Activation Of ATF6 Research Articles

Combinations of Tyrosine Kinase Inhibitor and ERAD Inhibitor Promote Oxidative Stress-Induced Apoptosis through ATF4 and KLF9 in Medullary Thyroid Cancer.

Medullary thyroid carcinoma (MTC) originates from the C cells of the thyroid gland, which secrete calcitonin. Lymph node and distant metastases are frequently present at diagnosis. Activating mutations of RET, a driver oncogene in MTC that encodes a tyrosine kinase receptor, prevents apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum (ER) stress. We hypothesized that the combination of a tyrosine kinase inhibitor (TKI) and an ATF4 inducer promotes cell death by triggering catastrophic oxidative stress and apoptotic cell death. Here, we report that the ER-associated protein degradation (ERAD) inhibitor eeyarestatin sensitized MTC cells to the TKIs, sunitinib and vandetanib, thereby leading to synergistic upregulation of ATF4 expression, accumulation of reactive oxygen species, and subsequent cell death. Genome-wide analysis of ATF4 interaction sites by chromatin immunoprecipitation (ChIP) sequencing revealed that among ATF4 target genes was KLF9 (Kruppel-like factor 9), which induces MTC apoptosis. ChIP assays revealed that ATF4 occupancy at the KLF9 promoter was increased in MTC cells treated with eeyarestatin or vandetanib alone and was further enhanced in cells treated with both drugs, leading to increased KLF9 transcription. Depletion of ATF4 by shRNA led to downregulation of KLF9 expression and prevented oxidative stress-induced cell death. Furthermore, we identified ATF4 target genes (LZTFL1, MKNK2, and SIAH1 with known tumor suppressor function) that were synergistically upregulated with the combination of TKI and ERAD inhibitor. IMPLICATIONS: These findings reveal a combination therapy that induces reactive oxygen species-dependent catastrophic cell death through induction of ATF4 and KLF9 transcriptional activity.

Pharmacologic ATF6 activation confers global protection in widespread disease models by reprograming cellular proteostasis

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo. Here we show, using a mouse model of myocardial ischemia/reperfusion, that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 is also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.

Endoplasmic Reticulum Stress and Autophagy.

In 1945, K. R. Porter et al. observed mouse embryonic fibroblasts (MEFs) and found that the cytoplasmic part of the cell had an unreported reticular structure, so it was named endoplasmic reticulum (ER). The major functions of the endoplasmic reticulum are: synthesis of intracellular proteins and the modification and processing of proteins. It is an important organelle in eukaryotic cells. It is a three-dimensional network structure in which complex and closed intracellular tubular intimal systems are intertwined. When cells are subjected to various strong stimulating factors such as nutrient deficiencies, Ca2+ metabolic imbalance, toxin stimulation, and sustained oxidative stress stimulation, the cell homeostasis will be broken. In order to survive, a series of cell self-protection event will be initiated including the endoplasmic reticulum stress (ERS). The UPR can further promote the expression of the proteins which can help the misfolded and unfolded proteins restore to its normal structure through the activation of PERK, IRE1, and ATF6. However, the co-working of UPR and the ubiquitin-proteasome system still cannot make the endoplasmic reticulum restoring to its normal state, when the stimuli persist or are too strong. The damaged endoplasmic reticulum can be partially engulfed by the autophagic vesicles for degradation when the ERS persists. The degraded endoplasmic reticulum fragments can be reassembled into a new endoplasmic reticulum to restore the normal state of it. Hence, it seems that the autophagy has become the last mean to restore the homeostasis of endoplasmic reticulum.

Abstract 16725: A Novel Role for MAGI1 in Disturbed Flow-Induced Endoplasmic Reticulum Stress in Endothelial Cells

Introduction: Atherosclerotic plaque predominantly develops in areas where endothelial cells (ECs) are exposed to disturbed flow (d-flow). Endoplasmic reticulum (ER) stress, characterized by the activation of ER transmembrane sensor ATF6 and transcription factor XBP-1, has been implicated in d-flow-induced atherogenesis. At d-flow regions, expression of both total and spliced XBP-1 and activation of ATF6 are increased, resulting in EC apoptosis. Additionally, over-expression of XBP-1 accelerates atherosclerotic formation. A recent genome-wide association study revealed a strong correlation between membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1), a scaffold protein that associates with the tight and adherens junctions, and chronic inflammatory diseases. Hypothesis: We hypothesize that MAGI1 is crucial in d-flow-induced EC inflammation, apoptosis, and atherogenesis via regulating ER stress signaling. Methods and Results: Microarray analysis revealed that MAGI1 depletion inhibits ER-stress related genes, including XBP-1. qRT-PCR results confirmed that MAGI1 depletion abolished d-flow-mediated expression of spliced and full-length XBP-1 which correlated with reduced d-flow-induced apoptosis. To examine the mechanism by which MAGI1 regulates ER stress signaling, we found that MAGI1 is associated with ATF6, and that MAGI1-ATF6 complex formation is increased by inflammatory stimuli including d-flow and thrombin. Because the cleaved form of ATF6 can translocate from ER and Golgi to the nucleus, where it binds to ER-stress response elements and upregulates XBP-1 expression, we asked if MAGI1 facilitates ATF6 nuclear translocation. We found that thrombin-mediated ATF6 nuclear translation is significantly inhibited by MAGI1 depletion. Conclusions: Our results indicate that d-flow elicits the formation and translocation of a MAGI1-ATF6 complex from the cytosol to the nucleus that leads to ER stress via XBP1 induction and EC apoptosis. Our study suggests a novel role for endothelial MAGI1 as a link between d-flow and ER stress, both of which are fundamental constituents of atherogenesis.

Pharmacologic ATF6 Activation Confers Global Protection in Widespread Disease Models by Reprogramming Cellular Proteostasis

Pharmacologic ATF6 Activation Confers Global Protection in Widespread Disease Models by Reprogramming Cellular Proteostasis

Role of the ISR-ATF4 pathway and its cross talk with Nrf2 in mitochondrial quality control.

Recent investigations have clarified the importance of mitochondria in various age-related degenerative diseases, including late-onset Alzheimer’s disease and Parkinson’s disease. Although mitochondrial disturbances can be involved in every step of disease progression, several observations have demonstrated that a subtle mitochondrial functional disturbance is observed preceding the actual appearance of pathophysiological alterations and can be the target of early therapeutic intervention. The signals from damaged mitochondria are transferred to the nucleus, leading to the altered expression of nuclear-encoded genes, which includes mitochondrial proteins (i.e., mitochondrial retrograde signaling). Mitochondrial retrograde signaling improves mitochondrial perturbation (i.e., mitohormesis) and is considered a homeostatic stress response against intrinsic (ex. aging or pathological mutations) and extrinsic (ex. chemicals and pathogens) stimuli. There are several branches of the mitochondrial retrograde signaling, including mitochondrial unfolded protein response (UPRMT), but recent observations increasingly show the importance of the ISR-ATF4 pathway in mitochondrial retrograde signaling. Furthermore, Nrf2, a master regulator of the oxidative stress response, interacts with ATF4 and cooperatively upregulates a battery of antioxidant and antiapoptotic genes while repressing the ATF4-mediated proapoptotic gene, CHOP. In this review article, we summarized the upstream and downstream mechanisms of ATF4 activation during mitochondrial stresses and disturbances and discuss therapeutic intervention against degenerative diseases by using Nrf2 activators.

Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (147) was previously identified (<xref ref-type="bibr" rid="bib35">Plate et al., 2016</xref>) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that 147-dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by 147 include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs 147-dependent induction of the ATF6-target gene, BiP, implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with 147. Thus, 147 is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.

Abstract 547: Pharmacologic ATF6 Activation Confers Global Protection in Widespread Disease Models by Reprogramming Cellular Proteostasis

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo . Here, using a mouse model of myocardial ischemia/reperfusion, we showed that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 was also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a first-in-class proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.

Abstract 2742: Additional mechanisms of action of ladiratuzumab vedotin contribute to increased immune cell activation within the tumor

Abstract Ladiratuzumab vedotin (LV, SGN-LIV1A) is an antibody-drug conjugate directed against the LIV-1 protein that is currently under investigation for treatment in metastatic breast cancer. This ADC consists of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a potent microtubule-disrupting agent. LV antitumor activity is thought to primarily be the result of intracellular payload release, leading to mitotic arrest and apoptotic cell death. While LV-induced cell death has been extensively studied, its potential immune modulatory activity has yet to be explored. Whereas normal apoptosis is nonimmunogenic, multiple chemotherapeutics have been shown to elicit a unique form of immunogenic cell death (ICD), characterized by the exposure of various proinflammatory intermediates prior to cell death. We have recently demonstrated that a CD30-directed ADC containing MMAE (brentuximab vedotin) was able to elicit ICD as a consequence of microtubule disruption and subsequent induction of endoplasmic reticulum stress. Induction of ICD in preclinical models of Hodgkin lymphoma results in directed antitumor immune responses, which is further potentiated by anti-PD-1 therapy. In this study, we provide evidence that ladiratuzumab vedotin induces apoptosis of tumor cells in a manner that is consistent with ICD. Treatment of LIV-1+ cells with LV resulted in the activation of all arms of the ER stress response, via the activation of ATF6 and phosphorylation of IRE1 and eIF2a. Severity of ER stress was confirmed by phosphorylation of the downstream effector Jun N-terminal kinase (JNK), and induction of the unfolded protein response. Critically, induction of C/EBP homologous protein (CHOP) indicated irreparable ER stress and subsequent apoptosis. Concurrent with ER stress induction but prior to cell death, LV-treated cells also released ATP and HMGB1 into the supernatant, hallmarks of ICD that are important for immune cell activation and recruitment into the tumor microenvironment. Critically, treatment of subcutaneously engrafted LIV1+ MCF7 cells generated a proinflammatory cytokine response within the tumor. Furthermore, intratumoral dendritic cells and macrophages displayed an inflammatory phenotype, highlighted by increased MHCII expression, providing evidence for improved immune activation and engagement. Via induction of ER stress, tumor cells killed by ladiratuzumab vedotin may initiate an antitumor immune response and provide a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens. Citation Format: Anthony T. Cao, Shaylin Higgins, Nicole Stevens, Shyra J. Gardai, Django Sussman. Additional mechanisms of action of ladiratuzumab vedotin contribute to increased immune cell activation within the tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2742.

Palmitate deranges erythropoietin production via transcription factor ATF4 activation of unfolded protein response

Lipotoxicity plays an important role in the progression of chronic kidney damage via various mechanisms, such as endoplasmic reticulum stress. Several studies proposed renal lipotoxicity in glomerular and tubular cells but the effect of lipid on renal erythropoietin (EPO)-producing (REP) cells in the interstitium has not been elucidated. Since renal anemia is caused by derangement of EPO production in REP cells, we evaluated the effect of palmitate, a representative long-chain saturated fatty acid, on EPO production and the endoplasmic reticulum stress pathway. EPO production was suppressed by palmitate (palmitate-conjugated bovine serum albumin [BSA]) or a high palmitate diet, but not oleic acid-conjugated BSA or a high oleic acid diet, especially under cobalt-induced pseudo-hypoxia both invitro and invivo. Importantly, suppression of EPO production was not induced by a decrease in transcription factor HIF activity, while it was significantly associated with endoplasmic reticulum stress, particularly transcription factor ATF4 activation, which suppresses 3'-enhancer activity of the EPO gene. ATF4 knockdown by siRNA significantly attenuated the suppressive effect of palmitate on EPO production. Studies utilizing inherited super-anemic mice (ISAM) mated with EPO-Cre mice (ISAM-REC mice) for lineage-labeling of REP cells showed that ATF4 activation by palmitate suppressed EPO production in REP cells. Laser capture microdissection confirmed ATF4 activation in the interstitial area of ISAM-REC mice treated with palmitate-conjugated BSA. Thus, endoplasmic reticulum stress induced by palmitate suppressed EPO expression by REP cells in a manner independent of HIF activation. The link between endoplasmic reticulum stress, dyslipidemia, and hypoxia may contribute to development and progression of anemia in CKD.

TARGETING ENDOPLASMIC RETICULUM STRESS AS A THERAPY TO MANAGE ABDOMINAL AORTIC ANEURYSM DISEASE

Objective: Abdominal aortic aneurysm (AAA) is a degenerative vascular disease with high morbidity and mortality. It has a prevalence of 4–8% in men over the age of 65 years that is increasing in association with the aging population. Clinical management of AAA is limited to elective surgical repair while an effective pharmacotherapy is awaited. Therefore, we aim to determine whether inhibition of endoplasmic reticulum stress is an effective therapy to reduce aortic wall degeneration occurring in AAA. Design and method: The endoplasmic stress activation was studied by real Time PCR, Western Blot and by immunostaining in a cohort of AAA samples from 100 patients compared with abdominal aorta from 20 healthy donors and in abdominal aorta of a mouse model of AAA (apolipoprotein E–deficient mice infused with angiotensin II for 4 weeks with osmotic minipumps). In males and females mice, the aortic diameter was monitored and recorded by ultrasonography at 0, 7, 14, 21 and 28 days of Ang II infusion and blood pressure was measured by tail-cuff method. The phenylbutyrate sodium or PBA and Tauroursodeoxycholic Acid or Tudca as classic endoplasmic reticulum stress inhibitors, were administered intraperitoneally for 3 days a week to hipertensive mice. Results: In human AAA the endoplasmic reticulum stress induction was assessed by enhanced expression of ATF6, IRE-1, XBP-1 and CHOP and by an increase in protein expression of active ATF6 and XBP1 and of the pro-apoptotic protein CHOP. This was accompanied by an exacerbated apoptosis in vascular wall measured by TUNEL and by cleaved caspase-3 and by an increase in NADPH oxidase expression and in superoxide anion production. In the abdominal aorta of the murine AAA model the gene expression of ATF4, GRP78 and IRE-1 was increased while no differences were found in ATF6 or CHOP between AAA and control mice. The administration of PBA and Tudca decreased the incidence of AAA by reducing the abdominal aortic dilatation, systolic blood pressure and aorta wall disorganization. Conclusions: Our results evidence that inhibition of ER stress is a potential therapy to reduce aortic dilatation and medial wall destruction in AAA.

Amyotrophic lateral sclerosis (ALS) and Alzheimer\u2019s disease (AD) are characterised by differential activation of ER stress pathways: focus on UPR target genes

The endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR), which is strongly activated in most neurodegenerative disorders. UPR signalling pathways mediated by IRE1α and ATF6 play a crucial role in the maintenance of ER homeostasis through the transactivation of an array of transcription factors. When activated, these transcription factors induce the expression of genes involved in protein folding and degradation with pro-survival effects. However, the specific contribution of these transcription factors to different neurodegenerative diseases remains poorly defined. Here, we characterised 44 target genes strongly influenced by XBP1 and ATF6 and quantified the expression of a subset of genes in the human post-mortem spinal cord from amyotrophic lateral sclerosis (ALS) cases and in the frontal and temporal cortex from frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD) cases and controls. We found that IRE1α-XBP1 and ATF6 pathways were strongly activated both in ALS and AD. In ALS, XBP1 and ATF6 activation was confirmed by a substantial increase in the expression of both known and novel target genes involved particularly in co-chaperone activity and ER-associated degradation (ERAD) such as DNAJB9, SEL1L and OS9. In AD cases, a distinct pattern emerged, where targets involved in protein folding were more prominent, such as CANX, PDIA3 and PDIA6. These results reveal that both overlapping and disease-specific patterns of IRE1α-XBP1 and ATF6 target genes are activated in AD and ALS, which may be relevant to the development of new therapeutic strategies.Graphical abstractThe endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR). Two major UPR signalling pathways are mediated by IRE1α and ATF6. Here, we demonstrate that these pathways activate differential gene sets in human post-mortem tissues derived from amyotrophic lateral sclerosis (ALS) compared to Alzheimer’s disease (AD) cases. Our results identify IRE1α and ATF6 specific targets that can have major implications in the development of new therapeutic strategies and potential biomarkers.

Inhibition of Sec61-dependent translocation by mycolactone uncouples the integrated stress response from ER stress, driving cytotoxicity via translational activation of ATF4

Mycolactone is the exotoxin virulence factor of Mycobacterium ulcerans that causes the neglected tropical disease Buruli ulcer. We recently showed it to be a broad spectrum inhibitor of Sec61-dependent co-translational translocation of proteins into the endoplasmic reticulum (ER). An outstanding question is the molecular pathway linking this to its known cytotoxicity. We have now used translational profiling to better understand the reprogramming that occurs in cells exposed to mycolactone. Gene ontology identified enrichment in genes involved in cellular response to stress, and apoptosis signalling among those showing enhanced translation. Validation of these results supports a mechanism by which mycolactone activates an integrated stress response meditated by phosphorylation of eIF2α via multiple kinases (PERK, GCN, PKR) without activation of the ER stress sensors IRE1 or ATF6. The response therefore uncouples the integrated stress response from ER stress, and features translational and transcriptional modes of genes expression that feature the key regulatory transcription factor ATF4. Emphasising the importance of this uncoupled response in cytotoxicity, downstream activation of this pathway is abolished in cells expressing mycolactone-resistant Sec61α variants. Using multiple genetic and biochemical approaches, we demonstrate that eIF2α phosphorylation is responsible for mycolactone-dependent translation attenuation, which initially protects cells from cell death. However, chronic activation without stress remediation enhances autophagy and apoptosis of cells by a pathway facilitated by ATF4 and CHOP. Our findings demonstrate that priming events at the ER can result in the sensing of stress within different cellular compartments.

Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

Autophagy, a regulated nutrient recycling program can affect both cell survival and cell death. Here, we show that Ormeloxifene (ORM), a selective estrogen receptor modulator approved for oral contraceptive use induces autophagic flux in ovarian cancer cells, which is activated by an ER stress response upstream of autophagy. The ER stress response is characterized by activation of IRE1α, PERK and ATF6 and is under regulation of JNK. Pharmacological inhibition of either autophagy or ER stress increased cell survival, as did silencing of autophagy proteins LC3 and Beclin 1, implying that ORM-induced autophagy is pro-death in nature. Ultrastructural observations of treated cells confirmed stages of autophagic maturation. Caspase-dependent apoptosis succeeded these events and was characterized by generation of reactive oxygen species and disruption of mitochondrial membrane potential. A concomitant inhibition of the Akt/mTOR axis was also observed with possible regulation of Akt by ORM. ORM inhibited tumor growth in ovarian xenograft model and displayed autophagic activity. In summary, in vitro and in vivo results reveal that ORM induces autophagy-associated cell death to attenuate proliferation of ovarian cancer cells. Our results demonstrate that using ORM in combination with ER stress and autophagy modulators could offer better therapeutic outcome in ovarian cancer.

TRIB3 limits FGF21 induction during in vitro and in vivo nutrient deficiencies by inhibiting C/EBP–ATF response elements in the Fgf21 promoter

Mammals must be able to endure periods of limited food availability, and the liver plays a central role in the adaptation to nutritional stresses. TRIB3 (Tribbles homolog 3) is a cellular stress-inducible gene with a liver-centric expression pattern and it has been implicated in stress response regulation and metabolic control. In the current article, we study the involvement of TRIB3 in responses to nutrient deficiencies, including fasting for up to 48 h in mice. We show that hepatic expression of Trib3 is increased after 48 h of fasting and mice with a targeted deletion of the Trib3 gene present elevated hepatic triglyceride content and liver weight at 48 h, along with an upregulation of lipid utilization genes in the liver. Further, hepatic and serum levels of the metabolic stress hormone FGF21 are considerably increased in 48-h-fasted Trib3 knockout mice compared to wild type. Trib3 deficiency also leads to elevated FGF21 levels in the mouse liver during essential amino acid deficiency and in cultured mouse embryonic fibroblasts during glucose starvation. Reporter assays reveal that TRIB3 regulates FGF21 by inhibiting ATF4-mediated, C/EBP–ATF site-dependent activation of Fgf21 transcription. Based on chromatin immunoprecipitation from mouse liver, the binding of TRIB3 and ATF4, a transcription factor known to physically interact with TRIB3, is significantly increased at the Fgf21 promoter following 48 h of fasting. Thus, under nutrient-limiting conditions that stimulate ATF4 activity, TRIB3 is implicated in the regulation of metabolic adaptation by restraining the transcription of Fgf21.

Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells

BackgroundBecause twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC’s anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC’s anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells.MethodsHow water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65.ResultsTC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3β-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of β-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis.ConclusionsOur results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Shiga Toxins Induce Apoptosis and ER Stress in Human Retinal Pigment Epithelial Cells.

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness and neurological abnormalities. Although numerous studies have defined apoptotic responses to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in a variety of cell types, the potential significance of Stx-induced apoptosis of photoreceptor and pigmented cells of the eye following intoxication is unknown. We explored the use of immortalized human retinal pigment epithelial (RPE) cells as an in vitro model of Stx-induced retinal damage. To the best of our knowledge, this study is the first report that intoxication of RPE cells with Stxs activates both apoptotic cell death signaling and the endoplasmic reticulum (ER) stress response. Using live-cell imaging analysis, fluorescently labeled Stx1 or Stx2 were internalized and routed to the RPE cell endoplasmic reticulum. RPE cells were significantly sensitive to wild type Stxs by 72 h, while the cells survived challenge with enzymatically deficient mutant toxins (Stx1A− or Stx2A−). Upon exposure to purified Stxs, RPE cells showed activation of a caspase-dependent apoptotic program involving a reduction of mitochondrial transmembrane potential (Δψm), increased activation of ER stress sensors IRE1, PERK and ATF6, and overexpression CHOP and DR5. Finally, we demonstrated that treatment of RPE cells with Stxs resulted in the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), suggesting that the ribotoxic stress response may be triggered. Collectively, these data support the involvement of Stx-induced apoptosis in ocular complications of intoxication. The evaluation of apoptotic responses to Stxs by cells isolated from multiple organs may reveal unique functional patterns of the cytotoxic actions of these toxins in the systemic complications that follow ingestion of toxin-producing bacteria.

Abstract IA13: A novel KRAS specific vulnerability in the nutrient stress response

Abstract Oncogenic KRAS has multiple effects on cellular functions. A relatively poorly understood role of oncogenic KRAS is in regulation of nutrient stress. As a strategy to identify new KRAS-specific tumor vulnerabilities, we sought to explore the role of oncogenic KRAS in mediating the response to nutrient stress. A key observation is that the gene expression signature of oncogenic KRAS is markedly different in standard tissue culture conditions compared to more physiologic levels of glutamine. Glutamine is a key nutrient for tumor cells that supports nucleotide and amino acid biosynthesis, replenishes TCA cycle intermediates and contributes to redox metabolism. We identified oncogenic KRAS as an important regulator of the response to glutamine deprivation in non-small cell lung cancer (NSCLC). A significant fraction of the gene expression signature mediated by KRAS in low glutamine is mediated by activation of ATF4. Activation of the ATF4 stress response pathway is dependent on PI3K and expression of NRF2 downstream of oncogenic KRAS in NSCLC. Through this mechanism, KRAS alters amino acid uptake and metabolism and sustains mTORC1 signaling during nutrient stress. We identified regulation of asparagine synthetase (ASNS) as a key effect of oncogenic KRAS signaling via ATF4 during glutamine deprivation. Preliminary data indicates that simultaneous blockade of PI3K and ASNS may be a useful therapeutic strategy in lung cancer. Our studies underscore an important role for KRAS in metabolism of NSCLC and identify a need for further studies that evaluate KRAS vulnerabilities under conditions of nutrient stress. Citation Format: Alejandro Sweet-Cordero. A novel KRAS specific vulnerability in the nutrient stress response [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA13.

Activation of unfolded protein response pathway is important for valproic acid mediated increase in immunoglobulin G productivity in recombinant Chinese hamster ovary cells

Process engineering to improve product quality and titers is gaining importance at late-stage cell culture process development. Valproic acid, a US Food and Drug Administration-approved histone deacetylase (HDAC) inhibitor, has been shown to improve cell culture performance with higher productivities and minimal effect on the product quality. However, the wider physiological impact of valproic acid on recombinant cells has not been investigated till date. In this study, we investigate the role of unfolded protein response pathway when immunoglobulin G (IgG)-secreting Chinese hamster ovary (CHO) cells are treated with valproic acid, resulting in a 3-fold increase in product titers and productivity. It is found that cells undergo an early transient endoplasmic reticulum (ER) stress on treatment with valproic acid, and subsequently adapt to perform as high producers. Induction of chaperones through enhanced XBP1 splicing activity and ATF6 activation suggests an increase in protein processing activity in these cells. We show that in addition to the enhanced recombinant mRNA expression of IgG heavy chain and light chain, the activation of unfolded protein response (UPR) pathway is critical to the increase in productivity of cells on valproic acid treatment. Further, upregulation of the UPR pathway is not through HDAC inhibition alone. To our knowledge, this is the first attempt to arrive at a phenotype-genotype mechanistic understanding of how valproic acid treatment enhances productivity in recombinant CHO cells.

ETS Related Gene mediated Androgen Receptor Aggregation and Endoplasmic Reticulum Stress in Prostate Cancer Development

Mechanistic studies of deregulated ERG in prostate cancer and other cancers continue to enhance its role in cancer biology and its utility as a biomarker and therapeutic target. Here, we show that ERG, through its physical interaction with androgen receptor, induces AR aggregation and endoplasmic reticulum stress in the prostate glands of ERG transgenic mice. Histomorphological alterations and the expression of ER stress sensors Atf6, Ire1α, Perk, their downstream effectors Grp78/BiP and eIF2α in ERG transgenic mouse prostate glands indicate the presence of chronic ER stress. Transient activation of apoptotic cell death during early age correlated well with the differential regulation of ER stress sensors, in particular Perk. Epithelial cells derived from ERG transgenic mouse prostates have increased prostasphere formation with resistance to radiation induced cell death. Continued activation of cell survival factors, Atf6 and Ire1α during chronic ER stress due to presence of ERG in prostate epithelium induces survival pathways and provides a selection pressure in the continuum of ERG dependent neoplastic process. These novel insights will enhance the understanding of the mechanistic functions of ERG in prostate tumor biology and towards development of early targeted therapeutic strategies for prostate cancer.