Activating Transcription Factor 4 Knockdown Research Articles

Malignant ascites supernatant enhances the proliferation of gastric cancer cells partially via the upregulation of asparagine synthetase.

Malignant ascites (MA) is a common manifestation of advanced gastric cancer (GC) with peritoneal metastasis (PM), which usually indicates a poor prognosis. The present study aimed to explore the effects of MA, a unique microenvironment of PM, on the proliferation of cancer cells and investigate the underlying mechanisms. Ex vivo experiments demonstrated that GC cells treated with MA exhibited enhanced proliferation. RNA sequencing indicated that asparagine synthetase (ASNS) was one of the differentially expressed genes in GC cells following incubation with MAs. Furthermore, the present study suggested that MA induced an upregulation of ASNS expression and the stimulatory effect of MA on cancer cell proliferation was alleviated upon ASNS downregulation. Activating transcription factor 4 (ATF4), a pivotal transcription factor regulating ASNS, was upregulated when cells were treated with MA supernatant. After ATF4 knockdown, the proliferation of MA-treated GC cells and the expression of ASNS decreased. In addition, the decline in the proliferation of the ATF4-downregulated AGS GC cell line was rescued by ASNS upregulation. The findings indicated that MA could promote the proliferation of GC cells via activation of the ATF4-ASNS axis. Hence, it may be a potential target for treating GC with PM and MA.

ATF4 knockdown in macrophage impairs glycolysis and mediates immune tolerance by targeting HK2 and HIF-1α ubiquitination in sepsis

Strengthened glycolysis is crucial for the macrophage pro-inflammatory response during sepsis. Activating transcription factor 4 (ATF4) plays an important role in regulating glucose and lipid metabolic homeostasis in hepatocytes and adipocytes. However, its immunometabolic role in macrophage during sepsis remains largely unknown. In the present study, we found that the expression of ATF4 in peripheral blood mononuclear cells (PBMCs) was increased and associated with glucose metabolism in septic patients. Atf4 knockdown specifically decreased LPS-induced spleen macrophages and serum pro-inflammatory cytokines levels in mice. Moreover, Atf4 knockdown partially blocked LPS-induced pro-inflammatory cytokines, lactate accumulation and glycolytic capacity in RAW264.7. Mechanically, ATF4 binds to the promoter region of hexokinase II (HK2), and interacts with hypoxia inducible factor-1α (HIF-1α) and stabilizes HIF-1α through ubiquitination modification in response to LPS. Furthermore, ATF4-HIF-1α-HK2-glycolysis axis launches pro-inflammatory response in macrophage depending on the activation of mammalian target of rapamycin (mTOR). Importantly, Atf4 overexpression improves the decreased level of pro-inflammatory cytokines and lactate secretion and HK2 expression in LPS-induced tolerant macrophages. In conclusion, we propose a novel function of ATF4 as a crucial glycolytic activator contributing to pro-inflammatory response and improving immune tolerant in macrophage involved in sepsis. So, ATF4 could be a potential new target for immunotherapy of sepsis.

Oxidized phospholipids facilitate calcific aortic valve disease by elevating ATF4 through the PERK/eIF2α axis.

In this study we sought to analyze the critical role of oxidized phospholipid (OxPL) in the progression of calcific aortic valve disease (CAVD) with the involvement of activating transcription factor 4 (ATF4). Differentially expressed genes related to CAVD were identified using bioinformatics analysis. Expression of ATF4 was examined in mouse models of aortic valve calcification (AVC) induced by the high cholesterol (HC) diet. Valvular interstitial cells (VICs) were then isolated from mouse non-calcified valve tissues, induced by osteogenic induction medium (OIM) and co-cultured with OxPAPC-stimulated macrophages. The effect of OxPLs regulating ATF4 on the macrophage polarization and osteogenic differentiation of VICs was examined with gain- and loss-of-function experiments in VICs and in vivo. In aortic valve tissues and OIM-induced VICs, ATF4 was highly expressed. ATF4 knockdown alleviated the osteogenic differentiation of VICs, as evidenced by reduced expression of bone morphogenetic protein-2 (BMP2), osteopontin (OPN), and osteocalcin. In addition, knockdown of ATF4 arrested the AVC in vivo. Meanwhile, OxPL promoted M1 polarization of macrophages and mediated osteogenic differentiation of VICs. Furthermore, OxPL up-regulated ATF4 expression through protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α) pathway. In conclusion, OxPL can potentially up-regulate the expression of ATF4, inducing macrophages polarized to M1 phenotype, osteogenic differentiation of VICs and AVC, thus accelerating the progression of CAVD.

ATF4 Transcriptionally Activates SHH to Promote Proliferation, Invasion, and Migration of Gastric Cancer Cells

Activating transcription factor 4 (ATF4) is a DNA-binding protein widely generated in mammals, which has two biological characteristics that bind the cAMP response element (CRE). The mechanism of ATF4 as a transcription factor in gastric cancer affecting the Hedgehog pathway remains unclear. Here, we observed that ATF4 was markedly upregulated in gastric cancer (GC) using immunohistochemistry and Western blotting assays in 80 paraffin-embedded GC samples and 4 fresh samples and para-cancerous tissues. ATF4 knockdown using lentiviral vectors strongly inhibited the proliferation and invasion of GC cells. ATF4 upregulation using lentiviral vectors promoted the proliferation and invasion of GC cells. We predicted that the transcription factor ATF4 is bound to the SHH promoter via the JASPA database. Transcription factor ATF4 is bound to the promoter region of SHH to activate the Sonic Hedgehog pathway. Mechanistically, rescue assays showed that ATF4 regulated gastric cancer cells' proliferation and invasive ability through SHH. Similarly, ATF4 enhanced the tumor formation of GC cells in a xenograft model.

Oridonin ameliorates acetaminophen-induced acute liver injury through ATF4/PGC-1α pathway.

Acetaminophen (APAP) overdose-induced acute liver injury (ALI) causes hepatocyte cell death, oxidative stress, and inflammation. Oridonin (Ori), a covalent NLRP3-inflammasome inhibitor, ameliorates APAP-induced ALI through an unclear molecular mechanism. This study found that Ori decreased hepatic cytochrome P450 2E1 level and increased glutathione content to prevent APAP metabolism, and then reduced the necrotic area, improved liver function, and inhibited APAP-induced proinflammatory cytokines and oxidative stress. Ori also decreased activating transcription factor 4 (ATF4) protein levels and increased peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) to reduce APAP-induced endoplasmic reticulum stress activation and mitochondrial dysfunction. Furthermore, western blot and luciferase assay found that ATF4 inhibited transcription in the PGC-1α promoter -507 to -495 region to reduce PGC-1α levels, while ATF4 knockdown neutralized the hepatoprotective effect of Ori. Molecular docking showed that Ori bound to ATF4's amino acid residue glutamate 302 through 6, 7, and 18 hydroxyl bands. Our findings demonstrated that Ori prevented metabolic activation of APAP and further inhibited the ATF4/PGC-1α pathway to alleviate APAP overdose-induced hepatic toxicity, which illuminated its potential therapeutic effects on ALI.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

ATF4 protects against sorafenib-induced cardiotoxicity by suppressing ferroptosis

Sorafenib (SOR) is an effective chemotherapy drug for hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. However, a long-standing clinical issue associated with SOR use is an increased risk of cardiotoxicity, but the underlying mechanisms remain obscure. Here we report that ferroptosis of cardiomyocytes is responsible for SOR-induced cardiotoxicity. The specific ferroptosis inhibitor ferrostatin-1 and deferoxamine mesylate, an iron chelator, significantly alleviate SOR-induced cardiac damage. RNA-sequencing revealed that endoplasmic reticulum (ER) stress and the unfolded protein response were predominately activated, which might be attributed to the lipid reactive oxygen species-mediated perturbation of the ER. Activating transcription factor 4 (ATF4) is one of the most significantly up-regulated genes, knockdown of ATF4 exacerbates cardiomyocyte ferroptosis induced by SOR, while overexpression of ATF4 promotes cell survival. Mice with AAV-mediated ATF4 knockdown exhibit lipid peroxidation and more severe cardiomyopathy. Further experiments demonstrated that ATF4 exerts its protective role by elevating SLC7A11 expression, a transport subunit of system Xc−, which promotes cystine uptake and glutathione biosynthesis. The cardioprotective effect of ATF4 was diminished by SLC7A11 knockdown in cardiomyocytes subjected to SOR treatment. Taken together, these findings show that ferroptosis of cardiomyocytes is an important cause of SOR-related cardiotoxicity. ATF4 acts as a key regulator to promote cardiomyocytes survival by up-regulation of SLC7A11 and suppression of ferroptosis.

Endoplasmic reticulum stress downregulates PGC-1\u03b1 in skeletal muscle through ATF4 and an mTOR-mediated reduction of CRTC2

BackgroundPeroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) downregulation in skeletal muscle contributes to insulin resistance and type 2 diabetes mellitus. Here, we examined the effects of endoplasmic reticulum (ER) stress on PGC-1α levels in muscle and the potential mechanisms involved.MethodsThe human skeletal muscle cell line LHCN-M2 and mice exposed to different inducers of ER stress were used.ResultsPalmitate- or tunicamycin-induced ER stress resulted in PGC-1α downregulation and enhanced expression of activating transcription factor 4 (ATF4) in human myotubes and mouse skeletal muscle. Overexpression of ATF4 decreased basal PCG-1α expression, whereas ATF4 knockdown abrogated the reduction of PCG-1α caused by tunicamycin in myotubes. ER stress induction also activated mammalian target of rapamycin (mTOR) in myotubes and reduced the nuclear levels of cAMP response element-binding protein (CREB)-regulated transcription co-activator 2 (CRTC2), a positive modulator of PGC-1α transcription. The mTOR inhibitor torin 1 restored PCG-1α and CRTC2 protein levels. Moreover, siRNA against S6 kinase, an mTORC1 downstream target, prevented the reduction in the expression of CRTC2 and PGC-1α caused by the ER stressor tunicamycin.ConclusionsCollectively, these findings demonstrate that ATF4 and the mTOR-CRTC2 axis regulates PGC-1α transcription under ER stress conditions in skeletal muscle, suggesting that its inhibition might be a therapeutic target for insulin resistant states.-LYtH6dWBUupjprW9VTowoVideo

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.

ATF4-dependent heme-oxygenase-1 attenuates diabetic nephropathy by inducing autophagy and inhibiting apoptosis in podocyte

Aim Podocyte injury plays an important role in diabetic nephropathy (DN), yet the underlying molecular mechanisms of podocyte injury in DN is not clear. Here, we investigated the role of activating transcription factor 4 (ATF4) and HO-1 in DN-induced podocyte injury. Methods Protein expression was measured by western blotting (WB) and immunofluorescence. Cellular apoptosis was quantified by flow cytometry. ATF4 siRNA knockdown and HO-1 overexpression in podocyte were employed to evaluate the role of ER stress in DN-induced apoptosis and autophagy response. Urinary protein levels, nephrin expression, serum creatinine and BUN were evaluated and glomerulosclerosis was quantified by Periodic Acid-Schiff staining. Results Expression of ATF4 was increased in podocytes exposed to serum from DN mice. ATF4 knockdown enhanced DN-induced podocyte apoptosis. HO-1 overexpression reduced the decline of DN-induced podocyte autophagy and inhibited apoptosis and the beneficial effects of HO-1 overexpression in DN were blocked by ATF4 knockdown. The diabetic mice were significantly ameliorated by HO-1 agonist hemin treatment. Conclusions ATF4 induces autophagy by enhancing the expression of HO-1, and inhibits podocyte apoptosis in DN. Treatment with the HO-1 agonist reduced proteinuria, apoptosis, and enhanced autophagy response, and thus improved renal function in DN mice.

ATF4 activation promotes hepatic mitochondrial dysfunction by repressing NRF1–TFAM signalling in alcoholic steatohepatitis

ObjectiveMitochondrial dysfunction plays a dominant role in the pathogenesis of alcoholic liver disease (ALD); however, the underlying mechanisms remain to be fully understood. We previously found that hepatic activating transcription...

IL-22 ameliorates LPS-induced acute liver injury by autophagy activation through ATF4-ATG7 signaling

Uncontrollable inflammatory response acts as a driver of sepsis-associated liver injury (SALI). IL-22 plays an important role in regulating inflammatory responses, but its role in SALI remains unknown. The aim of the study was to assess the association of serum IL-22 with SALI in pediatric patients and to enclose the underlying mechanisms of IL-22 involved in lipopolysaccharide (LPS) - induced acute liver injury (ALI) in mice. Serum IL-22 levels in patients with SALI were significantly lower than in septic patients without liver injury, and the area under receiver operating characteristic (ROC) curve of IL-22 for discriminating SALI was 0.765 (95% CI: 0.593–0.937). Pre-administration of recombinant murine IL-22 alleviated LPS-induced ALI in mice, and serum IL-6 levels and the mRNA levels of TNF-α, IL-1β, and IL-6 in livers were decreased in response to IL-22 pre-treatment in mice. More importantly, IL-22 pre-treatment activated hepatic autophagy mediated by activating transcription factor 4 (ATF4)-autophagy-related gene 7 (ATG7) signaling in vivo and in vitro in response to LPS administration. Moreover, knockdown of ATF4 in mice aggravated LPS-induced ALI, which was associated with suppressed ATG7-related autophagy. In addition, the protective effects of IL-22 on LPS-induced ALI was partially blocked by ATF4 knockdown, which was associated with lower expression of LC3II/I in the livers of ATF4 knockdown (HT or Atf4+/−) mice compared with wild-type mice (WT or Atf4+/+) mice. In conclusion, low serum IL-22 level is associated with SALI occurrence, and IL-22 pre-administration activates autophagy in hepatocytes and protects mice against LPS-induced ALI partially related to ATF4-ATG7 signaling pathway.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Abstract 6163: Integrated stress response contributes to triple negative breast cancer radioresistance by regulating glutathione biosynthesis

Abstract Triple negative breast cancer (TNBC) is one of the most malignant gynecological cancer worldwide. Radiotherapy (RT) is an effective option for TNBC local control. However, TNBC tends to develop a radioresistant phenotype which results in a poor response of TNBC to following therapy. In this study, we identified integrated stress response (ISR) as the most activated signaling in radioresistant MDA-MB-231, MDA-MB-436, and MDA-MB-468 cells. We found that constitutive phosphorylation of eIF2α in radioresistant TNBC cells promoted the activation of activating transcription factor 4 (ATF4) via increasing its translational efficiency, which elicited the transcription of a set of genes implicated in GSH biosynthesis, including glutamate-cysteine ligase catalytic subunit (GCLC), solute carrier family 7 member 11 (SLC7A11), and cystathionine-γ-lyase (CTH), leading to a higher intracellular level of reduced glutathione (GSH) and an efficient reactive oxygen species (ROS) scavenging after irradiation (IR). Treating radiosensitive TNBC cells with salubrinal, a selective inhibitor of eIF2α dephosphorylation, can mimic the stress-resistant phenotypes of their radioresistant counterparts. In contrast, eIF2α dephosphorylation or ATF4 knockdown accelerated IR-induced ROS accumulation and increased IR-induced cell apoptosis in radioresistant TNBC cells in vitro. In combination with eIF2α dephosphorylation, IR significantly reduced the size and weight of radioresistant TNBC xenograft tumors in vivo. These data reveal ISR as an important mechanism underlying TNBC radioresistance and provide a rationale for exploring eIF2 signaling as a novel therapeutic target for TNBC treatment. Citation Format: Xupeng Bai, Jie Ni, Yong Li, Julia Beretov. Integrated stress response contributes to triple negative breast cancer radioresistance by regulating glutathione biosynthesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6163.

KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis.

BackgroundMany studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Krüppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis.PurposeIn this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway.MethodsKLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs subcloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA` probe.ResultsEctopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio.ConclusionThe ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.

MicroRNA-214 contributes to regulation of necroptosis via targeting ATF4 in diabetes-associated periodontitis.

Diabetes and periodontal diseases have a mutual promoting relationship that induces severe tissue damage and cell death. The potential roles of microRNAs (miRNAs) and the type of cell death involved in diabetes-associated periodontitis are obscure. The gingival tissues of patients were obtained and MC3T3-E1 cells were costimulated with high glucose and lipopolysaccharide (LPS). Osseous morphometric analysis was evaluated with micro-CT, and histological characteristics were measured by hematoxylin/eosin and immunohistochemical staining. Cytokine secretion was confirmed by enzyme-linked immunosorbent assay, and reactive oxygen species (ROS) was measured using a DCFH-DA probe kit. Gene expression was measured by real-time quantitative reverse transcription PCR (qRT-PCR), and protein expression was assessed by Western blot and immunofluorescence analysis. The miR-214 level, receptor-interacting serine-threonine protein (RIP) 1, RIP3, and phospho-mixed lineage kinase domain-like (p-MLKL) protein expression were elevated in the inflamed gingival tissues of diabetes-associated periodontitis patients, with activating transcription factor 4 (ATF4) expression showing the opposite effect. The high glucose (22 mM) could not induce significant increase of RIP1, RIP3, and p-MLKL; however, the high glucose and LPS (500-1000 ng/mL) cotreatment resulted in increase in the number of RIP1, RIP3, and p-MLKL in MC3T3-E1 cells. NAC (ROS inhibitor) inhibited RIP1, RIP3, and increased ATF4; however, necrostatin-1 (Nec-1) (RIP1 inhibitor) specifically inhibited the protein expression of RIP1 and RIP3 and had no influence on ATF4. The use of antagomir-214 suppressed the expression of miR-214, RIP1, RIP3, and p-MLKL, but increased ATF4 protein level in glucose and LPS-induced cells. ATF4 knockdown by ATF4 small interfering RNA offset the effect of antagomir-214. RIP1- and RIP3-dependent necroptosis was confirmed in the inflamed gingival tissues of diabetes-associated periodontitis patients and high glucose- and LPS- cotreated cells. It was suggested that miR-214-targeted ATF4 participated in the regulation of necroptosis in vivo and in vitro.

Antitumor effect of ginsenoside Rg3 on gallbladder cancer by inducing endoplasmic reticulum stress-mediated apoptosis in vitro and in vivo.

Recent studies have highlighted the importance of the endoplasmic reticulum (ER) in apoptotic processes. In the present study, the traditional herbal medicine ginsenoside Rg3 was used to treat gallbladder cancer in vitro and in vivo. The underlying signaling mechanisms were investigated using various molecular biology techniques, including flow cytometry, western blot analysis, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). It was indicated that Rg3 exerted pro-apoptotic activity against the gallbladder cancer cell line GBC-SD through the ER stress-mediated signaling pathway. This was demonstrated by increased expression of phosphorylation of eukaryotic translation-initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein and lipocalin 2. In addition, eIF2α and ATF4 knockdown attenuated the pro-apoptotic effect of Rg3 by inhibiting reactive oxygen species. Furthermore, the results of RT-qPCR analysis indicated that long intergenic non-protein coding RNA-p21 was significantly upregulated following Rg3 treatment. In conclusion, the results of the present study demonstrated that Rg3 inhibited tumor growth in a GBC-SD gallbladder cancer xenograft, by upregulating the ER stress-mediated signaling pathway. Therefore, ER stress activation is suggested to mediate the antitumor effect of Rg3 in gallbladder cancer activity in vitro and in vivo.

Abstract 2426: Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy, caused by activating mutations of the RET proto-oncogene, with frequent lymph node and distant metastasis at diagnosis. Tyrosine kinase inhibitors (TKIs) that target the RET have proven effective for treatment of MTC, but resistance to TKIs evolves in half of the treated patients. We have previously shown that the expression levels of Activating Transcription Factor 4 (ATF4) which is a stress-induced transcription factor, significantly decreased or lost in half of the MTC tumors. We also demonstrated that forced expression of ATF4 or TKIs-induced level of ATF4 decreases survival of MTC cells by promoting degradation of RET, blocking the activation of RET downstream signaling pathways and subsequently induces apoptosis. More importantly, ATF4 knockdown by shRNA decreased the sensitivity to tyrosine kinase inhibitor-induced apoptosis. Further thyroid gland of Atf4-knockout mice showed C-cell hyperplasia, a precancerous lesion for MTC suggesting a potential tumor suppressor role of ATF4 in MTC. Stress-induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells, including MTC cells that secrete calcitonin. ATF4 promotes the induction of apoptosis under persistent stress conditions, although the mechanism is not clearly understood. We hypothesize that the combination of ATF4 inducer and TKIs causes an excessive cellular oxidative stress resulting in an activation of apoptosis, all of which may prevent resistance to TKIs. Here, we show that the ER-associated protein degradation (ERAD) inhibitor, eeyarestatin sensitizes MTC cells to the TKIs including sunitinib and vandetanib. The combination of eeyarestatin and TKIs causes a synergistic induction of ATF4 expression and its target genes, an accumulation of reactive oxygen species and subsequent cell death. Chromatin immunoprecipitation followed by sequencing assay (ChIP-seq) in MTC cells treated with eeyarestatin and immunoprecipitated with ATF4 and acetylated lysine 9 of histone 3, identified transcription Kruppel-like factor 9 (KLF9) gene as a transcriptional target of ATF4 activation. KLF9 plays a functional role in oxidative stress-induced cell death. Treatment with eeyarestatin and vandetanib alone or in combination increases the occupancy of ATF4 at the promoter of KLF9 gene and stimulates the expression of KLF9. Depletion of ATF4 by shRNA leads to downregulation of KLF9 expression and prevents oxidative stress-induced cell death. These findings suggest that induction of the ATF4/KLF9 axis causes oxidative stress leading to excessive ER stress, and subsequent cell death. Thus, combining TKIs and ERAD inhibitors that promote ATF4 levels could be effective therapeutic strategies for treating MTC and preventing resistance to TKIs. Citation Format: Rozita Bagheri-Yarmand, Krishna M. Sinha, Ling Li, Yue Lu, Robert F. Gagel. Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer [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 2426.

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.

Activating Transcription Factor 4 (ATF4) Regulates Neuronal Activity by Controlling GABABR Trafficking.

Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 downregulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF4's role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABAB receptors (GABABRs). Knocking down ATF4 results in significant reduction of GABABR-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABABR 1a and 1b subunits, indicating that ATF4 regulates GABABR trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABABR2s, several GABABR-coupled ion channels or β2 and γ2 GABAARs. Pharmacologic manipulations confirmed the relationship between GABABR functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 downregulation cited above are fully rescued by transcriptionally active, but not by transcriptionally inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABABR trafficking, we downregulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABABRs, which in turn modulates the excitability properties of neurons.SIGNIFICANCE STATEMENT GABAB receptors (GABABRs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABABRs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABABRs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 downregulation in hippocampal neurons reduces membrane-bound GABABR levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 downregulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABABRs.