ER Stress Inducer Thapsigargin Research Articles

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

RF32 | PSUN44 CARF Regulates Non-Alcoholic Fatty Liver Diseases (NAFLD) by Controlling ER Stress and Lipogenesis in Hepatic Cells.

Abstract Background Sedentary lifestyles and changes in diet are fueling the worldwide epidemic of obesity and the prevalence of NAFLD and insulin resistance (IR). It has been predicted that NAFLD and its more advanced form, nonalcoholic steatohepatitis (NASH) will be the next epidemic in chronic liver diseases, and NAFLD/NASH will soon replace viral hepatitis as the primary cause of end-stage liver failure and transplantation. NAFLD is a significant risk factor for IR and cardiovascular diseases, and it highlights the importance of finding a way to control the epidemic. CARF is a multifunctional stress-responsive gene and was found to be reduced in response to metabolic stress in the fatty liver of diet-induced obesity mice (DIO). We also showed that CARF expression was down-regulated in palmitate (PA)-treated HepG2 cells. In this study, we aim to understand the consequences of the reduced expression of CARF on the development of NAFLD. Methods We performed RNA seq, RT-PCR and western blotting (WB) to evaluate the effect of CARF knockdown on lipid metabolism in HepG2 cells. Differentially expressed genes were analyzed by gene sort enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to identify the metabolic pathways affected by CARF depletion. Apoptosis was assessed by TUNEL assay. To induce ER stress, HepG2 cells were treated with thapsigargin and evaluated its effect on CARF. Results By performing Sh-RNA mediated CARF knockdown, RNA-seq analysis, RT-PCR, western blotting (WB), we were able to show that ER-stress and de-novo lipogenesis pathways were significantly affected in HepG2 cells. We showed that GRP78, CHOP, PERK, ERN1a, genes associated with ER-stress were upregulated in CARF-depleted HepG2 cells. Additionally, thapsigargin-induced ER stress was found to reduce the expression of CARF along with the increased expression of ER stress marker genes. We also showed that overexpression of CARF mitigated the thapsigargin-induced ER stress suggesting that CARF protects against ER stress in HepG2 cells. In addition, the genes associated with triglyceride biosynthesis GPAT3, GPAM were significantly upregulated, indicating that lipogenesis was triggered by silencing of CARF in HepG2 cells. BODIPY staining and TG assay confirmed that silencing of CARF enhanced triglyceride biosynthesis in HepG2 cells. Furthermore, we showed that the expression of antioxidant genes GPX2, GPX3, and TXRND3 decreased, resulting in enhanced oxidative stress and higher apoptosis in CARF depleted cells. Conclusion We identified novel roles of CARF regulating cellular ER-Stress, lipid metabolism, and oxidative stress, and its impairment in response to metabolic stress could lead to the development of NAFLD in obese patients with metabolic abnormalities. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:42 p.m. - 12:47 p.m.

Multi-level inhibition of coronavirus replication by chemical ER stress

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Abstract 2026: KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness

Abstract Background: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and median survival of 20 months. Standard therapy consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide, and tumor-treating fields. Despite these therapies, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Glioma stem cells (GSCs) play a central role in GBM development and contribute to treatment resistance. Recently we showed that lysine-specific histone demethylase 1A (KDM1A/LSD1) is essential for GSCs stemness, and inhibition of KDM1A induces unfolded protein response (UPR) in GSCs. In this study, we tested the hypothesis that inhibition of KDM1A sensitizes GSCs to ER stress inducers by inducing UPR. Methods: KDM1A knockdown (KDM1A-KD) cells were generated using KDM1A specific shRNA. We studied the effect of KDM1A-KD or pharmacological KDM1A inhibitors (NCD38 and NCL-1) in combination with ER stress inducers (thapsigargin and brefeldin A) on GSCs viability using CellTiter-Glo assay. Stemness was determined using extreme limiting dilution analysis (ELDA) and sphere formation assays. Mechanistic studies were conducted using RNA-seq, ChIP, RT-qPCR, and Western blotting analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and ER stress inducer was studied using orthotopic models of GBM. Results: Cell viability assays demonstrated that knockdown or inhibition of KDM1A sensitized GSCs to ER stress inducers thapsigargin and brefeldin A. Furthermore, KDM1A-KD, NCD38, or NCL-1, in combination with ER stress inducers, significantly decreased the stemness and sphere-forming ability of GSCs. RNA-seq analysis revealed that UPR was activated after knockdown or inhibition of KDM1A in GSCs. Western blot and RT-qPCR analysis showed that a combination of KDM1A inhibitors and ER stress inducers increased UPR signaling in GSCs. ChIP analysis indicated that KDM1A inhibition enriched the active histone methylation mark (H3K4me2) at the promoter of UPR target genes. In vivo studies showed that KDM1A inhibition activates UPR in tumors and improved overall survival. Conclusions: Our results support that KDM1A knockdown or inhibition sensitizes GSCs to ER stress inducers and that the use of KDM1A inhibitors in conjunction with ER stress inducers is a potential novel therapy for GBM patients. Citation Format: Yi He, Prabhakar Pitta Venkata, Salvador Alejo, Yihong Chen, Bridgitte Palacios, Gabrielle Gray, Uday P. Pratap, Suryavathi Viswanadhapalli, Siyuan Zheng, Rajeshwar R. Tekmal, Andrew J. Brenner, Gangadhara R. Sareddy. KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2026.

The miR-200–Zeb1 axis regulates key aspects of β-cell function and survival in vivo

ObjectiveThe miR-200–Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as β-cell dedifferentiation and apoptosis contribute to the loss of functional β-cell mass and diabetes progression. Furthermore, EMT promotes the loss of β-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of β-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in β-cell function and survival in vivo. MethodsmiR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3′UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141–200c overexpression. ResultsMild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting β-cell identity, EMT, insulin secretion, cell–cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. ConclusionsThis study signifies the importance of the miR-200–Zeb1 axis in regulating key aspects of β-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing β-cell redifferentiation and maintaining β-cell identity in in vitro islet expansion.

41-OR: Roles of Transcription Factor EB (TFEB) in ß-Cell Function and Survival Under Stress

Autophagy can help protect stressed β-cells. We investigated if disruption of Transcription Factor EB (Tfeb), a transcriptional master-regulator of lysosomes and autophagy, contributes to β-cell failure and death under various ER stress-inducing conditions. Short-term β-cell autophagy-deficiency (inducible Atg5 knockout) reduced β-cell survival under hypoxic stress in vitro and impaired the ability of syngeneic marginal mass islet grafts to maintain glucose homeostasis in streptozotocin (STZ)-induced diabetic mice, with >80% of recipients of Atg5-deficient islets returning to diabetes within 10 days post-transplant (vs. 30% of controls, p=0.05). Hypoxic culture activated pro-apoptotic ER stress pathways, and confocal analysis of islet cells from LC3-RFP-EGFP autophagy reporter mice demonstrated that autophagic flux was interrupted. Hypoxia-induced impairment of autophagy was associated with reduced lysosomal cathepsin B activity and a significant loss of Tfeb, both of which could be partially rescued by Torin-1. Lipotoxicity and thapsigargin-induced ER-stress similarly reduced Tfeb protein and cell viability, suggesting ER stress-specific mechanisms may drive dysregulation of Tfeb and lysosomes. Conversely, overexpression of Tfeb:GFP reduced thapsigargin-induced death in MIN6 cells. To directly study the consequences of Tfeb loss we generated mice with Ins1Cre-driven, β-cell-specific, Tfeb deletion (TfebβKO). Notably, both male and female TfebβKO mice had normal glucose tolerance, but cultured Tfeb KO β-cells showed impaired lysosomal cathepsin B activity and TfebβKO mice had worsened glucose tolerance compared to Tfeb flox littermates following injection with multiple low-doses STZ. In summary, autophagy protects β-cells under hypoxia but is impaired under prolonged hypoxic stress, possibly through Tfeb dysregulation. Our data further suggest loss of Tfeb is a common feature of ER stress and that this loss exacerbates stress-induced β-cell failure and death. Disclosure Y. Zou: None. D. Pasula: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem, Stock/Shareholder; Self; Integrated Nanotherapeutics Inc. D. S. Luciani: None. Funding JDRF (2-2013-50)

Inflammation promotes adipocyte lipolysis via IRE1 kinase

Obesity associates with inflammation, insulin resistance, and higher blood lipids. It is unclear if immune responses facilitate lipid breakdown and release from adipocytes via lipolysis in a separate way from hormones or adrenergic signals. We found that an ancient component of ER stress, inositol-requiring protein 1 (IRE1), discriminates inflammation-induced adipocyte lipolysis versus lipolysis from adrenergic or hormonal stimuli. Our data show that inhibiting IRE1 kinase activity was sufficient to block adipocyte-autonomous lipolysis from multiple inflammatory ligands, including bacterial components, certain cytokines, and thapsigargin-induced ER stress. IRE1-mediated lipolysis was specific for inflammatory triggers since IRE1 kinase activity was dispensable for isoproterenol and cAMP-induced lipolysis in adipocytes and mouse adipose tissue. IRE1 RNase activity was not associated with inflammation-induced adipocyte lipolysis. Inhibiting IRE1 kinase activity blocked NF-κB activation, interleukin-6 secretion, and adipocyte-autonomous lipolysis from inflammatory ligands. Inflammation-induced lipolysis mediated by IRE1 occurred independently from changes in insulin signaling in adipocytes, suggesting that inflammation can promote IRE1-mediated lipolysis independent of adipocyte insulin resistance. We found no role for canonical unfolded protein responses or ABL kinases in linking ER stress to IRE1-mediated lipolysis. Adiponectin-Cre-mediated IRE1 knockout in mice showed that adipocyte IRE1 was required for inflammatory ligand-induced lipolysis in adipose tissue explants and that adipocyte IRE1 was required for approximately half of the increase in blood triglycerides after a bacterial endotoxin-mediated inflammatory stimulus in vivo. Together, our results show that IRE1 propagates an inflammation-specific lipolytic program independent from hormonal or adrenergic regulation. Targeting IRE1 kinase activity may benefit metabolic syndrome and inflammatory lipid disorders.

Abstract 1820: Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth

Abstract Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian ARTs are extracellular with glycosylphosphatidylinositol (GPI) anchors. ARTs can target cell surface proteins on bystander cells or soluble proteins in the tumor microenvironment, particularly P2RX7 on T cells1. We hypothesized that an evolutionarily conserved parallel protective role may be provided by ART1 expression in lung cancer cells, particularly during times of cellular stress2. A mouse KP1 lung tumor cell line with ART1 expression was established from KRASG12D/P53-/- mice and transduced with shRNA targeting ART1 (KP1-sh) or an empty vector (KP1-ev). For in vitro studies, KP1 WT cells were treated with an ER stress inducer thapsigargin, with radiation, or with chemotherapy. Using human materials, we evaluated ART1 expression in NSCLC tumors by whole tumor RT-PCR/RNAseq, immunofluorescence (IF), and immunohistochemistry (IHC). For evaluation of tumor growth in immune competent models, KP1 cells were injected into syngeneic C57BL/6 mice into the flank or intravenously to generate orthotopic lung tumors. Following in vitro treatment with thapsigargin, expression of ART1 increased 11.8-fold in KP1 WT cells (24 hrs). Similarly, following radiation (8Gy x 3,) ART1 expression increased 1.93-fold by RT-PCR and surface protein expression increased by 1.87 fold (p<0.0005) by IF in KP1 cells. Similar increases were seen following cisplatin (10uM, 24 hrs) treatment (1.87-fold). Radiation (2.26-fold) and cisplatin (25uM, 3.30-fold) also induced ART1 expression in human A549 cells (24 hrs). In a human tissue microarray of patients who received neoadjuvant chemotherapy or radiation therapy (n=200), all tumors expressed ART1 and 38% of strongly expressed ART1. We also compared pre-treatment biopsy and post-treatment surgical resection tumor samples (n=21) in an ongoing clinical trial (NCT02904954) of neoadjuvant durvalumab +/- stereotactic radiation (8Gy x 3). ART1 was expressed at low levels by RNAseq, but trended upward (4-fold) after treatment, particularly in those patients who received radiation therapy (6-fold). Expression trended higher in nonresponders. Of these tumors without major pathologic response (n=13), 44% had moderate-strong ART1 expression by IHC. In murine flank tumor models, KP1-ev cells grew significantly faster than KP1-sh cells (688 mm3 vs. 331mm3 at 4 weeks, p=0.004). Similarly, following tail vein injection, mice injected with KP1-ev cells had heavier tumor burdens by micro-CT scan and significantly greater total lung weights (p=0.036) at sacrifice than did mice injected with KP1-sh cells. In conclusion, ART1 is broadly expressed in human lung tumors and is upregulated by cellular stress invoked by common treatments such as radiation and chemotherapy. Knockdown of ART1 abrogates tumor growth and may be a potential therapeutic target for combination therapy in lung cancer patients. 1. PMID: 7930612 2. PMID: 25292337 Citation Format: Sumit Mukherjee, Erik Wennerberg, Clarey Hung, Najla Saadallah, Shashi Kariyawasam, Christopher Agrusa, Amanda Valeta, Nasser Altorki, Timothy McGraw, Sandra Demaria, Brendon Stiles. Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1820.

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

1767-P: Inflammatory Triggers of Lipolysis Act through IRE1 in Adipocytes

Hormones and inflammation can promote adipocyte lipolysis. Inflammation-induced insulin resistance can favor lipolysis, but the cellular mediators of unique lipolytic triggers are ill-defined. We found that ER stress discriminates inflammation-induced adipocyte lipolysis versus adrenergic-mediated lipolysis typical of hormones. Tauroursodeoxycholic acid (TUDCA) blocked adipocyte-autonomous lipolysis from multiple inflammatory ligands, including bacterial peptidoglycan (PGN), lipopolysaccharide (LPS) and tumor necrosis factor (TNF). TUDCA did not alter isoproterenol-induced lipolysis. Inhibiting inositol-requiring protein 1 (IRE1) kinase activity was sufficient to block lipolysis caused by inflammatory triggers and thapsigargin-induced ER stress. Tissue-specific deletion of IRE1 in mice confirmed that adipocyte-resident IRE1 was required for inflammatory ligand-induced lipolysis in adipose tissue. IRE1 kinase activity was dispensable for isoproterenol-induced lipolysis in adipocytes and adipose tissue. We found no role for typical unfolded protein responses as a mechanism linking ER stress to lipolysis, since IRE1 Rnase activity was not associated with changes in adipocyte lipolysis and adipose tissue from GRP78/BiP+/- mice had no change in lipolysis compared to littermate mice. Inhibiting IRE1 kinase activity blocked adipocyte NF-κB activation and Interleukin-6 (IL6) production in response to inflammatory ligands. However, inflammation-induced lipolysis mediated by IRE1 occurred independently from changes in insulin signaling in adipocytes, which further supported the concept that inflammatory triggers of lipolysis can work independent of hormone responses, including insulin resistance. Our results are consistent with IRE1-linked ER stress mediating an inflammation-induced lipolytic program independently from hormonal regulation of lipolysis. Targeting components of IRE1-kinase signaling may have value in obesity and inflammatory lipid disorders. Disclosure K.P. Foley: None. Y. Chen: None. K. Kwok: None. N.G. Barra: None. A.K. Tamrakar: None. Y. Liu: None. J.D. Schertzer: None. Funding Canadian Institutes of Health Research

Heparanase protects the heart against chemical or ischemia/reperfusion injury

Although cancer cells use heparanase for tumor metastasis, favourable effects of heparanase have been reported in the management of Alzheimer's disease and diabetes. Indeed, we previously established a protective function for heparanase in the acutely diabetic heart, where it conferred cardiomyocyte resistance to oxidative stress and apoptosis by provoking changes in gene expression. In this study, we tested if overexpression of heparanase can protect the heart against chemically induced or ischemia/reperfusion (I/R) injury. Transcriptomic analysis of Hep-tg hearts reveal that 240 genes related to the stress response, immune response, cell death, and development were altered in a pro-survival direction encompassing genes promoting the unfolded protein response (UPR) and autophagy, as well as those protecting against oxidative stress. The observed UPR activation was adaptive and not apoptotic, was mediated by activation of ATF6α, and when combined with mTOR inhibition, induced autophagy. Subjecting wild type (WT) mice to increasing concentrations of the ER stress inducer thapsigargin evoked a transition from adaptive to apoptotic UPR, an effect that was attenuated in Hep-tg mouse hearts. Consistent with these observations, when exposed to I/R, the infarct size and markers of apoptosis were significantly lower in the Hep-tg heart compared to WT. Finally, UPR and autophagy inhibitors reduced the protective effects of heparanase overexpression during I/R. Our data suggest that the mechanisms that underlie the role of heparanase in promoting cell survival could be uniquely beneficial to the heart by providing protection against cellular stresses, and could be useful for exploitation as a therapeutic target for the treatment of heart disease.

Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

BackgroundChronic ER stress and dysfunction is a hallmark of obesity and a critical contributor to metaflammation, abnormal hormone action and altered substrate metabolism in metabolic tissues, such as liver and adipocytes. Lack of STAMP2 in lean mice induces inflammation and insulin resistance on a regular diet, and it is dysregulated in the adipose tissue of obese mice and humans. We hypothesized that the regulation of STAMP2 is disrupted by ER stress. Methods3T3-L1 and MEF adipocytes were treated with ER stress inducers thapsigargin and tunicamycin, and inflammation inducer TNFα. The treatments effect on STAMP2 expression and enzymatic function was assessed. In addition, 3T3-L1 adipocytes and HEK cells were utilized for Stamp2 promoter activity investigation performed with luciferase and ChIP assays. ResultsER stress significantly reduced both STAMP2 mRNA and protein expression in cultured adipocytes whereas TNFα had the opposite effect. Concomitant with loss of STAMP2 expression during ER stress, intracellular localization of STAMP2 was altered and total iron reductase activity was reduced. Stamp2 promoter analysis by reporter assays and chromatin immunoprecipitation, showed that induction of ER stress disrupts C/EBPα-mediated STAMP2 expression. ConclusionThese data suggest a clear link between ER stress and quantitative and functional STAMP2-deficiency.

The toxicity of hydroxylated and carboxylated multi-walled carbon nanotubes to human endothelial cells was not exacerbated by ER stress inducer

Hydroxylated multi-walled carbon nanotubes (h-MWCNTs) and carboxylated MWCNTs (c-MWCNTs) have potential applications in biomedicine, but their toxicity to human endothelial cells under stressed conditions associated with chronic diseases was less studied in vitro. This study stressed human umbilical vein endothelial cells (HUVECs) with ER stress inducer thapsigargin (TG), and investigated the toxicity of h-MWCNTs and c-MWCNTs to normal and stressed HUVECs. h-MWCNTs and c-MWCNTs modestly reduced cellular viability, significantly promoted soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1) as well as intracellular ROS, and decreased the expression of transcription factor KLF2 and KLF4. Pre-treatment with TG significantly reduced cellular viability, promoted IL-6 and THP-1 monocyte adhesion, and increased the expression of a panel of ER stress genes. ANOVA indicated no interaction between MWCNTs and TG pre-treatment on most of the endpoints. It was concluded that the toxicity of h-MWCNTs and c-MWCNTs to HUVECs might not be exacerbated by ER stress inducer

Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis

One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin:insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1α and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre-treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1α and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses.

Unconventional SNARE in the ER 1 (Use1) is Expressed in Skeletal Muscle and is Upregulated in Response to Skeletal Muscle Atrophy

Muscle atrophy, or a loss in muscle size and strength, results from various physiological conditions and occurs when protein degradation outpaces protein synthesis. Unconventional SNARE in the ER 1 (Use1) protein, in which SNARE stands for soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor, was recently found to be highly upregulated in skeletal muscle cells undergoing atrophy. However, the mechanism of how Use1 is transcriptionally regulated and its potential role in skeletal muscle atrophy is not clear. Interestingly, we have cloned four distinct protein encoding splice‐variants in skeletal muscle, but the significance and functions of these isoforms remains to be determined. It is known that Use1 functions in the endomembrane system (EMS) and that it could play a role in ER stress and the unfolded protein response (UPS) as a consequence of skeletal muscle atrophy. Therefore, in order to simulate atrophy‐induced ER stress in cell culture, we treated cells with Tucamycin (TN) or Thapsigargin (TG) to determine if Use1 expression is impacted in response to UPS activation. If TN or TG induced ER stress results in an increase in Use1 protein, or an isoform switch, it may suggest that Use1 is an important marker of atrophy‐induced ER stress and that it could play an important role in the progression of skeletal muscle wasting. Since muscle atrophy may contribute to increased morbidity and mortality in response to chronic disease states, it is important to gain a better understanding of the factors that participate in the molecular cascade of muscle atrophy, which could lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.

Abstract 2858: Endoplasmic reticulum stress as possible target for therapy in glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most aggressive form of brain cancer with an overall survival of patients of only 1-2 years after diagnosis. The current standard treatment is largely ineffective due to high resistance and infiltrative nature of GBM. Cellular heterogeneity in the tumor and the presence of cancer stem cells in GBM (GSCs) are considered the main contributors to aggressiveness. Recently, different molecular subtypes have been found in GBM. Proneural (PN) and Mesenchymal (MES) GBM are most distinct and MES tumors are more aggressive and infiltrative. Drugs that induce Endoplasmic Reticulum Stress (ER stress)/Unfolded Protein Response (UPR) activation provide a potential novel therapy for cancer treatment. In this study we aim to characterize and explore the potential of ER stress-inducing drugs to target GBM. Methods: GBM neurospheres PN and MES were tested for sensitivity to the ER stress inducer Thapsigargin (Tg) by MTS. TGFβ was used to induce mesenchymal transition in PN GBMs. Limiting dilution assay was performed to analyze the spheroid formation capacity upon ER stress induction. Activation of UPR was determined by analyzing protein expression of key signals by Western Blotting or XBP1 splicing assay (RT-PCR). Caspase Glo 3/7 assay was performed as read-out for apoptosis. Results: GBM MES neurospheres cells were 2-fold more sensitive to the ER stress inducing drug Tg compared to PN cells. The reduction of cell viability upon ER stress induction coincided with induction of apoptosis (caspase 3/7 activation). Consistently MES GBM cells showed stronger increase in apoptosis activation. Tg-induced ER stress resulted in 2- 4-fold reduction in spheroid formation capacity of MES GBM cells and higher Tg concentration also reduced spheroid formation capacity in one of the PN GBM cell lines. Interestingly, TGFβ-induced mesenchymal transition sensitized particularly PN GBM cells to Tg. The activation of UPR was confirmed by induction of GRP78 and CHOP expression. In MES GBM cells particularly activation of the ATF6-UPR branch by Tg ER stress induction was detected and XBP1 splicing activation appeared to be stronger in PN cells. No significant differences were observed in PERK activation among the different GBM subtypes. Conclusion: ER stress induction appears to be effective for eradicating GBM neurospheres, particularly the aggressive MES subtype. ER stress also targets the GSC compartment in GBM. ATF6 is notably activated in MES GBM cells, while XBP1 seems more activated in PN GBM cells upon Tg-induced ER stress. The relationship between an UPR branch activation and the differential sensitivity for the ER stress induction will be further explored. Overall, targeting the ER stress response appears a promising approach to target GBM. Citation Format: Natalia M. Peñaranda Fajardo, Coby Meijer, Frank A. E. Kruyt. Endoplasmic reticulum stress as possible target for therapy in glioblastoma. [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 2858.

Bortezomib attenuates palmitic acid-induced ER stress, inflammation and insulin resistance in myotubes via AMPK dependent mechanism

Bortezomib is an anti-cancer agent that induces ER stress by inhibiting proteasomal degradation. However, the effects of bortezomib appear to be dependent on its concentration and cellular context. Since ER stress is closely related to type 2 diabetes, the authors examined the effects of bortezomib on palmitic acid (PA)-induced ER stress in C2C12 murine myotubes. At low concentrations (<20nM), bortezomib protected myotubes from PA (750μM)-induced ER stress and inflammation. Either tunicamycin or thapsigargin-induced ER stress was also reduced by bortezomib. In addition, reduced glucose uptake and Akt phosphorylation induced by PA were prevented by co-treating bortezomib (10nM) both in the presence or absence of insulin. These protective effects of bortezomib were found to be associated with reduced JNK phosphorylation. Furthermore, bortezomib-induced AMPK phosphorylation, and the protective effects of bortezomib were diminished by AMPK knockdown, suggesting that AMPK activation underlies the effects of bortezomib. The in vivo administration of bortezomib at nontoxic levels (at 50 or 200μg/kg, i.p.) twice weekly for 5weeks to ob/ob mice improved insulin resistance, increased AMPK phosphorylation, reduced ER stress marker levels, and JNK inhibition in skeletal muscle. The study shows that bortezomib reduces ER stress, inflammation, and insulin resistance in vitro and in vivo, and suggests that bortezomib has novel applications for the treatment of metabolic disorders.

SGK Kinase Activity in Multiple Myeloma Cells Protects against ER Stress Apoptosis via a SEK-Dependent Mechanism.

To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib. Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.

Neuroprotective Effects of Dexmedetomidine against Thapsigargin-induced ER-stress via Activity of α&lt;sub&gt;2&lt;/sub&gt;-adrenoceptors and Imidazoline Receptors

Dexmedetomidine is a potent and highly selective α2-adrenoceptor agonist with sedative, analgesic, and sympatholytic properties, though it also exhibits some affinity for imidazoline binding sites. In addition to its sedative effects, dexmedetomidine exerts neuroprotective effects under ischemic conditions. Invasive incidents such as ischemia or hypoxia induce dysfunctions in energy production or depletion of ATP as well as accumulation and aggregation of abnormal proteins in the endoplasmic reticulum (ER), leading to an ER-stress response. In the present study, we examined whether dexmedetomidine exerts inhibitory effects on apoptosis mediated by thapsigargin-induced ER-stress in SH-SY5Y cells, and proposed a possible underlying mechanism for its neuroprotective effects. We used thapsigargin (TG) to generate an ER-stress response in SH-SY5Y cells. SH-SY5Y cells were pretreated with Dex (1–1000 nM) or receptor antagonists (atipamezole, efaroxan, BU99006, and 2’,5’-dideoxyadenosine) for 1 hour before co-treatment with 1 mM TG for 20 hours. Co-incubation with dexmedetomidine suppressed thapsigargin-induced increases in cytosolic Ca2+, caspase-4 and -3 activity, eIF2α phosphorylation, and expression of ER-stress biomarkers. Dexmedetomidine treatment also decreased cAMP levels. In the presence of atipamezole or efaroxan, but not BU99006, inhibition of eIF2α phosphorylation and CHOP expression significantly increased following treatment with dexmedetomidine in thapsigargin-treated cells. However, pretreatment with BU99006 enhanced the increase in mitochondrial membrane potential associated with dexmedetomidine treatment. The results of the present study demonstrate that dexmedetomidine at clinically relevant concentrations suppresses ER-stress-induced apoptosis in SH-SY5Y cells. Some neuroprotective effects of dexmedetomidine may be mediated by α2-adrenoceptor and I1- and I2-receptors.

Crispr Sgrnas Genome-Wide Screen Identifies the Proteasome Regulatory Subunit PSMC6 As a Bortezomib Resistance Gene in Human Multiple Myeloma Cells

Background: The proteasome inhibitor Bortezomib (BTZ) is an efficient treatment option for both, newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite the effectiveness, most patients eventually acquire drug resistance for reasons not fully understoodMaterials and methods: To better understand BTZ resistance mechanism we used a CRISPR library (GeCKO V2) targeting 19052 human genes, trying to identify genes responsible for BTZ resistance. CRISPR sgRNAs targeting the ERN1-XBP1 pathway were used as positive controls. We first infected RPMI 8226 myeloma cell line expressing Cas9 with the CRISPR library packaged into lenti-vectors and selected for resistance to BTZ. Surviving cells were subjected to next generation sequencing. Based on the initial screen results we constructed a second CRISPR sgRNA library including 31 genes, each gene targeted with four sgRNAs. After the second round screening and subsequent sequencing, we selected the top 20 genes for individual validation.Results: Proteasome regulatory gene PSMC6 was identified as the only reproducible gene conferring BTZ resistance. Interestingly, the same gene was independently found by a second group using a CRISPR approach (Sheffer M et al. ASH Abstract 273, 2014). Resistance was reproducible using a PSMC6 knockout by three individual CRISPR sgRNAs targeting exonic regions and one pair of SgRNA targeting intron region flanking exon for the deletion of exon one. PSMC6 knockout was verified by PCR and Sanger sequencing. Sensitivity to BTZ was rescued by over expression of PSMC6 cDNA in RPMI 8226 cells harboring a deletion of PSMC6 exon 1. MM cells lacking PSMC6 also developed resistance against Carfilzomib. Resistance was reproduced on a second MM cell line, KMS11. We did not see any significant difference of toxicity in PSMC6 deleted cells for other chemicals tested (tunicamycin, staurosporine, dexamethasone and melphalan). We demonstrated that the sensitivity of chymotrypsin-like activity of proteasome against BTZ was significantly reduced in cells lacking PSMC6. Consequently, MM cells without the PSMC6 gene were relatively resistant to apoptosis induced by BTZ, which was verified by Western blot for caspase 8 degradation and luminescent assay for caspase 3/7 activities. Clinically we could not correlate the PSMC6 expression level with the outcome of BTZ treatment in BTZ naive patients using publically available gene expression data. We initially used CRISPR sgRNAs targeting ER stress pathway (ERN1 and XBP1) as a positive control. However we could not derive any resistant cells from the experiment. We also could not identify any sgRNAs targeting the ERN1-XBP1 pathway from our whole exome screen and next generation sequencing. Since this contradicts published data, we decided to knockout ERN1 and XBP1 genes individually. Clones of cells with successful knockout of ERN1 in three cell lines (RPMI 8226, KMS11 and JJN3) and XBP1 in two cell lines (RPMI 8226 and KMS11) were tested for response to BTZ and Carfilzomib , however we did not find any drug response difference between the knockout and parent cells. We also found that the ERN1-XBP1 knockout cells did not show difference in response to ERN1 specific inhibitors (4u8C and STF-038010) and ER stress inducers (tunicamycin and thapsigargin) compared to parental cells.Conclusions: Human multiple myeloma cells lacking the PSMC6 gene develop significant resistance to apoptosis induced by BTZ. We have however not found a correlation of PSMC6 expression levels with outcome to BTZ treatment in BTZ naïve patients. We are therefore currently investigating the PSMC6 mutation rate in relapsed MM patients after proteasome treatment. In contrast to previous reports showing that progenitor MM cells lacing XBP-1 or ERN-1 invoked BTZ resistance, we were not able to demonstrate a change in sensitivity after full CRISPR knock out of either ERN1 or XBP1. It has long been believed that ERN1-XBP1 pathway plays an important role for MM treatment, leading to the development of ERN1 specific inhibitors. However, we demonstrated that the toxicity of two ERN1-specific inhibitors appears independent of the ERN1-XBP1 pathway. We also demonstrated that the toxicity of two important ER stress inducers, tunicamycin and thapsigargin, is independent of the ERN1-XBP1 pathway. DisclosuresStewart:Celgene: Consultancy; Oncospire Inc.: Equity Ownership; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.