Endoplasmic Reticulum Stress-inducing Agents Research Articles

287 - The Redox Active Anti-Cancer Drug, Dp44mT, Induces Endoplasmic Reticulum Stress Activating Pro-Apoptotic Pathways of the Unfolded Protein Response in Cancer Cells

In the stressful tumor microenvironment, endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are activated in cancer cells promoting cancer cell survival. A novel strategy for anti-cancer drugs is to re-sensitise cancer cells to ER stress-induced apoptosis by altering the kinetics of the UPR. Chelators of the DpT thiosemicarbazone class form cellular redox active metal complexes and demonstrate potent in vitro and in vivo anti-cancer and anti-metastatic activity. The most potent of these agents is currently being examined in clinical trials for the treatment of patients with advanced solid tumors (NCT02688101). However, the mechanism by which these drugs alter the ER stress response and induce apoptosis is unknown. Consequently, studies assessed the mechanisms by which the potent DpT redox active agent, Dp44mT, alters the ER stress response, relative to the redox inactive chelator, DFO, and the classical ER stress-inducing agent, tunicamycin, in SK-N-MC neuroepithelioma and PANC1 pancreatic cancer cells. Our studies demonstrate that Dp44mT significantly: (1) increased activation of ER stress-associated pro-apoptotic signaling (i.e., p-eIF2α, ATF4 and CHOP); (2) increased phosphorylation of IRE1α and XBP1 mRNA splicing; (3) reduced the expression of molecules involved in ER stress-associated cell survival signaling ( e.g. , XBP1s and p58IPK); (4) activated the transcription factor, ATF6, and its downstream targets ( i.e. CHOP and BiP); and (5) activated pro-apoptotic p-CaMKII. In contrast, redox inactive DFO and the ER stress-inducing agent, tunicamycin, had little or no effect on the expression or phosphorylation of these proteins. Considering the role of reactive oxygen species (ROS) generation in the effects observed, it was demonstrated that the ROS scavenger, N -acetyl-l-cysteine, that supplements cellular glutathione could inhibit Dp44mT-mediated activation of the UPR, while the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced Dp44mT activity. In conclusion, ER stress is induced after iron sequestration by chelators, and this effect was enhanced by drugs such as Dp44mT that produce ROS. These results demonstrate that Dp44mT activates the pro-apoptotic pathways of the UPR, re-sensitizing cancer cells to apoptosis while inhibiting cell survival signals.

Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of resistance blood vessel structure and function.

Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.

The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

Endoplasmic reticulum (ER) stress triggers the integrated ER‐stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR during Leishmania amazonensis infection. Treatment of RAW 264.7 infected macrophages with the ER stress‐inducing agent thapsigargin (TG; 1 μM) increased L. amazonensis infectivity in an IFN1‐α receptor (IFNAR)‐dependent manner. In Western blot assays, we showed that L. amazonensis activates the inositol‐requiring enzyme (IRE1)/ X‐box binding protein (XBP)‐1‐splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for the Ifnb gene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)‐3 to the nucleus and a decrease in IFN1‐β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)‐1, that protect parasites from oxidative stress. We conclude that L. amazonensis activation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1‐β expression.—Dias‐Teixeira, K. L., Calegari‐Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X‐box binding protein 1 transcription factor. FASEB J. 30, 1557–1565 (2016). http://www.fasebj.org

The bZIP Transcription Factor HAC-1 Is Involved in the Unfolded Protein Response and Is Necessary for Growth on Cellulose in Neurospora crassa.

High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER) is exceeded, a pathway known as the unfolded protein response (UPR) is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications.

Protein kinase R-like endoplasmic reticulum kinase and glycogen synthase kinase-3α/β regulate foam cell formation

Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/β in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE(-/-) mice fed a diet supplemented with the GSK3α/β inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/β inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/β signaling promotes proatherogenic macrophage lipid accumulation.

Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease.

PKR-LIKE ENDOPLASMIC RETICULUM KINASE (PERK) AND GLYCOGEN SYNTHASE KINASE (GSK)-3α/β SIGNALING REGULATE ATHEROSCLEROSIS DEVELOPMENT

Recently, a role for endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/β has been proposed in atherogenesis; this study sought to delineate the underlying molecular mechanisms. Cultured THP1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin or palmitate in the presence or absence of inhibitors of the unfolded protein response (UPR) pathways (PERK, IRE, ATF6) or GSK3α/β (CT99021). ER stress enhanced GSK3α/β activity while inhibition of the PERK pathway, but not the IRE or ATF6 pathways, attenuated GSK3α/β activity. GSK3α/β inhibition did not affect adaptive components of the UPR but did repress the expression of factors downstream of PERK (ATF4 and CHOP). ER stress enhanced the mRNA expression of genes controlling lipid biosynthesis and uptake; while GSK3α/β inhibition attenuated this effect. GSK3α/β inhibition blocked ER stress-induced cholesterol accumulation in macrophages. To investigate the roles of macrophage GSK3α and GSK3β in a mouse model of atherosclerosis, LDLR-/- myeloid cell GSK3α knockout (LDLR-/-GSK3αflox/floxLyzMCre+/-) and GSK3β knockout (LDLR-/-GSK3βflox/floxLyzMCre+/-) mice were placed on a high fat diet. Myeloid cell GSK3α deficiency, but not GSK3β deficiency, attenuated atherogenesis. Our findings support a role for macrophage PERK - GSK3α signaling in atherosclerosis development.

UAP1 is overexpressed in prostate cancer and is protective against inhibitors of N-linked glycosylation.

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage.

Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus.

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforinmalin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/ quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a(-/-), Epm2b(-/-), and Epm2a(-/-) Epm2b(-/-) mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b(-/-) and Epm2a(-/-) Epm2b(-/-) cells) but not laforin (Epm2a(-/-) cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.

Ribosomal protein L19 overexpression activates the unfolded protein response and sensitizes MCF7 breast cancer cells to endoplasmic reticulum stress-induced cell death

Although first identified for their roles in protein synthesis, certain ribosomal proteins exert pleiotropic physiological functions in the cell. Ribosomal protein L19 is overexpressed in breast cancer cells by amplification and copy number variation. In this study, we examined the novel pro-apoptotic role of ribosomal protein L19 in the breast cancer cell line MCF7. Overexpression of RPL19 sensitized MCF7 cells to endoplasmic reticulum stress-induced cell death. RPL19 overexpression itself was not cytotoxic; however, cell death induction was enhanced when RPL19 overexpressing cells were incubated with endoplasmic reticulum stress-inducing agents, and this sensitizing effect was specific to MCF7 cells. Examination of the cell signaling pathways that mediate the unfolded protein response (UPR) revealed that overexpression of RPL19 induced pre-activation of the UPR, including phosphorylation of pERK-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), and activation of p38 MAPK-associated stress signaling. Our findings suggest that upregulation of RPL19 induces ER stress, resulting in increased sensitivity to ER stress and enhanced cell death in MCF7 breast cancer cells.

Abstract 659: PKR-Like Endoplasmic Reticulum Kinase and Glycogen Synthase Kinase-3α/ß Signaling Regulates Endoplasmic Reticulum Stress-Induced Foam Cell Formation

Background: Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. The molecular mechanisms by which conditions of ER stress promote pro-atherogenic processes are not understood. We have found that ER stress-inducing agents can activate glycogen synthase kinase (GSK)-3α/β, a protein involved in many metabolic pathways. The objective of this study is to investigate the role of GSK3α/β in pro-atherogenic ER stress signaling. Methods and Results: Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin or palmitate, in the presence or absence of the GSK3α/β inhibitor CT99021. GSK3α/β inhibition did not affect the adaptive unfolded protein response (UPR), but did block ER stress-induced lipid accumulation as well as the up regulation of genes associated with lipid biosynthesis and uptake. Using small molecule inhibitors of specific UPR pathways, we found that PERK, but not IRE1 or ATF6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition attenuated ER stress-induced expression of distal components of the PERK pathway, including CHOP and ATF4. Atherosclerotic plaques from ApoE-/- mice, fed a diet supplemented with the GSK3α/β inhibitor valproate, had reduced levels of CHOP within the macrophage foam cells. In primary mouse macrophages PERK inhibition blocked ER stress induced lipid accumulation whereas the overexpression of constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. Conclusions: Pharmacological inhibition of GSK3α/β attenuates ER stress-induced lipid accumulation and macrophage foam cell formation. These findings indicate that GSK3α/β is an important factor in the ER stress-PERK signaling pathway and may play a central role in the pro-atherogenic dysregulation of lipid metabolism.

Pkr‐like endoplasmic reticulum kinase (PERK) and glycogen synthase kinase‐3α/β signaling regulate atherosclerosis development (278.5)

Recently, a role for endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)‐3α/β has been proposed in atherogenesis; this study sought to delineate the underlying molecular mechanisms.Cultured THP1‐derived macrophages were treated with the ER stress‐inducing agents, glucosamine, thapsigargin or palmitate in the presence or absence of inhibitors of the unfolded protein response (UPR) pathways (PERK, IRE, ATF6) or GSK3α/β (CT99021). ER stress enhanced GSK3α/β activity while inhibition of the PERK pathway, but not the IRE or ATF6 pathways, attenuated GSK3α/β activity. GSK3α/β inhibition did not affect adaptive components of the UPR but did repress the expression of factors downstream of PERK (ATF4 and CHOP). ER stress enhanced the mRNA expression of genes controlling lipid biosynthesis and uptake; while GSK3α/β inhibition attenuated this effect. GSK3α/β inhibition blocked ER stress‐induced cholesterol accumulation in macrophages. To investigate the roles of macrophage GSK3α and GSK3β in a mouse model of atherosclerosis, LDLR‐/‐ myeloid cell GSK3α knockout (LDLR‐/‐GSK3αflox/floxLyzMCre+/‐) and GSK3β knockout (LDLR‐/‐GSK3βflox/floxLyzMCre+/‐) mice were placed on a high fat diet. Myeloid cell GSK3α deficiency, but not GSK3β deficiency, attenuated atherogenesis.Our findings support a role for macrophage PERK ‐ GSK3α signaling in atherosclerosis development.

ER Stress-Induced eIF2-alpha Phosphorylation Underlies Sensitivity of Striatal Neurons to Pathogenic Huntingtin

A hallmark of Huntington’s disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2α, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2α phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington’s disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2α phosphorylation by increasing the activity of PKR-like ER-localized eIF2α kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2α phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2α has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients.

Heat shock preconditioning protects against ER stress-induced apoptosis through the regulation of the BH3-only protein BIM

A mild heat shock (HS) preconditioning and acquisition of thermotolerance protects cells against a variety of cytotoxic agents that otherwise induce apoptosis. Here we tested whether there is a molecular link between HS preconditioning and endoplasmic reticulum (ER) stress-induced apoptosis. ER stress results from a loss of ER lumen homeostasis, culminating in an accumulation of unfolded/misfolded proteins in the ER and activation of unfolded protein response (UPR). Unresolved, ER stress leads to activation of BH3-only proteins, mitochondrial membrane permeabilization, caspase activation and apoptotic cell death. HS preconditioning (1h at 42°C) induced a rapid increase in HSPA1 (HSP70) levels which remained elevated for at least 48h post-HS. HS preconditioning significantly reduced BAX, caspase activation and apoptosis in cell cultures treated with the ER stress-inducing agents thapsigargin (TG) and tunicamycin (TM). HS-mediated protection was found to be due to regulation of the BH3-only protein BIM. Further, overexpression of HSPA1 could not mimic the effect of HS on BIM expression, suggesting that other HS factors may play a role in inhibiting ER stress-induced apoptosis by regulating BIM.

Abstract C284: IRE1 and PERK as targets of cellular adaptation and survival to hypoxia.

Abstract Hypoxia is an important and unique feature of the solid tumour microenvironment. Hypoxia promotes resistance to radiotherapy, aggressive disease phenotypes, and poor patient prognosis. Consequently, therapies directed against hypoxic cells are needed to improve patient outcome. Hypoxic stress leads to the activation of several adaptive pathways including the unfolded protein response (UPR). The UPR is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling pathways initiated by the ER-membrane resident proteins PERK, IRE1 and ATF6. Previous work using genetic models has implicated the PERK pathway, as well as downstream components of the IRE1 pathway (XBP1) as important mediators of hypoxia tolerance and tumor response to therapy. Here we employed short hairpin RNAs (shRNAs) and recently developed small-molecule inhibitors of IRE1 (4μ8c) and PERK (GSK-compound 39) to investigate the relative importance of these two pathways in promoting hypoxia tolerance and their potential as therapeutic targets. We tested the pathway activity of PERK and IRE1 by quantitative PCR and western blotting. Hypoxia tolerance was measured by in vitro clonogenic assays. First, knockdown experiments using two different shRNAs showed that IRE1 is essential for survival of HCT116 colorectal cancer cells during mild hypoxia while PERK was essential in both normoxia and hypoxia. Interestingly, the IRE1 knockdown cells had a significantly altered glycolytic metabolism, a known mechanism of hypoxia adaptation. Using our small-molecule IRE1 inhibitor, we show that 4μ8c is a potent and non-toxic inhibitor of IRE1 RNase activation in response to both hypoxia and other ER stress-inducing agents. This compound effectively inhibited IRE1 induced activation of the downstream target genes in both HCT116 colorectal cancer and KP4 pancreatic cancer cell lines under hypoxia. However, despite potent inhibition of IRE1 activation and the, 4μ8c had no effect on cell proliferation or clonogenic survival of HCT116 and KP4 cells during exposure to hypoxia or anoxia. Similarly, 4μ8c inhibition of IRE1 did not sensitize cells to other ER stress inducing agents. In contrast, genetic inactivation of PERK signaling or treatment with the highly selective and potent PERK inhibitor GSK-compound 39 substantially reduced tolerance to hypoxia, anoxia and other ER stress inducing agents. Our results demonstrate that although hypoxia potently activates both PERK and IRE1 arms of the UPR pathway, the PERK arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress. Furthermore, our results suggest that newly developed and orally available PERK inhibitors such as GSK-compound 39 are selectively toxic to hypoxic cells, and thus warrant further development for use in combination therapies with radiation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C284. Citation Format: Dan Cojocari, Ravi Vellanki, Brandon Sit, Marianne Koritzinsky, Bradly G. Wouters. IRE1 and PERK as targets of cellular adaptation and survival to hypoxia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C284.

Investigating the Role of Glycogen Synthase Kinase (GSK)-3α/β in Pro-Atherogenic Endoplasmic Reticulum (ER) Stress Signaling

Atherosclerosis is a major underlying cause of cardiovascular disease. Our lab and others have identified a role for endoplasmic reticulum (ER) stress in the dysregulation of lipid metabolism and the development of atherosclerosis, however the molecular mechanisms by which conditions of ER stress promote the pro-atherogenic processes are not understood. Recently, we have found that ER stress-inducing agents can activate glycogen synthase kinase (GSK)-3 α and β, homologous kinases involved in the regulation of cell metabolism.

Endoplasmic Reticulum stress reduces COPII vesicle formation and modifies Sec23a cycling at ERESs

Exit from the Endoplasmic Reticulum (ER) of newly synthesized proteins is mediated by COPII vesicles that bud from the ER at the ER Exit Sites (ERESs). Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred to as ER stress. Previously, we demonstrated that ER stress rapidly impairs the formation of COPII vesicles. Here, we show that membrane association of COPII components, and in particular of Sec23a, is impaired by ER stress-inducing agents suggesting the existence of a dynamic interplay between protein folding and COPII assembly at the ER.

Nelfinavir and bortezomib inhibit mTOR activity via ATF4-mediated sestrin-2 regulation

Endoplasmic reticulum (ER) stress and autophagy are two basic cell survival mechanisms often occurring in concert. Extensive ER stress in cancer cells deliberately induced by chemotherapeutic drugs may lead to growth arrest and cell death. However, the link between ER stress and autophagy is not well understood. In this study, the treatment of cancer cells with ER stress-inducing drug nelfinavir resulted in the expression of endogenous mTOR inhibitor sestrin-2 (SESN2). Upregulation of SESN2 expression was associated with expression of ER stress markers ATF4, ATF3, and CHOP. SESN2 upregulation also occurred in cells treated with the proteasome inhibitor bortezomib. Ectopic expression of ATF4, but not of ATF3 or CHOP, caused transcriptional upregulation of SESN2 expression, indicating expressional regulation of SESN2 by ATF4. Transient overexpression of ectopic SESN2 resulted in mTOR inhibition and autophagy, confirming a link between ER stress, SESN2 upregulation, and mTOR inhibition. Accordingly, cancer cells treated with the ER stress-inducing agent nelfinavir showed reduced mTOR activity and associated increases in the expression levels of ATF4 and SESN2. These results show that ATF4-regulated SESN2 expression presents a new link between ER stress and mTOR inhibition and autophagy. mTOR inhibition by nelfinavir, which is currently in clinical trials for cancer patients, may also explain its observed ability to induce autophagy, growth arrest, and radiosensitization in cancer cells.

New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1α signaling is essential for promoting adaptation and survival to hypoxia

Background and purposeThe unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. MethodsWe utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. ResultsUsing knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. ConclusionsOur results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts.