly reduced in AoV tissue of patients with aortic stenosis compared with controls. Tubacin treatment or HDAC6 silencing markedly promoted osteoblastic differentiation accompanied by endoplasmic reticulum stress activation in VICs. The HDAC6 inhibition-induced osteogenic pathway was mediated by endoplasmic reticulum stress/ATF4 pathway as indicated by tauroursodeoxycholic acid pretreatment or ATF4 silencing. Finally, alizarin red staining showed that HDAC6 inhibition promoted osteoblastic differentiation of VICs, which could be suppressed by tauroursodeoxycholic acid. ConclusionsHDAC6 inhibition promotes AoV calcification via an endoplasmic reticulum stress/ATF4-mediated osteogenic pathway. HDAC6 may be a novel target for AoV calcification prevention and treatment.

Activating Transcription Factor 4 Modulates TGFβ-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697-709. ©2018 AACR.

Inhibition of endoplasmic reticulum stress by intermedin1-53 attenuates angiotensin II-induced abdominal aortic aneurysm in ApoE KO Mice.

Endoplasmic reticulum stress (ERS) is involved in the development of abdominal aortic aneurysm (AAA). Since bioactive peptide intermedin (IMD)1-53 protects against AAA formation, here we investigated whether IMD1-53 attenuates AAA by inhibiting ERS. AAA model was induced by angiotensin II (AngII) in ApoE KO mouse background. AngII-treated mouse aortas showed increased ERS gene transcription of caspase12, eukaryotic translation initiation factor 2a (eIf2a) and activating transcription factor 4(ATF4).The protein level of ERS marker glucose regulated protein 94(GRP94), ATF4 and C/EBP homologous protein 10(CHOP) was also up-regulated by AngII. Increased ERS levels were accompanied by severe VSMC apoptosis in human AAA aorta. In vivo administration of IMD1-53 greatly reduced AngII-induced AAA and abrogated the activation of ERS. To determine whether IMD inhibited AAA by ameliorating ERS, we used 2 non-selective ERS inhibitors phenyl butyrate (4-PBA) and taurine (TAU). Similar to IMD, PBA, and TAU significantly reduced the incidence of AAA and AAA-related pathological disorders. In vitro, AngII infusion up-regulated CHOP, caspase12 expression and led to VSMC apoptosis. IMD siRNA aggravated the CHOP, caspase12-mediated VSMC apoptosis, which was abolished by ATF4 silencing. IMD infusion promoted the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in aortas in ApoE KO mice, and the AMPK inhibitor compound C abolished the protective effect of IMD on VSMC ERS and apoptosis induced by AngII. In conclusion, IMD may protect against AAA formation by inhibiting ERS via activating AMPK phosphorylation.

Sorafenib induces renal cell carcinoma apoptosis via upregulating activating transcription factor 4.

Previous studies have shown sorafenib to function as a multitargeted tyrosine kinase inhibitor in different tumors. However, whether sorafenib improves renal cell carcinoma (RCC) through activating transcription factor 4 (ATF4) has never been explored. In the current study, we showed that sorafenib could suppress RCC cell viability in a time- and dose-dependent manner. Furthermore, sorafenib is demonstrated to enhance the mRNA and protein levels of ATF4. Meanwhile, overexpression of ATF4 was demonstrated to induce ACHN cell cycle arrest and cell apoptosis. Moreover, treatment with sorafenib could enhance the expression of CCAAT/enhancer-binding protein-homologous protein (CHOP) and p53 upregulated modulator of apoptosis (PUMA), thereby leading to ACHN cell apoptosis. More importantly, silencing of ATF4 could largely abolish sorafenib-induced upregulation of CHOP and PUMA in ACHN cells. Meanwhile, sorafenib-induced cell apoptosis may be dependent on the activation of ATF4 since knockdown of ATF4 partially reversed sorafenib-induced ACHN cell apoptosis. In summary, the present study demonstrates that sorafenib activates ATF4-CHOP-PUMA pathway in RCC cells, resulting in enhanced ER stress-related cell apoptosis.

BRAF-mutated cells activate GCN2-mediated integrated stress response as a cytoprotective mechanism in response to vemurafenib

In BRAF-mutated melanoma cells, the BRAF inhibitor, vemurafenib, induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) and subsequent induction of activating transcription factor 4 (ATF4), the central regulation node of the integrated stress response (ISR). While the ISR supports cellular adaptation to various stresses, the role of vemurafenib-triggered ISR has not been fully characterized. Here, we showed that in response to vemurafenib, BRAF-mutated melanoma and colorectal cancer cells rapidly induced the ISR as a cytoprotective mechanism through activation of general control nonderepressible 2 (GCN2), an eIF2α kinase sensing amino acid levels. The vemurafenib-triggered ISR, an event independent of downstream MEK inhibition, was specifically prevented by silencing GCN2, but not other eIF2α kinases, including protein kinase-like endoplasmic reticulum kinase, which transmits endoplasmic reticulum (ER) stress. Consistently, the ER stress gatekeeper, GRP78, was not induced by vemurafenib. Interestingly, ATF4 silencing by siRNA rendered BRAF-mutated melanoma cells sensitive to vemurafenib. Thus, the GCN2-mediated ISR can promote cellular adaptation to vemurafenib-induced stress, providing an insight into the development of drug resistance.

Activating transcription factor 4 (ATF4) modulates post-synaptic development and dendritic spine morphology.

The ubiquitously expressed activating transcription factor 4 (ATF4) has been variably reported to either promote or inhibit neuronal plasticity and memory. However, the potential cellular bases for these and other actions of ATF4 in brain are not well-defined. In this report, we focus on ATF4's role in post-synaptic synapse development and dendritic spine morphology. shRNA-mediated silencing of ATF4 significantly reduces the densities of PSD-95 and GluR1 puncta (presumed markers of excitatory synapses) in long-term cultures of cortical and hippocampal neurons. ATF4 knockdown also decreases the density of mushroom spines and increases formation of abnormally-long dendritic filopodia in such cultures. In vivo knockdown of ATF4 in adult mouse hippocampal neurons also reduces mushroom spine density. In contrast, ATF4 over-expression does not affect the densities of PSD-95 puncta or mushrooom spines. Regulation of synaptic puncta and spine densities by ATF4 requires its transcriptional activity and is mediated at least in part by indirectly controlling the stability and expression of the total and active forms of the actin regulatory protein Cdc42. In support of such a mechanism, ATF4 silencing decreases the half-life of Cdc42 in cultured cortical neurons from 31.5 to 18.5 h while knockdown of Cdc42, like ATF4 knockdown, reduces the densities of mushroom spines and PSD-95 puncta. Thus, ATF4 appears to participate in neuronal development and plasticity by regulating the post-synaptic development of synapses and dendritic mushroom spines via a mechanism that includes regulation of Cdc42 levels.

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

ATF4 Protects Against Neuronal Death in Cellular Parkinson's Disease Models by Maintaining Levels of Parkin

Parkinson's disease (PD) is a common neurodegenerative disorder, for which there are no effective disease-modifying therapies. The transcription factor ATF4 (activating transcription factor 4) is induced by multiple PD-relevant stressors, such as endoplasmic reticulum stress and oxidative damage. ATF4 may exert either protective or deleterious effects on cell survival, depending on the paradigm. However, the role of ATF4 in the pathogenesis of PD has not been explored. We find that ATF4 levels are increased in neuromelanin-positive neurons in the substantia nigra of a subset of PD patients relative to controls. ATF4 levels are also upregulated in neuronal PC12 cells treated with the dopaminergic neuronal toxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). To explore the role of ATF4 in cell survival in PD-relevant contexts, we either silenced or overexpressed ATF4 in cellular models of PD. In neuronal PC12 cells, silencing of ATF4 enhanced cell death in response to either 6-OHDA or MPP+. Conversely, overexpression of ATF4 reduced cell death caused by dopaminergic neuronal toxins. ATF4 was also protective against 6-OHDA-induced death of cultured mouse ventral midbrain dopaminergic neurons. We further show that parkin, a gene associated with autosomal recessive PD, plays a critical role in ATF4-mediated protection. After treatment with 6-OHDA or MPP+, parkin protein levels fall, despite an increase in mRNA levels. ATF4 silencing exacerbates the toxin-induced reduction of parkin, whereas ATF4 overexpression partially preserves parkin levels. Finally, parkin silencing blocked the protective capacity of ATF4. These results indicate that ATF4 plays a protective role in PD through the regulation of parkin.