ATF4 Protein Expression Research Articles

Abstract 5835: Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin

Abstract Developmental transcription programs are epigenetically regulated by multi-protein complexes including the MLL-containing trithorax (TrxG) complexes, which methylate H3K4 to promote gene transcription. We recently reported that the TrxG protein MLL1 and its binding partner menin are overexpressed by and function as oncogenes in Ewing sarcoma. Moreover, small molecule inhibition of the menin-MLL1 protein-protein interaction leads to loss of menin and MLL1 protein expression and to inhibition of growth and tumorigenicity. For the current study we investigated the mechanistic basis of menin-MLL1 mediated oncogenic activity in Ewing sarcoma. Bru-seq analysis was performed to identify changes in nascent gene transcription in Ewing sarcoma cells following exposure to the menin-MLL interaction inhibitor MI-503. Identified targets were validated by qRT-PCR, western blot, immunocytochemistry and by interrogation of published databases. Loss of function was achieved by lentiviral shRNAs and by inhibitor treatment. Exposure of three independent Ewing sarcoma cell lines to MI-503 resulted in significant and reproducible modulation of nearly 100 genes. Bioinformatics analysis of MI-503-regulated genes revealed that the serine synthesis pathway (SSP) was the most significantly impacted pathway downstream of MI-503 treatment. The SSP has recently emerged as a major mediator of tumorigenicity in multiple human cancers. qRT-PCR confirmed reduced expression of SSP enzyme-encoding genes PHGDH, PSAT1 and PSPH in MI-503 treated cells. To determine if the SSP contributes to Ewing sarcoma pathogenesis we evaluated expression of SSP genes and proteins in tumors and cell lines. Our findings showed that Ewing sarcomas express high levels of PHGDH, PSAT1, and PSPH and interrogation of the Cancer Cell Line Encyclopedia revealed that PHGDH expression is higher in Ewing sarcoma than in any other cancer in the database. Studies of Ewing sarcoma cells in serine and glycine free media showed that cells retain viability but that exposure to the PHGDH inhibitor, CBR-5884, inhibits survival in both standard and serine/glycine-free conditions, confirming the importance of the pathway to tumor pathogenesis. To determine how menin-MLL1 promote SSP activation we performed loss of function studies and discovered that knockdown of menin, but not of MLL1, leads to a significant reduction in SSP gene expression, implicating menin as the primary mediator of SSP gene activation. Transcriptional up regulation the SSP pathway has been linked to ATF4 in non-small cell lung cancer. Significantly, ATF4 protein expression is high in Ewing sarcoma cells and ATF4 transcript and protein expression are down regulated following MI-503 treatment and menin knockdown. Together these data demonstrate that the SSP is highly active in Ewing sarcoma and that its activation is induced, at least in part, by a novel menin-ATF4 transcriptional axis. Citation Format: Laurie Kathleen Svoboda, Selina Shiqing Teh, Sudha Sud, Armand Bankhead, Brian Magnuson, Mats Ljungman, Costas Lyssiotis, Tomasz Cierpicki, Jolanta Grembecka, Elizabeth R. Lawlor. Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5835. doi:10.1158/1538-7445.AM2017-5835

Endoplasmic Reticulum Stress PERK-ATF4-CHOP Pathway Is Associated with Hypothalamic Neuronal Injury in Different Durations of Stress in Rats.

The hypothalamus, which is the initial part of the hypothalamic-pituitary-adrenal (HPA) axis, plays a critical role in regulating stress in the central nervous system. The present study aimed to determine whether endoplasmic reticulum stress in hypothalamic neurons is differentially stimulated by varying durations of stress exposure, which ultimately leads to pathological changes in neurons by affecting HPA axis function. There is a need for better morphological evidence of the mechanisms involved in stress-induced neuron injury. A stress model was established in rats by restraining for 8 h and forced ice-water swimming for 5 min each day. The stress-inducing process lasted for 1, 3, 7, 14, and 21 days. Enzyme-linked immunosorbent assay (ELISA) was used to assay serum glucocorticoid levels. Thionine staining was used to observe morphological changes in hypothalamic neurons. Immunohistochemistry and microscopy-based multicolor tissue cytometry (MMTC) was used to detect changes in expression of endoplasmic reticulum stress protein GRP78, ATF4, and CHOP. Serum glucocorticoid levels significantly increased after 3 days of stress exposure and the levels peaked by 7 days. By 21 days, however, the levels were significantly decreased. Thionine staining revealed that prolonged stress exposure resulted in hypothalamic neurons with edema, a lack of Nissl bodies, and pyknotic neurons. Immunohistochemistry and MMTC showed that increasing stress periods significantly decreased GRP78 expression, although ATF4 and CHOP protein expression significantly increased. Stress resulted in pathological changes and significant dynamic changes because of endoplasmic reticulum stress in rat hypothalamic neurons. These results suggested that the endoplasmic reticulum stress PERK-ATF4-CHOP pathway may be associated with hypothalamic neuronal injury.

Roles of the Exogenous H2S-Mediated SR-A Signaling Pathway in Renal Ischemia/ Reperfusion Injury in Regulating Endoplasmic Reticulum Stress-Induced Autophagy in a Rat Model

This study aims to explore the effects of the exogenous hydrogen sulfide (H2S)-mediated scavenger receptor A (SR-A) signaling pathway on renal ischemia/reperfusion injury (IRI) by regulating endoplasmic reticulum (ER) stress-induced autophagy in rats. A total of 48 normal Sprague-Dawley (SD) rats and SR-A knockout rats were selected and divided into six groups (n = 8): wild-type (WT) + sham, WT + ischemia-reperfusion (I/R), WT + I/R + NaHS, SR-A-/- + sham, SR-A-/- + I/R and SR-A-/- + I/R + NaHS. The concentrations of urinary protein, blood urea nitrogen (BUN), serum creatinine (SCR), malondialdehyde (MDA) and H2S in renal tissue were detected. qRT-PCR and Western blotting were used to detect the mRNA and protein levels of IL-6, TGF-β, SR-A, LC3I, LC3II, P62, PERK, ATF6 and IRE1 pathway-related genes. A TUNEL assay was used to detect cell apoptosis. Electron microscopy was applied to observe the structure of renal autophagosomes. Compared with the WT + sham group, in the rates of the WT + I/R group, the urine volume, urinary protein, BUN, SCR and MDA concentrations, the mRNA and protein expression of IL-6, TGF-β, LC3II/I, and ER stress pathway-related genes, the cell apoptosis index, and the number of autophagosomes were significantly increased 24 h after I/R, while P62 and SR-A protein expression and SOD and H2S concentrations were significantly decreased (all P < 0.05). The levels of renal injury, autophagy and ER stress pathway-related genes were decreased in the WT + I/R + NaHS group but were increased in the SR-A-/- + I/R group relative to the WT + I/R group. No significant differences were observed in the urine volume; the concentrations of urinary protein, BUN, SCR and MDA; the SOD activity; the mRNA and protein expression of IL-6, TGF-β, SR-A, GRP78, SR-A, GPR94, ATF4, IRE1, XBP1, ATF6, and eIF2α; the cell apoptosis index; or the number of autophagosomes in rats of the SR-A-/- + I/R and SR-A-/- + I/R + NaHS groups (all P > 0.05). These results demonstrate that the exogenous H2S-mediated SR-A signaling pathway reduces renal IRI injury by up-regulating ER stress-induced autophagy in rats.

Rifampicin-induced injury in L02 cells is alleviated by 4-PBA via inhibition of the PERK-ATF4-CHOP pathway

Endoplasmic reticulum (ER) stress-induced cell injury plays an important role in the development of drug-induced liver injury (DILI). However, little is known about the contribution of ER stress to RFP-induced cell injury. In our study, L02 cells were treated with different concentrations of RFP for different time intervals, and cell apoptosis, the survival rate, and the gene and protein expression of GRP78, PERK, ATF4, and CHOP were measured. Additionally, L02 cells were transfected with CHOP-siRNA or a CHOP-over expression plasmid or administered 4-PBA before treatment with RFP. We found that RFP increased the cell apoptosis rate, decreased cell survival, and increased the protein and gene levels of GRP78, PERK, ATF4 and CHOP in both a dose-dependent and a time-dependent manner. Following the transient knockdown of CHOP and treatment with RFP, cell apoptosis decreased and the survival rate increased. Overexpression of CHOP produced the opposite effects. Treatment with 4-PBA decreased the protein and gene expression of GRP78, PERK, ATF4 and CHOP. Additionally, 4-PBA reduced cell apoptosis, increased cell survival and decreased the level of ALT, AST, AKP, LDH and ATP in the cell culture supernatant. These results indicate that 4-PBA alleviates RFP-induced injury in L02 cells via inhibition of the PERK-ATF4-CHOP pathway.

Abstract 3660: ATF4 is a putative tumor suppressor gene in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p&amp;lt;0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3660.

A novel curcuminoid exhibits enhanced antitumor activity in nasopharyngeal carcinoma.

Curcumin shows growth-inhibition against tumor cells through multi-target mechanisms. Nevertheless, the poor stability and pharmacokinetics considerably limit its clinical functions. Increased effort has been put into the chemical alteration of curcumin to find potential analogues with improved bioavailability and antitumor activities. In this study, the antitumor activity of a novel curcuminoid (B63) in nasopharyngeal carcinoma (NPC) was examined. The MTT and colony formation assays were used to detect NPC cell viability and proliferation. Flow cytometry was used to detect cell cycle distribution. The Annexin V/PI staining assay and cleavage PARP and cleavage caspase-3 expression were used to examine apoptosis. Western blotting was used to examine the protein expression of endoplasmic reticulum (ER) stress pathway markers, XBP-1, ATF-4 and CHOP. The suppressive effect of B63 on tumor growth was examined in vivo by subcutaneously inoculated NPC in a tumor model using nude mice. Treatment with B63 potentially caused growth inhibition and apoptosis in NPC cells in a dose- and time-responsive manner. Its antitumor effect was associated with the ER stress activation. Nevertheless, the same dose of curcumin did not activate ER stress. In addition, knockdown of Chop attenuated B63-induced cell viability inhibition, suggesting that the apoptotic pathway is ER stress-dependent. The tumor volume and weight were significantly reduced by pretreating the NPC cells with B63 before implantation in the in vivo mouse model. B63 exhibited a more potent antitumor action than curcumin in NPC. These observations on the novel compound B63 indicate a novel candidate for NPC therapy.

Abstract A34: The stress response transcription factor ATF4 mediates cytoprotective pathways in c-Myc overexpressing cells

Abstract The proto-oncogene c-Myc is often deregulated in human tumors and its overexpression associates with poor prognosis in patients. While c-Myc is a potent activator of pro-tumorigenic pathways, it can also induce anti-tumorgenic pathways such as apoptosis. However, in c-Myc driven cancers the anti-tumorigenic state is evaded via a yet poorly understood pro-survival mechanism. We previously reported that activation of the PERK arm of the Unfolded Protein Response (UPR) is one such pro-survival mechanism induced by c-Myc to bypass c-Myc induced apoptosis. We also demonstrated enhanced activation of the PERK/p-eIF2α arm of the UPR in a mouse model of lymphoma and in B-cells from human lymphoma patients compared to normal tissues. ATF4, one of the main downstream effectors of PERK, regulates amino acid metabolism, antioxidant response and autophagy. Using the CRISPR/CAS9 genome-editing tool we were able to ablate portions of the ATF4 gene in DLD-1, human colon carcinoma cell line, preventing ATF4 protein expression. Here, we show ATF4 expression is induced in Myc overexpressing ATF4-WT DLD-1 cells. Activation of c-Myc in ATF4-deficient DLD-1 cells resulted in decreased clonogenic survival and reduced autophagy. Addition of Trolox, an antioxidant, conferred protection in ATF4 deficient cells. These findings indicate that the transcription factor ATF4 activates pro-survival pathways such as antioxidant response and autophagy during c-Myc overexpression to promote survival. Therefore, therapeutically targeting these pathways would switch the balance towards cell death in c-Myc dependent tumors. Citation Format: Feven Tameire Tameire, Costas Koumenis. The stress response transcription factor ATF4 mediates cytoprotective pathways in c-Myc overexpressing cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A34.

Preconditioning with Endoplasmic Reticulum Stress Ameliorates Endothelial Cell Inflammation

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

A new curcumin analogue exhibits enhanced antitumor activity in nasopharyngeal carcinoma

The aim of the present study was to evaluate the antitumor effects of the curcumin analogue GL63 on radioresistant nasopharyngeal carcinoma (NPC) CNE2R cells and parental CNE2 cells. The cell viability and proliferation of NPC cells were detected by MTT assay and colony formation assay. The suppressive effect on tumor growth was examined using in vivo subcutaneously inoculated NPC tumor models using nude mice. The cell cycle distribution was detected using flow cytometry. Apoptosis was examined by Hoechst 33342 and Annexin V/PI staining assay. The protein expression of endoplasmic reticulum (ER) stress pathway markers, XBP-1, ATF-4 and CHOP, were examined by western blotting. A growth inhibitory effect was observed following treatment with GL63 in a dose-dependent manner and was more potent when compared to curcumin. GL63 at 5 µM induced significant G2/M arrest and apoptosis in NPC. The tumor-suppressive activity of GL63 in NPC xenograft models was more potent when compared to curcumin. Furthermore, GL63 induced an ER stress response, upregulation of CHOP, XBP-1 and ATF-4 expression, while the same concentration of curcumin had no effect on ER stress. These results suggest that GL63 has more potent antitumor activity than curcumin, which is associated with activation of ER stress, induction of G2/M arrest and apoptosis in NPC cells.

Endoplasmic Reticulum Stress Implicated in the Development of Renal Fibrosis

Endoplasmic reticulum (ER) stress-associated apoptosis plays a role in organ remodeling after insult. The effect of ER stress on renal tubular damage and fibrosis remains controversial. This study aims to investigate whether ER stress is involved in tubular destruction and interstitial fibrosis in vivo. Renal cell apoptosis was proven by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain and poly-ADP ribose polymerase expression in the unilateral ureteral obstruction (UUO) kidney. ER stress was evoked and confirmed by the upregulation of glucose-regulated protein 78 (GRP78) and the common Lys-Asp-Glu-Leu (KDEL) motif of ER retention proteins after UUO. ER stress-associated proapoptotic signals, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2-associated × protein (BAX) expression, caspase-12 and c-Jun N-terminal kinase (JNK) phosphorylation, were activated in the UUO kidney. Prolonged ER stress attenuated both unsplicing and splicing X-box binding protein 1 (XBP-1) protein expression, but continued to activate inositol-requiring 1α (IRE1α)-JNK phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α subunit (eIF2α), activating transcription factor (ATF)-4, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleavage activating transcription factor 6 (cATF6)-CHOP signals, which induce ER stress-related apoptosis but attenuate adaptive unfolded protein responses in UUO kidneys. However, renal apoptosis and fibrosis were attenuated in candesartan-treated UUO kidney. Candesartan was associated with maintenance of XBP-1 expression and attenuated ATF4, cATF6 and CHOP protein expression. Taken together, results show that overwhelming ER stress leads to renal cell apoptosis and subsequent fibrosis; and candesartan, at least in part, restores renal integrity by blocking ER stress-related apoptosis. Reducing ER stress may present a way to attenuate renal fibrosis.

5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells

5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism. Human hepatocellular carcinoma cell lines Hep3B, Huh-7, and Hep G2 and human embryo liver L-02 cells were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cell death was examined using Flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, caspase-8, and caspase-9 were measured using ELISA. Intracellular ROS was measured by FCM using the fluorescent probe DCHF-DA, and the expression of DR4, DR5, CHOP, GPR78, and ATF4 proteins was analyzed using Western blot. Our results demonstrated subtoxic concentrations of DMF sensitize HCC cells to TRAIL-induced apoptosis and induce the death receptor 5 (DR5) expression level, accompanying the generation of reactive oxygen species (ROS) and the upregulation of CHOP, GPR78, and ATF4 protein expression. Pretreatment with N-acetylcysteine (NAC) inhibited DMF-induced upregulation of DR5, CHOP, GPR78, and ATF4 protein expression and blocked the cotreatment-induced apoptosis. Furthermore, DMF-mediated sensitization of HCC cells to TRAIL was reduced by administration of a blocking antibody or small interfering RNAs for DR5, salubrinal, an inhibitor of ER stress, and the small interfering RNAs for CHOP. However, DMF could not induce the upregulation of DR5 expression, generation of ROS, and sensitization of TRAIL-induced apoptotic cell death in human embryo liver L-02 cells or normal human peripheral blood mononuclear cells (PBMCs). The present study demonstrates that DMF selectively enhances TRAIL-induced apoptosis by ROS-stimulated ER-stress triggering CHOP-mediated DR5 upregulation in HCC.

Endoplasmic Reticulum Stress Response Mediated by the PERK-eIF2α-ATF4 Pathway Is Involved in Osteoblast Differentiation Induced by BMP2

To avoid excess accumulation of unfolded proteins in the endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are collectively termed the ER stress response. Double stranded RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. Phosphorylated eIF2α specifically promotes the translation of the transcription factor ATF4. ATF4 plays important roles in osteoblast differentiation and bone formation. Perk−/− mice are reported to exhibit severe osteopenia, and the phenotypes observed in bone tissues are very similar to those of Atf4−/− mice. However, the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenesis is unclear. Phosphorylated eIF2α and ATF4 protein levels were attenuated in Perk−/− calvariae, and the gene expression levels of osteocalcin (Ocn) and bone sialoprotein (Bsp), which are targets for ATF4, were also down-regulated. Treatment of wild-type primary osteoblasts with BMP2, which is required for osteoblast differentiation, induced ER stress, leading to an increase in ATF4 protein expression levels. In contrast, the level of ATF4 in Perk−/− osteoblasts was severely diminished. The results indicate that PERK signaling is required for ATF4 activation during osteoblast differentiation. Perk−/− osteoblasts exhibited decreased alkaline phosphatase activities and delayed mineralized nodule formation relative to wild-type cultures. These abnormalities were almost completely restored by the introduction of ATF4 into Perk−/− osteoblasts. Taken together, ER stress occurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4 signaling pathway followed by the promotion of gene expression essential for osteogenesis, such as Ocn and Bsp.

Abstract 297: Activating transcription factor 4 negatively regulates the mammalian target of rapamycin via Redd1 expression in response to 2-deoxyglucose

Abstract Aerobic glycolysis is one of major metabolic changes found in cancer cells and is a valid target for cancer therapy. The nonmetabolizable glucose analogue, 2-deoxyglucose (2-DG) interferes with glucose metabolism and enhances chemotherapy/radiotherapy in cancer cells. We have investigated the mechanism of action by which 2-DG inhibits mammalian target of rapamycin (mTOR) activity. We found that 2-DG treatment led to decreased mTOR activity which was regulated by induction of Redd1 protein in both lung cancer cells and cervical cancer cells. We also found 2-DG treatment led to increased ATF4 protein expression and transcriptional activity. Suppression of ATF4 using siRNAs led to decrease of 2-DG induced Redd1 expression. Our data suggested that 2-DG induced Redd1 expression is regulated by the ATF4, leading to mTOR inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 297.

Synthesis and biological analysis of a new curcumin analogue for enhanced anti-tumor activity in HepG 2 cells

The aim of the present study was to investigate the apoptosis of human hepatocellular carcinoma cell line HepG 2 induced by a new curcumin analogue, GL63. HepG 2 cells were treated with increasing doses of GL63 and curcumin for 48 h. The proliferation of cells was detected with MTT. The apoptosis were examined by flow cytometry. The caspase-3 activity was detected by western blotting. ER calcium stores were assessed by the fluorescent calcium indicator fura-2/AM. The protein expression of ER stress pathway, GRP78, XBP-1, ATF-4 and CHOP were examined with western blotting. Growth inhibitory effect was observed for treatment with GL63 in a dose-dependent manner and with more potential than curcumin. GL63 at 20 microM induced significant apoptosis in HepG 2 cells. Furthermore, GL63 induced the ER stress response, up-regulation of CHOP, XBP-1, ATF-4 and GRP78 expression in a dose-dependent, while curcumin had no effect on ER stress. These results suggest that GL63 has more potent anti-tumor activity than curcumin, which is associated with activation of ER stress and induction of apoptosis in HepG 2 cells.

An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1-3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) alpha-subunit (Ser(52))]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser(240/244)) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h (P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2 alpha phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA (P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.

Abstract B164: Role of endoplasmic reticulum stress in mechanism of action of antitumor quinols

Abstract The novel quinol PMX 290 is an experimental anti-tumor agent with potent activity against colon, renal and breast tumor cell lines in vitro (GI50 &amp;lt; 200 nM). PMX 290 disrupts thioredoxin/ thioredoxin reductase redox regulation and inhibits HIF-1 signaling. A pro-drug of PMX 290 is currently under development at Pharminox Ltd, UK. Here we present data suggesting an additional mechanism of action for PMX 290 highlighting a role for the induction of endoplasmic reticulum stress. Treatment of HCT116 colon or TK-10 renal carcinoma cells with PMX 290 leads to the induction of a distinct vacuolar phenotype 4 – 6h after addition of the compound. These perinuclear vacuoles were identified as swollen endoplasmic reticulum (ER) as their membranes stain positively with a fluorescent ER dye. Furthermore when a fluorescent analogue of PMX 290 is added to cells it co-localized with the ER dye. Microarray data from HCT116 cells treated with PMX 290 demonstrated the induction of a panel of mRNAs for genes involved in ER stress. These included GADD34, TRIB3, DNAJB9, BiP and CHOP as well as several genes upregulated by ATF4. Uncharacteristically for a quinol there was no increase in thioredoxin reductase mRNA levels although other markers of oxidative stress were upregulated (e.g. heme oxygenase 1). The gene array results suggest that the PERK-eIF2alpha ER stress pathway is activated by PMX 290. Upon activation by ER stress PERK phosphorylates eIF2alpha which switches off general translation and allows the expression of the transcription factor ATF4. This switch to reduced protein synthesis and expression of ATF4 transcribed chaperone proteins lowers the load on ER and increases protein folding efficiency thus alleviating ER stress. A transient increase in eIF2aplha phosphorylation and a concurrent increase in BiP levels were detected byWestern blot in response to PMX 290 in HCT116 cells. In conclusion, ER stress appears to play an important role in the anti-tumor activity of PMX 290 and represents a novel mechanism of action for this class of compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B164.

Abstract 1394: Sublethal Levels of Aldehydes Augmented Cardiac Anti-Oxidant Defense through Activation of eIF2 α -ATF4 Pathway via GCN2 Kinase

[Introduction] Despite an increase in the levels of aldehydes, the heart from aldehyde dehydrogenase ( ALDH ) 2*2 -transgenic (Tg) mice, loss of function model of ALDH, exhibited a greater tolerance to oxidative stress via activation of amino acid metabolism leading to glutathione biosynthesis. This study was designed to identify the signaling cascades responsible for the activation of amino acid metabolism by aldehydes. [Methods &amp; Results] (1) Phosphorylation of α -subunit of eukaryotic translation initiation factor 2 (eIF2 α ) and subsequent translational activation of ATF4 have been shown to induce amino acid metabolism as a common response to a wide variety of stressors. Consistent with this, phosphorylation levels of eIF2 α and protein expression of ATF4 were increased in ALDH2*2 -Tg hearts. (2) Among four eIF2 α kinases, general control non-depressible (GCN)2 kinase, a sensor for amino acid insufficiency, was activated in ALDH2*2 -Tg heart. (3) Quantification of intracellular amino acid demonstrated that free histidine concentration in ALDH2*2 -Tg heart was selectively reduced by 50% compared to that in non-Tg littermates. (4) To clarify the functional significance of observed reduction in histidine, ALDH2*2 -Tg mice were fed a high histidine diet. The phosphorylation levels of eIF2 α and the protein levels of ATF4 were diminished by 50% in ALDH2*2 -Tg mice fed the high histidine diet, in agreement with the normalization of histidine concentration. Accordingly, both enhanced tolerance to ischemia-reperfusion injury and elevated levels of glutathione were partially diminished in the heart from ALDH2*2 -Tg mice fed the high histidine diet compared to ALDH2*2 -Tg mice fed normal chow. (5) In culture, exposure to 4-hydroxy-2-nonenal (4-HNE) phosphorylated GCN2 and eIF2 α and increased protein levels of ATF4 in a time-dependent manner. (6) siRNA-mediated knockdown of GCN2 abrogated 4-HNE-induced induction of amino acid metabolic genes. [Conclusions] Activation of eIF2 α -ATF4 pathway via GCN2 kinase might be of special importance in the transcriptional control that coordinately promotes amino acid metabolism in response to aldehydes. Intracellular depletion of free histidine is at least partly involved in the activation of GCN2 kinase by aldehydes.

Impairment of pancreatic islet beta cell function induced by intermittent high glucose through oxidative and endoplasmic reticulum stress: experiment with rat pancreatic islet beta cells

To investigate the effect of intermittent high glucose (IHG) on the pancreatic islet beta-cell function and mechanism thereof. Rat pancreatic islet p-cells of the line INS-1 were cultured and randomly divided into 3 groups: IHG group exposed to fluctuating concentrations of glucose, stable high glucose (SHG) group exposed to 16. 7 mmol/L glucose, and control group exposed to normal concentration (5.5 mmol/L) glucose. 24, 48, and 72 hours later radioimmunoassay was used to detect the insulin secretion index (ISI). 72 h later, the concentration of insulin in the cells was detected with radioimmunoassay. The contents of oxidative stress markers, nitrotyrosine (NT) and 8-hydroxy-2-deoxyguanosine (8-OHdG) were detected. Real-time PCR was used to detect the mRNA expression of peroxiredoxin 1 (PDX-1), ATF-4, one of the transcription factors of the family bZIP, and insulin. Western blotting was used to detect the protein expression of ATF-4. The ISI of the IHG and SHG groups decreased time-dependently, The ISI of IHG and SHG groups were 0.64 +/- 0.11 and 1.31 +/- 0. 04 respectively, both significantly lower than that of the control group (1.67 +/- 0.23, both P < 0.05). The intracellular insulin contents of the IHG and SHG groups were (10.91 +/- 0.14) and (11.08 +/- 0.03) +/- U/microg respectively, both significantly lower than that of the control group [(12.37 +/- 0.37) microU/microg, both P < 0.05]. The intracellular concentrations of 8-OHdG and NT of the SHG and IHG groups, were significantly higher than those of the control group (all P < 0.01), and those of the IHG group were significantly higher than those of the SHG group (both P < 0.05). The mRNA and protein expression levels of ATF-4 of the IHG group were all significantly higher than those of the control group (all P < 0.05) and those of the IHG group were significantly higher than those of the SHG group (both P < 0.05). IHG and SHG induce severe impairment in pancreatic islet beta cell functions, especially IHG, which is closely associated with the aggravation of oxidative stress and endoplasmic reticulum stress triggered by intermittent high glucose.

Coping with the stress: Expression of ATF4, ATF6, and downstream targets in organs of hibernating ground squirrels

Perturbation of the endoplasmic reticulum (ER) protein folding apparatus via any one of several environmental or metabolic stresses rapidly triggers a complex program of cellular responses that is termed the unfolded protein response (UPR). Stresses that trigger this response in mammals can include low temperature, hypoxia, ischemia, and oxidative stress. All of these can be natural features of mammalian hibernation, and hence the UPR might be integral to long term survival in a state of cold torpor. The present study analyzes changes in gene and/or protein expression of multiple markers of the UPR in tissues of euthermic (control) versus hibernating ground squirrels, Spermophilus tridecemlineatus. Immunoblot analysis of ATF4 protein expression revealed strong increases of 1.9- to 2.5-fold in brown adipose tissue, skeletal muscle, and brain during hibernation. However, transcript levels of atf4 were unchanged or lowered which suggests that ATF4 protein levels were regulated at the translational level. Subcellular localization studies showed that ATF4 translocated into the nucleus during hibernation, as did its cofactor, the phosphorylated form of CREB-1, which rose by 25- to 39-fold in nuclear extracts of brain and skeletal muscle of torpid animals. The responses of other proteins involved in the UPR including p-PERK, ATF6, GADD153, and GADD34 were also evaluated. The data suggest that ATF4 up-regulation may play an important role in coordinating gene expression responses that support the hibernating phenotype.

The Oxidative Stressor Arsenite Activates Vascular Endothelial Growth Factor mRNA Transcription by an ATF4-dependent Mechanism

Aberrant retinal expression of vascular endothelial growth factor (VEGF) leading to neovascularization is a central feature of age-related macular degeneration and diabetic retinopathy, two leading causes of vision loss. Oxidative stress is suggested to occur in retinal tissue during age-related macular degeneration and diabetic retinopathy and is suspected in the mechanism of VEGF expression in these diseases. Arsenite, a thiol-reactive oxidative stressor, induces VEGF expression by a HIF-1alpha-independent mechanism. Previously, we demonstrated that homocysteine, an endoplasmic reticulum stressor, increases VEGF transcription by a mechanism dependent upon activating transcription factor ATF4. Because ATF4 is expressed in response to oxidative stress, we hypothesized that ATF4 was also responsible for increased VEGF transcription in response to arsenite. We now show that arsenite increased steady state levels of VEGF mRNA and activated transcription from a VEGF promoter construct. Arsenite induced eIF2alpha phosphorylation, resulting in increased ATF4 protein levels. Inactivation or loss of ATF4 greatly diminished the VEGF response to arsenite treatment. Overexpression of ATF4 was sufficient to activate the VEGF promoter, and arsenite cooperated with exogenous ATF4 to further activate the promoter. A complex containing ATF4 binds a DNA element at +1767 bp relative to the VEGF transcription start site, and DNA binding activity is increased by arsenite treatment. In addition, the ability of a thiol antioxidant, N-acetylcysteine, to inhibit the effect of arsenite on VEGF expression coincided with its ability to inhibit phosphorylation of eIF2alpha and ATF4 protein expression. Thus, arsenite-induced up-regulation of VEGF gene transcription occurs by an ATF4-dependent mechanism.