Knockdown Of ATF4 Research Articles

Copper exposure induces mitochondrial dysfunction and hepatotoxicity via the induction of oxidative stress and PERK/ATF4 -mediated endoplasmic reticulum stress

Copper is an essential trace element, and excessive exposure could result in hepatoxicity, however, the underlying molecular mechanisms remain incompletely understood. The present study is aimed to investigate the molecular mechanisms of copper sulfate (CuSO4) exposure-induced hepatoxicity both in vivo and in vitro. In vitro, HepG2 and L02 cells were exposed to various doses of CuSO4 for 24 h. Cell viability, ROS production, oxidative stress biomarkers, mitochondrial functions, ultrastructure, intracellular calcium (Ca2+) concentration, and the expression of proteins related to mitochondrial apoptosis and endoplasmic reticulum (ER) stress were assessed. In vivo, C57BL/6 mice were treated with CuSO4 at doses of 10 and 30 mg/kg BW/day and co-treated with 4-PBA at 100 mg/kg BW/day for 35 days. Subsequently, liver function, histopathological features, and protein expression were evaluated. Results found that exposure to CuSO4 at concentrations of 100–400 μM for 24 h significantly decreased the viabilities of HepG2 and L02 cells and it was in a dose-dependent manner. Additionally, CuSO4 exposure induced significant oxidative stress and mitochondrial dysfunction in HepG2 cells, which were partially ameliorated by the antioxidant N-acetylcysteine (NAC). Furthermore, CuSO4 exposure prominently triggered ER stress, as evidenced by the upregulation of GRP94, GRP78, phosphorylated forms of PERK and eIF2α, and CHOP proteins in livers of mice and HepG2 cells. NAC treatment significantly inhibited CuSO4 exposure -induced ER stress in HepG2 cells. Pharmacological inhibition of ER stress through co-treatment with 4-PBA and the PERK inhibitor GSK2606414, as well as genetic knockdown of ATF4, partially mitigated CuSO4-induced cytotoxicity in HepG2 cells by reducing mitochondrial dysfunction and inhibiting the mitochondrial apoptotic pathway. Moreover, 4-PBA treatment significantly attenuated CuSO4-induced caspase activation and hepatoxicity in mice. In conclusion, these results reveal that CuSO4-induced hepatotoxicity involves mitochondrial dysfunction and ER stress by activating oxidative stress induction and PERK/ATF4 pathway.

Inhibiting the compensatory elevation of xCT collaborates with disulfiram/copper-induced GSH consumption for cascade ferroptosis and cuproptosis

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the fourth leading cause of cancer-related death globally, which is characterized by complicated pathophysiology, high recurrence rate, and poor prognosis. Our previous study has demonstrated that disulfiram (DSF)/Cu could be repurposed for the treatment of HCC by inducing ferroptosis. However, the effectiveness of DSF/Cu may be compromised by compensatory mechanisms that weaken its sensitivity. The mechanisms underlying these compensatory responses are currently unknown. Herein, we found DSF/Cu induces endoplasmic reticulum stress with disrupted ER structures, increased Ca2+ level and activated expression of ATF4. Further studies verified that DSF/Cu induces both ferroptosis and cuproptosis, accompanied by the depletion of GSH, elevation of lipid peroxides, and compensatory increase of xCT. Comparing ferroptosis and cuproptosis, it is interesting to note that GSH acts at the crossing point of the regulation network and therefore, we hypothesized that compensatory elevation of xCT may be a key aspect of the therapeutic target. Mechanically, knockdown of ATF4 facilitated the DSF/Cu-induced cell death and exacerbated the generation of lipid peroxides under the challenge of DSF/Cu. However, ATF4 knockdown was unable to block the compensatory elevation of xCT and the GSH reduction. Notably, we found that DSF/Cu induced the accumulation of ubiquitinated proteins, promoted the half-life of xCT protein, and dramatically dampened the ubiquitination–proteasome mediated degradation of xCT. Moreover, both pharmacologically and genetically suppressing xCT exacerbated DSF/Cu-induced cell death. In conclusion, the current work provides an in-depth study of the mechanism of DSF/Cu-induced cell death and describes a framework for the further understanding of the crosstalk between ferroptosis and cuproptosis. Inhibiting the compensatory increase of xCT renders HCC cells more susceptible to DSF/Cu, which may provide a promising synergistic strategy to sensitize tumor therapy and overcome drug resistance, as it activates different programmed cell death.

Dual targeting agent Thiotert inhibits the progression of glioblastoma by inducing ER stress-dependent autophagy

Glioblastoma (GBM) is the most aggressive and lethal type of tumor in the central nervous system, characterized by a high incidence and poor prognosis. Thiotert, as a novel dual targeting agent, has potential inhibitory effects on various tumors. Here, we found that Thiotert effectively inhibited the proliferation of GBM cells by inducing G2/M cell cycle arrest and suppressed the migratory ability in vitro. Furthermore, Thiotert disrupted the thioredoxin (Trx) system while causing cellular DNA damage, which in turn caused endoplasmic reticulum (ER) stress-dependent autophagy. Knockdown of ER stress-related protein ATF4 in U251 cells inhibited ER stress-dependent autophagy caused by Thiotert to some extent. Orthotopic transplantation experiments further showed that Thiotert had the same anti-GBM activity and mechanism as in vitro. Conclusively, these results suggest that Thiotert induces ER stress-dependent autophagy in GBM cells by disrupting redox homeostasis and causing DNA damage, which provides new insight for the treatment of GBM.

DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells

As one of the most important phthalates, di-isononyl phthalate (DINP) has been widely used as a common plasticizer in the food and personal care products sectors. In our previous study, we found that DINP can induce autophagy of ovarian granulosa cells; while the underlying mechanism is unclear. In the study, we showed that DINP exposure could induce autophagy of ovarian granulosa cells and KGN cells, accompanied with the increase in the mRNA and protein level of DDIT4. Furthermore, overexpression of DDIT4 were shown to induce autophagy of KGN cells; while knockdown of DDIT4 inhibited DINP-induced autophagy, implying that DDIT4 played an important role in DINP-induced autophagy of ovarian granulosa cells. There were three putative binding sites of transcription factor ATF4 in the promoter region of DDIT4 gene, suggesting that DDIT4 might be regulated by ATF4. Herein, we found that overexpression of ATF4 could upregulate the expression of DDIT4 in KGN cells, while knockdown of ATF4 inhibited its expression. Subsequently, ATF4 was identified to bind to the promoter region of DDIT4 gene and promote its transcription. The expression of ATF4 was also increased in the DINP-exposed granulosa cells, and ATF4 overexpression promoted autophagy of KGN cells; whereas knockdown of ATF4 alleviated DINP-induced upregulation of DDIT4 and autophagy of the cells. Taken together, DINP triggered autophagy of ovarian granulosa cells through activating ATF4/DDIT4 signals.

TXNDC12 inhibits lipid peroxidation and ferroptosis

Ferroptosis is a type of regulated cell death characterized by lipid peroxidation and subsequent damage to the plasma membrane. Here, we report a ferroptosis resistance mechanism involving the upregulation of TXNDC12, a thioredoxin domain-containing protein located in the endoplasmic reticulum. The inducible expression of TXNDC12 during ferroptosis in leukemia cells is inhibited by the knockdown of the transcription factor ATF4, rather than NFE2L2. Mechanistically, TXNDC12 acts to inhibit lipid peroxidation without affecting iron accumulation during ferroptosis. When TXNDC12 is overexpressed, it restores the sensitivity of ATF4-knockdown cells to ferroptosis. Moreover, TXNDC12 plays a GPX4-independent role in inhibiting lipid peroxidation. The absence of TXNDC12 enhances the tumor-suppressive effects of ferroptosis induction in both cell culture and animal models. Collectively, these findings demonstrate anendoplasmic reticulum-based anti-ferroptosis pathway in cancer cells with potential translational applications.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

UORF-Mediated Translational Regulation of ATF4 Serves as an Evolutionarily Conserved Mechanism Contributing to Non-Small-Cell Lung Cancer (NSCLC) and Stress Response.

Diseases and environmental stresses are two distinct challenges for virtually all living organisms. In light of evolution, cellular responses to diseases and stresses might share similar molecular mechanisms, but the detailed regulation pathway is not reported yet.We obtained the transcriptomes and translatomes from several NSCLC (non-small-cell lung cancer) patients as well as from different species under normal or stress conditions. We found that the translation level of gene ATF4 is remarkably enhanced in NSCLC due to the reduced number of ribosomes binding to its upstream open reading frames (uORFs). We also showed the evolutionary conservation of this uORF-ATF4 regulation in the stress response of other species. Molecular experiments showed that knockdown of ATF4 reduced the cell growth rate while overexpression of ATF4 enhanced cell growth, especially for the ATF4 allele with mutated uORFs. Population genetics analyses in multiple species verified that the mutations that abolish uATGs (start codon of uORFs) are highly deleterious, suggesting the functional importance of uORFs.Our study proposes an evolutionarily conserved pattern that enhances the ATF4 translation by uORFs upon stress or disease. We generalized the concept of cellular response to diseases and stresses. These two biological processes may share similar molecular mechanisms.

Activated amino acid response pathway generates apatinib resistance by reprograming glutamine metabolism in non-small-cell lung cancer

The efficacy of apatinib has been confirmed in the treatment of solid tumors, including non-small-cell lung cancer (NSCLC). However, the direct functional mechanisms of tumor lethality mediated by apatinib and the precise mechanisms of drug resistance are largely unknown. In this study, we demonstrated that apatinib could reprogram glutamine metabolism in human NSCLC via a mechanism involved in amino acid metabolic imbalances. Apatinib repressed the expression of GLS1, the initial and rate-limiting enzyme of glutamine catabolism. However, the broken metabolic balance led to the activation of the amino acid response (AAR) pathway, known as the GCN2/eIF2α/ATF4 pathway. Moreover, activation of ATF4 was responsible for the induction of SLC1A5 and ASNS, which promoted the consumption and metabolization of glutamine. Interestingly, the combination of apatinib and ATF4 silencing abolished glutamine metabolism in NSCLC cells. Moreover, knockdown of ATF4 enhanced the antitumor effect of apatinib both in vitro and in vivo. In summary, this study showed that apatinib could reprogram glutamine metabolism through the activation of the AAR pathway in human NSCLC cells and indicated that targeting ATF4 is a potential therapeutic strategy for relieving apatinib resistance.

Induction of DR5-Dependent Apoptosis by PGA2 through ATF4-CHOP Pathway.

Prostaglandin (PG) A2, a cyclopentenone PG, induced apoptosis in both HCT116 and HCT116 p53 −/− cells. Although PGA2-induced apoptosis in HCT116 cells was dependent on the p53-DR5 pathway, the mechanism underlying PGA2-induced apoptosis in HCT116 p53 −/− cells remains unknown. In this study, we observed that PGA2 caused an increase of mRNA expression of DR5 and protein expression even in HCT116 p53 −/− cells, accompanied by caspase-dependent apoptosis. Knockdown of DR5 expression by RNA interference inhibited PGA2-induced apoptosis in HCT116 p53 −/− cells. Parallel to the induction of apoptosis, PGA2 treatment upregulated expression of genes upstream of DR5 such as ATF4 and CHOP. Knockdown of CHOP prevented DR5-dependent cell death as well as the expression of DR5 protein. Furthermore, knockdown of ATF4 by RNA interference decreased both mRNA and protein levels of CHOP and DR5, thereby suppressing PGA2-induced cell death. Consistently, the DR5 promoter activity increased by PGA2 was not stimulated when the CHOP binding site in the DR5 promoter was mutated. These results collectively suggest that PGA2 may induce DR5-dependent apoptosis via the ATF4-CHOP pathway in HCT116 p53 null cells.

Determining the role of ATF4 in the regulation of mitochondrial remodeling during myotube differentiation and contractile activity

ATF4 is a transcriptional regulator that is selectively induced in response to cellular stress conditions, such as exercise, through the activation of the integrated stress response (ISR). Specifically, in the context of mitochondrial‐specific stress, ATF4 is induced as a key component of the mitochondrial unfolded protein response (UPRmt) and is suggested to upregulate various organellar chaperones and proteases that both preserve, and promote, mitochondrial function. In response to the stress brought about by contractile activity, ATF4 has been implicated in regulating skeletal muscle health by mediating the various signaling events associated with mitochondrial quality control (MQC), including i) mitochondrial biogenesis (expansion), ii) the mitophagy‐lysosomal clearance of damaged and thus potentially harmful organelles, or iii) by activating the mitochondrial unfolded protein response (UPRmt) as an intermediate response to acute cellular stress. However, it remains to be determined whether ATF4 is necessary for mitochondrial adaptations in skeletal muscle. Therefore, our aim was to determine whether ATF4 is required for the maintenance of mitochondrial function and adaptation following an acute 3h bout of contractile activity (ACA), or after repeated bouts (4 days; CCA) in C2C12 myotubes in which ATF4 was either overexpressed (OE) or knocked down (KD) via lentiviral transduction of plasmids containing the ATF4 open reading frame, or siRNA, respectively. Knockdown of ATF4 promoted elongated myotube formation following 5 days of differentiation, whereas ATF4 OE contributed to the opposite effect in which shorter myotubes were observed relative to the control condition. Induction of PGC‐1α mRNA following ACA in ATF4 KD myotubes was attenuated, suggesting diminished drive for mitochondrial biogenesis in the absence of ATF4. The mRNA expression of ATF5, a downstream target of ATF4, was induced both in response to ACA as well as with ATF4 OE, and was reduced in the absence of ATF4. Mitophagy flux, measured by mitochondrial‐localized LC3‐II, was upregulated in ATF4 OE and KD myotubes basally, and was augmented in both control and ATF4 OE cells following ACA, but not in the absence of ATF4. Furthermore, ATF4 OE revealed decrements in mitochondrial content indicated by 20%, and 40%, reductions in VDAC and COX I protein expression relative to control, while 1.2‐1.5‐fold increases in these markers were observed when ATF4 was knocked down. However, following CCA, COX I and VDAC protein content were increased 3‐4‐fold in ATF4 OE cells, while there was no observable increase in mitochondrial content in ATF4 KD myotubes. Together, these data highlight a potential role of ATF4 in regulating basal mitochondrial content and further suggest that ATF4 may be required for contractile activity‐induced increases in mitochondrial content.

Knockdown of ecdysteroid synthesis genes results in impaired molting and high mortality in Bactericera cockerelli (Hemiptera: Triozidae).

RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction. Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10dpi, whereas dsD24 ingestion resulted in 65% mortality by 10dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP. Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.

Mitochondrial Electron Transport Chain Inhibition Promotes Resistance to Proteasome Inhibitors in Multiple Myeloma

INTRODUCTIONProteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax.METHODSWe have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15).RESULTSWe find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy.CONCLUSIONWe show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. DisclosuresLonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

Dihydroartemisinin triggers ferroptosis in primary liver cancer cells by promoting and unfolded protein response‑induced upregulation of CHAC1 expression.

Dihydroartemisinin (DHA), an artemisinin derivate, has been investigated as a potential antitumor drug in primary liver cancer (PLC). Ferroptosis is a form of iron-dependent cell death that can be driven by lipid peroxidation inducers. The present study aimed to determine whether and how DHA could promote the death of PLC cells by inducing ferroptosis. In total, four PLC cell lines with different p53 statuses, including Hep3B (p53 null), Huh7 (p53 mutant), PLC/PRF/5 (p53 mutant) and HepG2 (p53 wild-type), were treated with various concentrations of DHA. The effects of DHA on all three branches of the unfolded protein response (UPR) were evaluated. To deactivate the UPRs, small interfering RNA was used to knockdown the expression of activating transcription factor (ATF)4, X-box binding protein 1 (XBP1) or ATF6 in PLC cells. The effect of DHA on the promoter activity of Chac glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) was evaluated using a dual luciferase reporter assay. The results revealed that DHA-induced death in PLC cells was irrelevant of the p53 status. PLC cells exposed to DHA displayed classic features of ferroptosis, such as increased lipid reactive oxygen species and malondialdehyde levels, an iron overload, and decreased activity or expression of glutathione (GSH), glutathione peroxidase 4, solute carrier family (SLC) 7 member 11 and SLC family 3 member 2. The antitumor effects of DHA in PLC cells were significantly weakened by two typical ferroptosis inhibitors, ferrostatin-1 and deferoxamine mesylate salt, whereas the antitumor effects were augmented following iron overload. Furthermore, DHA activated all three branches of the UPR (eukaryotic translation initiation factor 2 α kinase 3/eukaryotic translation initiation factor 2A/ATF4, inositol-requiring transmembrane kinase/endoribonuclease 1α/XBP1 and ATF6 branches) in vitro. Notably, DHA-induced ferroptosis was significantly attenuated following the knockdown of ATF4, XBP1 or ATF6 expression. In addition, the promoter activity of CHAC1, a gene capable of degrading GSH, was enhanced by DHA, but weakened when the aforementioned three UPR transcription factors were knocked down. In conclusion, the findings of the present study suggested that DHA may effectively induce ferroptosis in PLC cells through the activation of anti-survival UPRs and the upregulation of CHAC1 expression.

EZH2i EPZ-6438 and HDACi vorinostat synergize with ONC201/TIC10 to activate integrated stress response, DR5, reduce H3K27 methylation, ClpX and promote apoptosis of multiple tumor types including DIPG

ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.

Magnesium implantation or supplementation ameliorates bone disorder in CFTR-mutant mice through an ATF4-dependent Wnt/β-catenin signaling

Magnesium metal and its alloys are being developed as effective orthopedic implants; however, the mechanisms underlying the actions of magnesium on bones remain unclear. Cystic fibrosis, the most common genetic disease in Caucasians caused by the mutation of CFTR, has shown bone disorder as a key clinical manifestation, which currently lacks effective therapeutic options. Here we report that implantation of magnesium-containing implant stimulates bone formation and improves bone fracture healing in CFTR-mutant mice. Wnt/β-catenin signaling in the bone is enhanced by the magnesium implant, and inhibition of Wnt/β-catenin by iCRT14 blocks the magnesium implant to improve fracture healing in CFTR-mutant mice. We further demonstrate that magnesium ion enters osteocytes, increases intracellular cAMP level and activates ATF4, a key transcription factor known to regulate Wnt/β-catenin signaling. In vivo knockdown of ATF4 abolishes the magnesium implant-activated β-catenin in bones and reverses the improved-fracture healing in CFTR-mutant mice. In addition, oral supplementation of magnesium activates ATF4 and β-catenin as well as enhances bone volume and density in CFTR-mutant mice. Together, these results show that magnesium implantation or supplementation may serve as a potential anabolic therapy for cystic fibrosis-related bone disease. Activation of ATF4-dependent Wnt/β-catenin signaling in osteocytes is identified as a previously undefined mechanism underlying the beneficial effect of magnesium on bone formation.

Abstract 1156: The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines

Abstract First-in-class small molecule ONC201 is an anti-cancer agent that activates the TRAIL pathway through ATF4 and the integrated stress response (ISR). ONC201 demonstrated durable tumor regressions and prolonged disease stabilization in some patients with histone H3K27M-mutated diffuse midline glioma (DMG). The H3K27M-mutation reduces H3K27 methylation. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. We hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of the H3K27M-mutation, particularly in tumor types beyond DMG. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, glioblastoma, and DMG with single agent ONC201, EZH2 inhibitor EPZ-6438 or PF-06821497, or the combination of ONC201 plus EPZ-6438 or ONC201 plus PF-06821497. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activation widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4. We determined TRAIL pathway activity by analysis of DR5, and apoptosis by analysis of PARP cleavage. The effect of EPZ-6438 or PF-06821497on the target was validated by immunoblotting of H3K27me3. We knocked down ATF4 with siRNA to evaluate the role of ISR in cell death. Our results demonstrate that ONC201 synergistically reduced cell viability in combination with EZH2i, induced integrated stress response and apoptosis in combination with EZH2i. There was greater induction of ATF4 and DR5 in combinatorial therapy-treated tumor cells versus monotherapy. Knockdown of ATF4 reduced the apoptosis induced by combination therapy. EPZ-6438 or PF-06821497 decreased H3K27 tri-methylation. Our study results unravel potent synergy between ONC201 and EZH2i, highlight the role of ISR in the synergy, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Varun V. Prabhu, Joshua E. Allen, Attila A. Seyhan, Wafik S. El-Deiry. The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1156.

Tanshinone IIA enhances susceptibility of non-small cell lung cancer cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5.

Natural killer (NK) cells have a great potential in cancer immunotherapy. However, their therapeutic efficacy is clinically limited owing to cancer cell immune escape. Therefore, it is urgently necessary to develop novel method to improve the antitumor immunity of NK cells. In the present study, it was found that the natural product tanshinone IIA (TIIA) enhanced NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells. TIIA in combination with adoptive transfer of NK cells synergistically suppressed the tumor growth of NSCLC cells in an immune-incompetent mouse model. Furthermore, TIIA significantly inhibited the tumor growth of Lewis lung cancer (LLC) in an immune-competent syngeneic mouse model, and such inhibitory effect was reversed by the depletion of NK cells. Moreover, TIIA increased expressions of ULBP1 and DR5 in NSCLC cells, and inhibition of DR5 and ULBP1 reduced the enhancement of NK cell-mediated lysis by TIIA. Besides, TIIA increased the levels of p-PERK, ATF4 and CHOP. Knockdown of ATF4 completely reversed the up-regulation of ULBP1 and DR5 by TIIA in all detected NSCLC cells, while knockdown of CHOP only partly reduced these enhanced expressions in small parts of NSCLC cells. These results demonstrated that TIIA could increase the susceptibility of NSCLC cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5, suggesting that TIIA had a promising potential in cancer immunotherapy, especially in NK cell-based cancer immunotherapy.

Homeostasis of the ER redox state subsequent to proteasome inhibition

Endoplasmic reticulum (ER) maintains within, an oxidative redox state suitable for disulfide bond formation. We monitored the ER redox dynamics subsequent to proteasome inhibition using an ER redox probe ERroGFP S4. Proteasomal inhibition initially led to oxidation of the ER, but gradually the normal redox state was recovered that further led to a reductive state. These events were found to be concomitant with the increase in the both oxidized and reduced glutathione in the microsomal fraction, with a decrease of total intracellular glutathione. The ER reduction was suppressed by pretreatment of a glutathione synthesis inhibitor or by knockdown of ATF4, which induces glutathione-related genes. These results suggested cellular adaptation of ER redox homeostasis: (1) inhibition of proteasome led to accumulation of misfolded proteins and oxidative state in the ER, and (2) the oxidative ER was then reduced by ATF4 activation, followed by influx of glutathione into the ER.

The acute transcriptional responses to dietary methionine restriction are triggered by inhibition of ternary complex formation and linked to Erk1/2, mTOR, and ATF4

The initial sensing of dietary methionine restriction (MR) occurs in the liver where it activates an integrated stress response (ISR) that quickly reduces methionine utilization. The ISR program is regulated in part by ATF4, but ATF4’s prototypical upstream regulator, eIF2α, is not acutely activated by MR. Bioinformatic analysis of RNAseq and metabolomics data from liver samples harvested 3 h and 6 h after initiating MR shows that general translation is inhibited at the level of ternary complex formation by an acute 50% reduction of hepatic methionine that limits formation of initiator methionine tRNA. The resulting ISR is induced by selective expression of ATF4 target genes that mediate adaptation to reduced methionine intake and return hepatic methionine to control levels within 4 days of starting the diet. Complementary in vitro experiments in HepG2 cells after knockdown of ATF4, or inhibition of mTOR or Erk1/2 support the conclusion that the early induction of genes by MR is partially dependent on ATF4 and regulated by both mTOR and Erk1/2. Taken together, these data show that initiation of dietary MR induces an mTOR- and Erk1/2-dependent stress response that is linked to ATF4 by the sharp, initial drop in hepatic methionine and resulting repression of translation pre-initiation.

Magnolol Enhances the Therapeutic Effects of TRAIL through DR5 Upregulation and Downregulation of c-FLIP and Mcl-1 Proteins in Cancer Cells.

Magnolol is a biologically active compound, isolated from the Chinese herb Magnolia, that regulates antiproliferative, anticancer, antiangiogenic and antimetastatic activities. We found that magnolol sensitizes TRAIL-induced apoptotic cell death via upregulation of DR5 and downregulation of cellular FLICE-inhibitory protein (c-FLIP) and Mcl-1 in cancer cells, but not in normal cells. Mechanistically, magnolol increased ATF4-dependent DR5 expression at the transcription level, and knockdown of ATF4 markedly inhibited magnolol-induced DR5 upregulation. Silencing DR5 with siRNA prevented combined treatment with magnolol and TRAIL-induced apoptosis and PARP cleavage. Magnolol induced proteasome-mediated Mcl-1 downregulation, while magnolol-induced c-FLIP downregulation was regulated, at least in part, by lysosomal degradation. Our results revealed that magnolol enhanced TRAIL-induced apoptosis via ATF4-dependent DR5 upregulation and downregulation of c-FLIP and Mcl-1 proteins.