ATF4 Expression Research Articles

Selenium alleviates cadmium-induced oxidative stress, endoplasmic reticulum stress, and apoptosis in L8824 cells

Cadmium (Cd) is a toxic pollutant in industrial production that induces organ damage and apoptosis, While, selenium (Se) has the biological function of antagonizing Cd toxicity. Hence, to gain further insight into the protective mechanisms of selenium against Cd-induced damage in Ctenopharyngodon idella liver (L8824) cells, L8824 were exposed to 5 μM, 15 μM, 25 μM cadmium chloride for 24 h after pre-incubation with 25 μM sodium selenite for 9 h. Cell proliferation and morphological changes, the levels of reactive oxygen species (ROS) and antioxidant enzyme activity, mitochondrial membrane potential (MMP), endoplasmic reticulum stress (ERS)-related pathway genes expression, intracellular calcium levels and apoptosis were assessed to explore the protective effect of selenium in Cd-induced L8824 cell damage. The results showed that Cd caused decreased cell viability, ROS accumulation, reduced activity of antioxidant enzymes (SOD, CAT GPx and T-AOC) and apoptosis in L8824 cells. The incubation of Se prominently ameliorated cell proliferation, activated the Keap1-Nrf2 pathway, and restored antioxidant enzyme activity. Furthermore, the expression of grp78, perk, eif-2α, atf4, chop bax, jnk, caspase-3 and caspase-9 was significantly upregulated after Cd exposure, while the expression of bcl-2 was significantly downregulated. Se supplementation alleviated Cd-induced ERS and apoptosis. Moreover, Cd-induced elevation of intracellular Ca2+ levels were alleviated by dantrolene and 2-APB, suggesting that intracellular calcium disorders were caused by Ca2+ released by RyR and IP3R-mediated ER. The results of this study suggested that Cd could induce oxidative stress, ERS, mitochondrial damage and evoke apoptosis, whereas Se had protective effects in preventing Cd induced damage by inhibiting ERS, maintaining intracellular calcium homeostasis, enhancing the antioxidant capacity of L8824 cells and downregulating the Keap1/Nrf2 pathway.

The Protective Effect of Ginkgolide K on Astrocytes Exposed to Myelin Debris in vitro and Demyelination in vivo

Background Ginkgolide K ( GK) has a protective effect on neurons and myelin sheath, and the function of astrocytes in phagocytizing myelin debris has attracted extensive attention in remyelination. Objectives This study focuses on the impact of GK on the phagocytosis of myelin debris by astrocytes and explores the possibility of treating demyelination. Materials and Methods Male C57BL/6 mice were used to establish a cuprizone (CPZ)-induced demyelination model. After being fed a normal or CPZ diet for 4 weeks, mice were intraperitoneally injected with PEG400 or GK for 14 consecutive days. GL261 and primary astrocytes were exposed to myelin debris. Immunohistochemistry/immunocytochemistry staining, western blot, RT-PCR, and other methods were used to detect the relevant indicators. Results Astrocytes engulfed myelin debris, leading to astrocyte reactivity with increased p-NF-kB/p65 and ATF6 expression and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression, which was reversed by GK. Subsequently, astrocytes stimulated by myelin debris underwent self-apoptosis with increased expression of caspase-3 and BCL2-associated X (Bax), which was inhibited by GK. Simultaneously, GK efficiently promoted astrocytes to increase the production of neurotrophic ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF), speculating that increased CNTF/bFGF, decreased p-NF-kB/p65/ATF6, and up-regulated Nrf2 should participate in the protection of astrocyte apoptosis and build a beneficial microenvironment for myelin regeneration. Conclusion The results suggest that GK may have the potential to treat demyelination by promoting debris clearance and improving the brain microenvironment. However, further studies are needed to understand the physiological and pathological consequences of astrocytic phagocytosis and to investigate the possibility of using GK as a therapeutic application.

Zinc deficiency causes oxidative stress, endoplasmic reticulum stress, apoptosis and inflammation in hepatocytes in grass carp.

A lack of the trace element zinc (Zn) in freshwater environments causes slow growth and malnutrition and affects the normal biological functions of organisms. In this study, a Zn deficiency model of grass carp hepatocytes was established with TPEN. Acetylcysteine (NAC) was used as an inhibitor. TPEN was added to L8824cell culture medium, and LDH, AST, ALT, and AKP activities were enhanced in a Zn-deficient environment, leading to abnormal hepatopancreas function. Fluorescence microscopy showed an increase in ROS levels, and antioxidant enzyme activity assays revealed that SOD, CAT, GSH-PX, and T-AOC activities were decreased, indicating oxidative stress caused by Zn deficiency. The RT‒PCR results showed that the mRNA expression of GRP78, PERK, EIF2α, ATF4, and Chop was increased due to the addition of TPEN. Calcium kits showed increased Ca2+ levels. The RT‒PCR results showed that the mRNA expression levels of Caspase-12, Caspase-9, Caspase-3, and PARP apoptotic were increased due to the addition of TPEN. RT‒PCR and ELISA showed that the expression levels of interleukin-1β (IL-1β), interleukin-8 (IL-8), tumour necrosis factor (TNF-α), and inducible nitric oxide synthase (iNOS) were increased. This led to the conclusion that Zn deficiency in the freshwater environment caused inflammation and apoptosis in hepatocytes in grass carp. For the first time, apoptosis caused by endoplasmic reticulum stress in grass carp hepatocytes due to Zn deficiency was studied in the context of Ca2+. The present study provided some insight into the adverse effects of Zn deficiency in freshwater environments on fish.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

Loss of PERK function promotes ferroptosis by downregulating SLC7A11 (System Xc⁻) in colorectal cancer

Ferroptosis, a genetically and biochemically distinct form of programmed cell death, is characterised by an iron-dependent accumulation of lipid peroxides. Therapy-resistant tumor cells display vulnerability toward ferroptosis. Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) play a critical role in cancer cells to become therapy resistant. Tweaking the balance of UPR to make cancer cells susceptible to ferroptotic cell death could be an attractive therapeutic strategy. To decipher the emerging contribution of ER stress in the ferroptotic process, we observe that ferroptosis inducer RSL3 promotes UPR (PERK, ATF6, and IRE1α), along with overexpression of cystine-glutamate transporter SLC7A11 (System Xc-). Exploring the role of a particular UPR arm in modulating SLC7A11 expression and subsequent ferroptosis, we notice that PERK is selectively critical in inducing ferroptosis in colorectal carcinoma. PERK inhibition reduces ATF4 expression and recruitment to the promoter of SLC7A11 and results in its downregulation. Loss of PERK function not only primes cancer cells for increased lipid peroxidation but also limits in vivo colorectal tumor growth, demonstrating active signs of ferroptotic cell death in situ. Further, by performing TCGA data mining and using colorectal cancer patient samples, we demonstrate that the expression of PERK and SLC7A11 is positively correlated. Overall, our experimental data indicate that PERK is a negative regulator of ferroptosis and loss of PERK function sensitizes colorectal cancer cells to ferroptosis. Therefore, small molecule PERK inhibitors hold huge promise as novel therapeutics and their potential can be harnessed against the apoptosis-resistant condition.

Transducin Beta-Like 2 is a Potential Driver Gene that Adapts to Endoplasmic Reticulum Stress to Promote Tumor Growth of Lung Adenocarcinoma.

Endoplasmic reticulum (ER) stress has a close relation with cancer progression. Blocking the adaptive pathway of ER stress could be an anticancer strategy. Here, we identified an ER stress-related gene, Transducin beta-like 2 (TBL2), an ER-localized type I transmembrane protein, on increased chromosome 7q as a candidate driver gene of lung adenocarcinoma (LUAD). The association between TBL2 mRNA expression and prognostic outcomes and clinicopathological factors was analyzed using The Cancer Genome Atlas (TCGA) datasets of LUAD and lung squamous cell carcinoma (LUSC). Localization of TBL2 in tumor tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) was conducted using TCGA dataset. In vitro cell proliferation assays were performed using TBL2 knockdown LUAD cells, LUSC cells, and LUAD cells overexpressing TBL2. Apoptosis and ATF4 expression (ER stress marker) were evaluated by western blotting. TBL2 was overexpressed in LUAD and LUSC cells. Multivariate analysis indicated high TBL2 mRNA expression was an independent poor prognostic factor of LUAD. GSEA revealed high TBL2 expression was positively correlated to the ER stress response in LUAD. TBL2 knockdown attenuated LUAD cell proliferation under ER stress. TBL2 inhibited apoptosis in LUAD cells under ER stress. TBL2 knockdown reduced ATF4 expression under ER stress. TBL2 may be a novel driver gene that facilitates cell proliferation, possibly by upregulating ATF4 expression followed by adaptation to ER stress, and it is a poor prognostic biomarker of LUAD.

Selenium nanoparticles reduce oxidative stress-induced cardiomyocyte apoptosis in ascites syndrome in broiler chickens via the ATF6-DR5 signaling pathway

Broiler ascites syndrome (AS) is one of the main diseases threatening the health of broilers. It is well documented that myocardial hypertrophy and failure is one of the key mechanisms of broiler ascites syndrome. Therefore, prevention of cardiac hypertrophy and failure would be one goal to reduce broiler ascites syndrome incidence. Myocardial hypertrophy and failure are closely related to endoplasmic reticulum stress (ERS) in cardiac myocytes, and the endoplasmic reticulum stress signaling system (ATF6-DR5) is one of the important pathways of myocardial apoptosis. Excessive hypertrophy will affect the heart muscle’s normal contraction and diastole function, and the heart will turn from compensated to decompensate thus causing myocardial injury. Myocardial apoptosis is a core component of the pathological changes of this myocardial injury. Nano-selenium is a kind of red elemental selenium nanoparticle. Due to its excellent physical, chemical and biological properties, it has attracted extensive academic attention in recent years. It has been proven to have excellent antioxidant, antibacterial, antitumor, antihypertrophic, and antiapoptotic abilities. Herein, nano-selenium (1 µmol/L) can inhibit hydrogen peroxide (H2O2)-induced oxidative stress in broiler primary cardiomyocytes, and at the same time reduce cardiomyocyte apoptosis. In vivo, nano-selenium can reduce broiler myocardial injury-related enzyme indicators (AST, CK and LDH), and alleviate myocardial injury. It can also activate the antioxidant enzyme system (SOD, GSH-Px and CAT) and reduce MDA, and make the recovery of T-AOC ability in the organization. Meanwhile, nano-selenium can down-regulate the genes and proteins expression of ATF-6, GRP-78, CHOP and caspase 12 in the ERS-related signaling pathway, and inhibit that of downstream-related caspase 3, Bax and caspase 9, and increase that of the downstream anti-apoptotic Bcl-2, thereby maintaining the homeostasis of the endoplasmic reticulum and alleviating cardiomyocyte apoptosis. It can be seen that nano-selenium can protect the damaged myocardium in the broiler ascites caused by high-salt drinking by regulating the ATF6-DR5 signaling pathway. This study was performed in chickens and cardiomyocyte cells and attempted to demonstrate that selenium nanoparticles can protect the damaged myocardium in broiler ascites. This paper provides a new idea for preventing and treating broiler ascites syndrome.

Effect of eIF2α in Neuronal Injury Induced by High Glucose and the Protective Mechanism of Resveratrol.

Diabetes mellitus (DM) is a type of metabolic disease characterized by chronic hyperglycemia, which can lead to different degrees of cognitive decline. Therefore, it is crucial to explore the molecular biological mechanisms of neuronal injury. In this study, we investigated the effect of high glucose on eIF2α expression and the mechanism of neuronal injury, and on this basis, the protective mechanism of resveratrol is explored. Treatment with 50 mM high glucose in cortical neurons increased the levels of eIF2α phosphorylation; the expressions of ATF4 and CHOP increased. ISRIB alleviated high glucose-induced neuronal injury by reducing eIF2α phosphorylation when neurons were pretreated with ISRIB before high glucose treatment. Compared with the high glucose-treated group, resveratrol pretreatment reduced eIF2α phosphorylation, the levels of its downstream molecules ATF4 and CHOP, and LDH release. Resveratrol reduced the level of cortical eIF2α phosphorylation and the expression of its downstream molecules in DM mice and improved the ability of spatial memory and learning in DM mice without affecting anxiety and motor performance. Meanwhile, resveratrol modulated the expression of Bcl-2 protein and also effectively decreased the DM-induced up-regulation of Bax, caspase-3, p53, p21, and p16. Taken together, these results suggested that high glucose caused neuronal injury through the eIF2α/ATF4/CHOP pathway which was inhibited by ISRIB and resveratrol. The present study indicates that eIF2α is the new target for the treatment of high glucose-induced neuronal injury, and resveratrol is a potential new medicine to treat diabetes encephalopathy.

Stress responses to warming in Japanese flounder (Paralichthys olivaceus) from different environmental scenarios

Japanese flounder (Paralichthys olivaceus) is one of cold-water species widely farmed in Asia. In recent years, the increased frequency of extreme weather events caused by global warming has led to serious impact on Japanese flounder. Therefore, it is crucial to understand the effects of representative coastal economic fish under increasing water temperature. In this study, we investigated the histological and apoptosis responses, oxidative stress and transcriptomic profile in the liver of Japanese flounder exposed to gradual temperature rise (GTR) and abrupt temperature rise (ATR). The histological results showed liver cells in ATR group were the most serious in all three groups including vacuolar degeneration and inflammatory infiltration, and had more apoptosis cells than GTR group detected by TUNEL staining. These further indicated ATR stress caused more severe damage than GTR stress. Compared with control group, the biochemical analysis showed significantly changes in two kinds of heat stress, including GPT, GOT and D-Glc in serum, ATPase, Glycogen, TG, TC, ROS, SOD and CAT in liver. In addition, the RNA-Seq was used to analyze the response mechanism in Japanese flounder liver after heat stress. A total of 313 and 644 differentially expressed genes (DEGs) were identified in GTR and ATR groups, respectively. Further pathway enrichment of these DEGs revealed that heat stress affected cell cycle, protein processing and transportation, DNA replication and other biological processes. Notably, protein processing pathway in the endoplasmic reticulum (ER) was enriched significantly in KEGG and GSEA enrichment analysis, and the expression of ATF4 and JNK was significantly up-regulated in both GTR and ATR groups, while CHOP and TRAF2 were high expressed in GTR and ATR groups, respectively. In conclusion, heat stress could cause tissue damage, inflammation, oxidative stress and ER stress in the liver of Japanese flounder. The present study would provide insight into the reference for the adaptive mechanisms of economic fish in face of increasing water temperature caused by global warming.

Comparing the effects of microgravity and amyotrophic lateral sclerosis on mouse dorsal root ganglia

Introduction: Microgravity (MG) exposure causes motor deficits and decreased neuronal activity, effects that resemble the ones observed in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Several recent studies have shown that exposure to MG and ALS also impacts the sensory systems. Yet, the role of sensory impairment in this degenerative process of exposure to MG and ALS remains unknown. In this study, we aimed at elucidating how the sensory system is affected by exposure to MG and ALS.Methods: To this end, we compared gene expression in the mouse lumbar dorsal root ganglia (DRG) of MG-exposed animals with that of control animals that remained under artificial gravity conditions. We then investigated the effects of the human superoxide dismutase 1 (SOD1) G93A mutation in a mouse model of ALS (SOD1G93A mice) on gene expression in the DRG.Results: The overlap of genes with negatively correlated expression was greater than those with positively correlated expression between the DRG of MG-exposed and SOD1G93A mice. Additionally, genes related to Imoonglia (characteristics of both immune and glial cells) and macrophage increased in response to MG exposure, while satellite glial cell genes were expressed in response to SOD1 mutation. Next, we examined genes related to sensory neuron subtypes in the DRG. We found altered gene expression in genes related to proprioceptive and mechanoreceptive neurons in the DRG of MG-exposed and SOD1G93A mice. Remarkably, the expression of Atf3 and genes related to nociceptive neurons in the DRG of SOD1G93A mice at postnatal day (P) 120 was considerably altered, whereas MG-exposed and SOD1G93A mice at P30 presented little changes.Discussion: These results indicate that exposure to MG and ALS affect gene expression in genes related to neurons and non-neuronal cells in the DRG, with significant differences between the effects of MG and the SOD1 mutation. Elucidation of the impact of exposure to MG and ALS pathogenesis in the DRG, including identification of the molecular pathways that regulate DRG dysfunction, will help better understand the differences in vulnerability and the triggering processes of impaired motor function associated with MG and ALS.

1491-P: ER Stress Drives the VEGF Expression in Monocytic Cells by Mechanisms Involving CHOP/ROS/HIF-1alpha/NRF2 and NF-kappaB/p38 MAPK Pathways

Obesity is marked by metabolic inflammation and by metabolic impairment caused by increased endotoxin, free fatty acids, and vascular endothelial growth factor (VEGF) levels; and involves the endoplasmic reticulum (ER) stress as well. However, it remains unclear whether the ER stress can induce/amplify VEGF expression in metabolically-stressed monocytic cells; and if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 monocytic cells by treating cells separately with lipopolysaccharide (LPS), palmitic acid (PA), and oleic acid (OA), in presence/absence of ER stressor thapsigargin (TG). Gene expression of VEGF/ER stress markers was assessed by qRT-PCR, protein expression by ELISA, ROS by DCFH-DA assay, phosphorylation of HIF-1α, NF-κB, ERK1/2, and p38 MAPK by immunoblotting, and the insulin response in stressed cells by glucose-uptake assay. Regarding clinical analyses, adipose VEGF gene and protein expression was detected using qRT-PCR and IHC, respectively, while plasma hs-CRP, TNF-α, MDA, and OX-LDL levels were measured by ELISA. The experimental data show that a cooperative interaction between the metabolic and ER stresses led to the expression of VEGF, ROS, CHOP, ATF6, SOD2, and NRF2 (P˂0.05), as well as stimulated the CHOP and NRF2 promoter activities in reporter cells (P˂0.05). However, the glucose uptake was not impaired. The VEGF expression was dependent on phosphorylation of HIF-1α, NF-κB, and p38 MAPK; and the inhibitors of NF-κB/MAPK pathways as well as antioxidants or ROS scavengers suppressed the VEGF production. Further, individuals with obesity showed increased VEGF expression which associated positively with plasma levels of hs-CRP, TNF-α, MDA, and OX-LDL (P≤0.05). Overall, our findings support a cooperativity model in which the ER and metabolic stresses interact to augment VEGF expression in monocytic cells via the mechanisms involving CHOP/ROS/HIF-1α/NRF2 and NF-κB/p38 MAPK pathways. Disclosure S.A.K.Sindhu: None. F.Alzaid: None. F.Almulla: None. R.Ahmad: None. N.Akhter: None. A.Wilson: None. H.Arefanian: None. A.Al madhoun: None. R.S.Thomas: None. S.P.Kochumon: None. F.Alrashed: None. F.Bahman: None. Funding Kuwait Foundation for the Advancement of Sciences (RA2015-027, RA2010-003, RAHM-2019-022)

1778-P: Endoplasmic Reticulum Stress Leads to Pancreatic Beta-Cell Failure by Suppressing cAMP Signaling via ATF4

Type 2 diabetes (T2D) is characterized by β-cell dysfunction and loss. Endoplasmic reticulum (ER) stress responses, especially the PERK-ATF4 pathway, promote β-cell disorders. The role of the PERK-ATF4 pathway in β-cell dysfunction is still unknown. We report that ATF4 induces PDE4D production which impairs β-cell function by downregulating cAMP signaling. Transgenic expression of ATF4 in β-cells caused early β-cell dysfunction and loss, resembling accelerated T2D. ATF4 expression enhanced PDE4D, decreased cAMP signaling, and inhibited incretin and glucose responses in β-cells. In leptin receptor-deficient diabetic (db/db) mice, β-cells displayed higher nuclear ATF4 localization along with elevated PDE4D expression. Inhibiting PDE4 activity with selective pharmacological agents increased β-cell function in db/db and ATF4 transgenic mice. In summary, ER stress promotes β-cell failure via ATF4-mediated PDE4D synthesis, proposing PDE4D as a therapeutic target for safeguarding β-cell function in T2D. Disclosure J. Lee: None. Funding National Research Foundation of Korea (2019R1A4A102972413, 2020M3A9D8038660)

Role of PERK-mediated pathway in the effect of mild hypothermia after cerebral ischaemia/reperfusion.

Hypothermia is an effective method of reducing brain injury caused by a variety of neurological insults. It is aimed to elucidate whether a change in the expression of PERK-mediated pathway proteins is an indicator of the neuroprotective effect of mild hypothermia after cerebral ischaemia/reperfusion. One hundred and ninety-two male C57BL/6 mice were randomly divided into three groups: a sham group, a cerebral normothermic ischaemia/reperfusion (I/R) group and a cerebral hypothermic I/R group. A cerebral ischaemia model was established by ligating the bilateral common carotid artery for 15 min. Mice in the hypothermia group stayed in a cage that was set at 33°C, sprayed with a spray of 70% ethanol, and blown with two high-speed fans. The state of neurons was assessed on micropreparations stained with haematoxylin-eosin and TUNEL. The expressions of GRP78, p-perk, p-eif2α, ATF4 and CHOP were measured by western blot analysis 6, 12, 24 and 72 h after reperfusion. The number of surviving cells was significantly higher in the hypothermia group than in the group without hypothermia (p < .05). The GRP78 expression in the hypothermia group was statistically higher (p < .05) than in the ischaemia/reperfusion group. Optical densities of p-perk, p-eif2α and ATF4 in hippocampus CA1 neurons ischaemia were statistically significantly lower in the hypothermia group than in the ischaemia/reperfusion group (p < .05). The CHOP expression in the hypothermia group was statistically lower (p < .05) than in the ischaemia/reperfusion group. Mild hypothermia for 6 h promoted moderate neuroprotection by mediating the expression of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP.

Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3.

Hepatocellular carcinoma (HCC) is a common cancer worldwide, and sorafenib is a first-line drug for the treatment of advanced liver cancer. Resistance to sorafenib has become a major challenge in the treatment of hepatocellular carcinoma, however, studies have shown that metformin can promote ferroptosis and sorafenib sensitivity. Therefore, the aim of this study was to investigate the promotion of ferroptosis and sorafenib sensitivity by metformin via ATF4/STAT3 in hepatocellular carcinoma cells. Hepatocellular carcinoma cells Huh7 and Hep3B and induced sorafenib resistance (SR) Huh7/SR and Hep3B/SR cells were used as in vitro cell models. Cells were injected subcutaneously to establish a drug-resistant mouse model. CCK-8 was used to detect cell viability and sorafenib IC50. Western blotting was used to detect the expression of relevant proteins. BODIPY staining was used to analyze the lipid peroxidation level in cells. A scratch assay was used to detect cell migration. Transwell assays were used to detect cell invasion. Immunofluorescence was used to localize the expression of ATF4 and STAT3. Metformin promoted ferroptosis in hepatocellular carcinoma cells through ATF4/STAT3, decreased sorafenib IC50, increased ROS and lipid peroxidation levels, decreased cell migration and invasion, inhibited the expression of the drug-resistant proteins ABCG2 and P-GP in hepatocellular carcinoma cells, and thus inhibited sorafenib resistance in hepatocellular carcinoma cells. Downregulating ATF4 inhibited the phosphorylated nuclear translocation of STAT3, promoted ferroptosis, and increased the sensitivity of Huh7 cells to sorafenib. Metformin was also shown in animal models to promote ferroptosis and sorafenib sensitivity in vivo via ATF4/STAT3. Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3, and it inhibits HCC progression.

ATRT-12. HYPERACTIVATING THE INTEGRATED STRESS RESPONSE WITH PROTEASOME INHIBITION IN AT/RT

Abstract Atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive pediatric brain tumor with a poor prognosis. AT/RT has increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. Elevated expression of ATF4 indicates activation of the ISR. Transient or low-level expression of this transcription factor protects cells from stress, while sustained, high-level activation results in cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. Because AT/RT has a high baseline level of ATF4, we hypothesized that this tumor would be susceptible to ISR activators such as ixazomib. After determining the IC50 of ixazomib, AT/RT cell lines were treated with increasing concentrations of the drug. Cleaved caspase-3 immunofluorescence showed a significant increase of apoptosis (CHLA06 p&amp;lt;0.0001 by ANOVA). We are currently testing ixazomib in combination with idarubicin, a brain-penetrant anthracycline, and gemcitabine, a brain-penetrant nucleoside analog. Synergy testing in CHLA06 showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 with inhibitory concentrations of ixazomib and idarubicin in the low nanomolar range (scores of 10 or above indicate synergy). Similarly, ixazomib and gemcitabine synergized to suppress CHLA06 growth and induce apoptosis (67% Annexin V+ cells in combination compared to 14 and 24% Annexin V+ with single agent treatment). In the future, we will determine if ixazomib selectively kills AT/RT while sparing normal cells in iPSC brain organoids. We will also test ixazomib in orthotopic xenografts of AT/RT. These results suggest that ixazomib, especially in combination with idarubicin, gemcitabine or other ISR activators, has the potential to serve as an effective therapy for AT/RT.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n= 72) and tumoral (n= 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of β-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

Shikonin suppresses small cell lung cancer growth via inducing ATF3-mediated ferroptosis to promote ROS accumulation

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Induction of Unfolded Protein Response by Tannic Acid Triggers Apoptosis in MDA-MB-231 Breast Cancer Cells.

Endoplasmic reticulum (ER) stress can reduce cell survival and enhances the apoptosis of cancer cells. Plant polyphenols like tannic acid trigger ER stress and apoptosis and therefore can be a novel agent for the treatment of cancer. In this study, we investigated the effect of tannic acid on survival, migration, colony formation, ER stress pathway, and apoptosis of the MDA-MB-231 breast cancer cells. The MTT assay was performed to investigate the effect of tannic acid on the cell survival of breast cancer cells. We used the qPCR method to reveal the effect of tannic acid on the Bak, CHOP, ATF4, P21, MMP-2, and Bcl-2 expression. Also, colony formation, cell migration, and Hoechst staining assays were employed. The results of the MTT test showed that tannic acid reduced the cell survival rate. In the qPCR assay, we found that tannic acid decreased the expression levels of MMP-2, Bcl-2, ATF4, and CHOP genes, paradoxically, enhanced the expression of Bak and P21 genes. The colony formation and cell migration assays indicated that tannic acid significantly diminished breast cancer cell proliferation and migration, respectively. In the apoptosis assay, tannic acid increased the number of apoptotic cells. Tannic acid increases the rate of cell death but decreases viability and cell migration. Moreover, tannic acid induces apoptosis in breast cancer cells. Overall, our study demonstrates that tannic acid induces ER stress by increasing the genes which are playing role in ER stress pathway. These results show that tannic acid can be used as an effective agent for breast cancer treatment.

ERVW-1 Activates ATF6-Mediated Unfolded Protein Response by Decreasing GANAB in Recent-Onset Schizophrenia.

Schizophrenia, a mental disorder, afflicts 1% of the worldwide population. The dysregulation of homeostasis in the endoplasmic reticulum (ER) has been implicated in schizophrenia. Moreover, recent studies indicate that ER stress and the unfolded protein response (UPR) are linked to this mental disorder. Our previous research has verified that endogenous retrovirus group W member 1 envelope (ERVW-1), a risk factor for schizophrenia, is elevated in individuals with schizophrenia. Nevertheless, no literature is available regarding the underlying relationship between ER stress and ERVW-1 in schizophrenia. The aim of our research was to investigate the molecular mechanism connecting ER stress and ERVW-1 in schizophrenia. Here, we employed Gene Differential Expression Analysis to predict differentially expressed genes (DEGs) in the human prefrontal cortex of schizophrenic patients and identified aberrant expression of UPR-related genes. Subsequent research indicated that the UPR gene called XBP1 had a positive correlation with ATF6, BCL-2, and ERVW-1 in individuals with schizophrenia using Spearman correlation analysis. Furthermore, results from the enzyme-linked immunosorbent assay (ELISA) suggested increased serum protein levels of ATF6 and XBP1 in schizophrenic patients compared with healthy controls, exhibiting a strong correlation with ERVW-1 using median analysis and Mann-Whitney U analysis. However, serum GANAB levels were decreased in schizophrenic patients compared with controls and showed a significant negative correlation with ERVW-1, ATF6, and XBP1 in schizophrenic patients. Interestingly, in vitro experiments verified that ERVW-1 indeed increased ATF6 and XBP1 expression while decreasing GANAB expression. Additionally, the confocal microscope experiment suggested that ERVW-1 could impact the shape of the ER, leading to ER stress. GANAB was found to participate in ER stress regulated by ERVW-1. In conclusion, ERVW-1 induced ER stress by suppressing GANAB expression, thereby upregulating the expression of ATF6 and XBP1 and ultimately contributing to the development of schizophrenia.