Reticulum Stress Response Genes Research Articles

Interplay of toxic metal levels and endoplasmic reticulum stress gene profile in type 2 diabetes mellitus

The increasing global prevalence of type 2 diabetes mellitus (T2DM) necessitates investigating its complex etiology. This study aimed to explore the relationship between exposure to toxic metals, expression of endoplasmic reticulum stress response (ERSR) genes, and various biochemical parameters, including glycated hemoglobin (HbA1c), insulin resistance (HOMA-IR)/sensitivity (QUICKI), lipid profile, and estimated glomerular filtration rate (eGFR) in T2DM patients. T2DM patients and control subjects were matched for age, gender, and lifestyle factors. Biochemical parameters, toxic metal levels, and ERSR gene expression were analyzed using inductively coupled plasma mass spectrometry (ICPMS) and quantitative reverse transcription PCR (qRT-PCR), respectively. T2DM patients exhibit dysregulated lipid profiles and significantly higher fasting blood sugar (FBS), HbA1c, and insulin levels (all p < 0.0001). The insulin sensitivity was lower in T2DM patients (0.32 ± 0.09) than in the control group (0.35 ± 0.02, p = 0.02). Insulin resistance was significantly higher in the T2DM group (5.38 ± 3.15) than in the control group (1.98 ± 0.86, p = 0.0001). Nickel (4.75 ± 2.45 ppb, p < 0.0001) and arsenic (1.85 ± 1.78 ppb, p < 0.0001) levels were significantly elevated in T2DM patients. There was significant upregulation of ER stress genes: GRP78, CHOP, IRE1, ATF4, ATF6, and XBP1 (all p < 0.0001), while PERK was significantly down regulated (0.68-fold, p < 0.0001). Nickel levels were positively correlated with HOMA-IR (r = 0.49, p < 0.0001) and HbA1c (r = 0.35, p = 0.002). Arsenic levels were correlated with insulin (r = 0.34, p < 0.0001), insulin resistance (r = 0.51,p < 0.0001), HbA1c (r = 0.53, p < 0.0001), Arsenic levels (β = 0.37, p < 0.001), XBP1 (β = 0.36, p < 0.0001) independently associated with HbA1c.This study has revealed a significant association between arsenic exposure and the upregulation of XBP1 at the onset of T2DM. The overexpression of XBP1 and high levels of arsenic were independently associated with HbA1c and insulin resistance.

Construction of a prognostic model for colon cancer by combining endoplasmic reticulum stress responsive genes

Endoplasmic reticulum stress may affect the occurrence and development of cancer. However, its effect on the prognosis of colon cancer (CC) patients is not clear yet. Herein, based on TCGA database, we screened 15 endoplasmic reticulum stress responsive genes (ERSRGs) associated with the prognosis of CC patients by Cox regression. By LASSO and multivariate Cox regression analyses, a prognostic risk assessment model involving 12 genes (DNAJB2, EIF4A1, YPEL4, COQ10A, IRX3, ASPHD1, NTRK2, TRIM39, XBP1, GRIN2B, LRRC59, and RORC) was built. The survival curves indicated that patients in the low-risk group had good prognosis. ROC curves demonstrated a good performance of this 12-gene prognostic model, and the Riskscore could be considered as an independent prognostic factor. Patients in low-risk group benefit more from immune checkpoint inhibitor and immune checkpoint blockade (ICB) treatment. Besides, the enrichment analysis suggested a remarkable difference in Ca2+ signaling in both groups. Finally, based on the cMAP database, we identified several potential drugs that could target high-risk groups, such as Dasatinib, GNF-2, Saracatinib, and WZ-1-84. To sum up, our research constructed an ERSRGs-characteristic prognostic model. The model is a promising biomarker for prediction of clinical outcomes and immune therapy response of CC patients. SignificanceBased on the transcriptomic data of colon cancer in the TCGA database, this study screens 12 endoplasmic reticulum stress-related genes (ERSRGs), including DNAJB2, EIF4A1, YPEL4, COQ10A, IRX3, ASPHD1, NTRK2, TRIM39, XBP1, asphD1, NTRK2. GRIN2B, LRRC59, and RORC, and a prognostic model was constructed. This model can be used as a predictor of prognosis and immunotherapy response in colon cancer patients. At the same time, model-based prediction of drugs can also be a potential option for colon cancer treatment in the future.

Targeting PERK and GRP78 in colorectal cancer: Genetic insights and novel therapeutic approaches

Colorectal cancer (CRC) ranks among the leading causes of cancer-related deaths worldwide. Enhancing CRC diagnosis and prognosis requires the development of improved biomarkers and therapeutic targets. Emerging evidence suggests that the unfolded protein response (UPR) plays a pivotal role in CRC progression, presenting new opportunities for diagnosis, treatment, and prevention. This study hypothesizes that genetic variants in endoplasmic reticulum (ER) stress response genes influence CRC susceptibility. We examined the frequencies of SNPs in PERK (rs13045) and GRP78/BiP (rs430397) within a South Iranian cohort.We mapped the cellular and molecular features of PERK and GRP78 genes in colorectal cancer, observing their differential expressions in tumor and metastatic tissues. We constructed co-expression and protein-protein interaction networks and performed gene set enrichment analysis, highlighting autophagy as a significant pathway through KEGG. Furthermore, the study included 64 CRC patients and 60 control subjects. DNA extraction and genotyping were conducted using high-resolution melting (HRM) analysis. Significant differences in PERK and GRP78 expressions were observed between CRC tissues and controls. Variations in PERK and GRP78 genotypes were significantly correlated with CRC risk. Utilizing a Multi-Target Directed Ligands approach, a dual PERK/GRP78 inhibitor was designed and subjected to molecular modeling studies. Docking experiments indicated high-affinity binding between the proposed inhibitor and both genes, PERK and GRP78, suggesting a novel therapy for CRC.These findings highlight the importance of understanding genetic backgrounds in different populations to assess CRC risk. Polymorphisms in UPR signaling pathway elements may serve as potential markers for predicting CRC susceptibility, paving the way for personalized therapeutic strategies.

Pancreatic islet remodeling in cotadutide-treated obese mice

Obesity and type 2 diabetes mellitus (T2DM) cause morphofunctional alterations in pancreatic islet alpha and beta cells. Therefore, we hypothesize that the new GLP-1/Glucagon receptor dual agonist cotadutide may benefit islet cell arrangement and function. Twelve-week-old C57BL/6 male mice were fed a control diet (C, 10 % kJ fat) or a high-fat diet (HF, 50 % kJ fat) for ten weeks. Then, the animals were divided into four groups for an additional 30 days and daily treated with subcutaneous cotadutide (30 nmol/kg) or vehicle: C, CC (control+cotadutide), HF, and HFC (high-fat+cotadutide). Cotadutide led to weight loss and reduced insulin resistance in the HFC group, increasing insulin receptor substrate 1 and solute carrier family 2 gene expressions in isolated islets. Also, cotadutide enhanced transcriptional factors related to islet cell transdifferentiation, decreasing aristaless-related homeobox and increasing the paired box 4 and 6, pancreatic and duodenal homeobox 1, v-maf musculoaponeurotic fibrosarcoma oncogene family protein A, neurogenin 3, and neurogenic differentiation 1. In addition, cotadutide improved the proliferating cell nuclear antigen, NK6 homeobox 1, B cell leukemia/lymphoma 2, but lessening caspase 3. Furthermore, cotadutide mitigated the endoplasmic reticulum (ER) stress-responsive genes, reducing transcription factor 4, DNA-damage-inducible transcript 3, and growth arrest and DNA-damage-inducible 45. In conclusion, our data demonstrated significant beneficial actions of cotadutide in DIO mice, such as weight loss, glycemic control, and insulin resistance improvement. In addition, cotadutide counteracted the pathological adaptive cellular arrangement of the pancreatic islet in obese mice, improving the markers of the transdifferentiating pathway, proliferation, apoptosis, and ER stress.

First In Vivo Insights on the Effects of Tempol-Methoxycinnamate, a New UV Filter, as Alternative to Octyl Methoxycinnamate, on Zebrafish Early Development

The demand for organic UV filters as active components in sunscreen products has rapidly risen over the last century, as people have gradually realized the hazards of overexposure to UV radiation. Their extensive usage has resulted in their ubiquitous presence in different aquatic matrices, representing a potential threat to living organisms. In this context, the need to replace classic UV filters such as octyl methoxycinnamate (OMC), one of the most popular UV filters reported to be a potential pollutant of aquatic ecosystems, with more environmentally friendly ones has emerged. In this study, using zebrafish, the first in vivo results regarding the effect of exposure to tempol-methoxycinnamate (TMC), a derivative of OMC, are reported. A comparative study between TMC and OMC was performed, analyzing embryos exposed to similar TMC and OMC concentrations, focusing on morphological and molecular changes. While both compounds seemed not to affect hatching and embryogenesis, OMC exposure caused an increase in endoplasmic reticulum (ER) stress response genes, according to increased eif2ak3, ddit3, nrf2, and nkap mRNA levels and in oxidative stress genes, as observed from modulation of the sod1, sod2, gpr, and trx mRNA levels. On the contrary, exposure to TMC led to reduced toxicity, probably due to the presence of the nitroxide group in the compound's molecular structure responsible for antioxidant activity. In addition, both UV filters were docked with estrogen and androgen receptors where they acted differently, in agreement with the molecular analysis that showed a hormone-like activity for OMC but not for TMC. Overall, the results indicate the suitability of TMC as an alternative, environmentally safer UV filter.

Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress.

Abnormally high serum homocysteine levels have been associated with several disorders, including obesity, cardiovascular diseases or neurological diseases. Leptin is an anti-obesity protein and its action is mainly mediated by the activation of its Ob-R receptor in neuronal cells. The inability of leptin to induce activation of its specific signaling pathways, especially under endoplasmic reticulum stress, leads to the leptin resistance observed in obesity. The present study examined the effect of homocysteine on leptin signaling in SH-SY5Y neuroblastoma cells expressing the leptin receptor Ob-Rb. Phosphorylation of the signal transducer and activator of transcription (STAT3) and leptin-induced STAT3 transcriptional activity were significantly inhibited by homocysteine treatment. These effects may be specific to homocysteine and to the leptin pathway, as other homocysteine-related compounds, namely methionine and cysteine, have weak effect on leptin-induced inhibition of STAT3 phosphorylation, and homocysteine has no impact on IL-6-induced activation of STAT3. The direct effect of homocysteine on leptin-induced Ob-R activation, analyzed by Ob-R BRET biosensor to monitor Ob-R oligomerization and conformational change, suggested that homocysteine treatment does not affect early events of leptin-induced Ob-R activation. Instead, we found that, unlike methionine or cysteine, homocysteine increases the expression of the endoplasmic reticulum (ER) stress response gene, a homocysteine-sensitive ER resident protein. These results suggest that homocysteine may induce neuronal resistance to leptin by suppressing STAT3 phosphorylation downstream of the leptin receptor via ER stress.

Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples, and identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) response gene, XBP1, indicating the role of ER stress in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cells (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4μ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deep insights into the immune atlas in advanced BTC, which might be instrumental in exploring innovative immunotherapy strategies.

Analysis of gene expression in monocytes of immunized pigs after infection with homologous or heterologous African swine fever virus.

African swine fever is a deadly disease of pigs caused by the large DNA virus (ASFV). Despite intensive research, little is known about the molecular mechanisms of ASFV pathogenesis. Transcriptome analysis of host and viral genes in infected macrophages revealed changes in expression of genes involved in various biological processes, including immune response, inflammatory response and apoptosis. To understand the mechanisms of virus pathogenesis, we used transcriptome analysis to identify the differences in gene expression between peripheral blood monocytes (PBMCs) isolated from pigs immunized with attenuated Congo ASFV strain (KK262), and then infected in vitro with virulent homologous Congo strain (K49) or heterologous Mozambique strain (M78). We found that overexpression of IFN-γ was detected only in cells infected with M78, although the expression of interferon-stimulated genes was increased in both types of cells. In addition, up-regulation of pro-inflammatory cytokines and chemokines was found in PBMCs infected with the heterologous strain M78, in contrast to the cells infected with K49. These data may indicate the beginning of an early immune response in cells infected with a heterologous, but not homologous strain. Transcriptome analysis revealed down-regulation of genes involved in endocytosis and phagocytosis in cells infected with the K49 strain, but not in PBMCs infected with M78. On the contrary, we detected activation of endoplasmic reticulum stress response genes in cells infected with a homologous strain, but not in cells infected with a heterologous strain. This study is the first attempt to determine the differences in the response to ASF infection between homologous and heterologous strains at the cellular level. Our results showed that not only genes of the immune response, but also genes involved in endocytosis and cellular stress response may be important for the formation of cross-protective immunity. This data may be useful for vaccine development or testing of candidate vaccines.

Elongin functions as a loading factor for Mediator at ATF6α-regulated ER stress response genes

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.

Adipocyte-driven unfolded protein response is a shared transcriptomic signature of metastatic prostate carcinoma cells

A critical unknown in the field of skeletal metastases is how cancer cells find a way to thrive under harsh conditions, as exemplified by metastatic colonization of adipocyte-rich bone marrow by prostate carcinoma cells. To begin understanding molecular processes that enable tumor cells to survive and progress in difficult microenvironments such as bone, we performed unbiased examination of the transcriptome of two different prostate cancer cell lines in the absence or presence of bone marrow adipocytes. Our RNAseq analyses and subsequent quantitative PCR and protein-based assays reveal that upregulation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) genes is a shared signature between metastatic prostate carcinoma cell lines of different origin. Pathway analyses and pharmacological examinations highlight the ER chaperone BIP as an upstream coordinator of this transcriptomic signature. Additional patient-based data support our overall conclusion that ER stress and UPR induction are shared, important factors in the response and adaptation of metastatic tumor cells to their micro-environment. Our studies pave the way for additional mechanistic investigations and offer new clues towards effective therapeutic interventions in metastatic disease.

Single nucleotide polymorphisms (SNPs) in endoplasmic reticulum (ER) stress response genes to predict first-line treatment outcome in patients (pts) with metastatic colorectal cancer (mCRC): Data from the MAVERICC and FIRE-3 trials.

103 Background: ER stress, triggered by the disruption of intracellular protein homeostasis, promotes the activation of a highly conserved adaptive program called unfolded protein response (UPR). UPR signaling has been reported to contribute to tumor initiation and progression, as well as microenvironment remodeling and chemoresistance in multiple cancer types, including CRC. Several studies point to a direct role of the UPR in tumor angiogenesis and an interaction with EGFR signaling. We therefore hypothesized that genetic variants in ER stress response genes may predict first-line treatment outcome in mCRC pts. Methods: The impact on outcome of 17 functional SNPs in 8 core genes of the ER stress response pathway ( IRE1, PERK, ATF6, XBP1, CHOP, GRP78, GADD34, ATF4) was analyzed on a total of 560 pts enrolled in two independent randomized first-line trials: MAVERICC (FOLFIRI/bevacizumab, [bev] n = 163; FOLFOX6/bev, n = 161), and FIRE-3 (FOLFIRI/bev, n = 107; FOLFIRI/cetuximab [cet], n = 129). Genomic DNA from blood samples of pts was genotyped through the OncoArray, a custom array manufactured by Illumina. The association between SNPs and clinical outcomes was evaluated using Cox regression and log-rank tests. The SNP*treatment interaction was assessed in both trials. Results: IRE1 rs16947383 any T allele variant was associated with shorter progression free survival (PFS) and overall survival (OS) compared to the C/C genotype in pts treated with FOLFIRI/bev in the MAVERICC trial both in uni- and multivariate analysis: 10.5 vs 14.1 months for PFS, adjusted hazard ratio (HR) 1.83, 95% confidence interval (CI) 1.12-2.99, adjusted P-value = 0.02; 23.8 vs 38.4 months for OS, adjusted HR 2.12, 95%CI 1.1-4.09, adjusted P-value = 0.02. The association with OS was validated in the FIRE-3 FOLFIRI/bev arm where any T allele carriers showed significantly shorter OS compared to the C/C genotype in both uni- and multivariate analysis: 21.1 vs 44.2 months, adjusted HR 2.06, 95%CI 1.01-4.18, adjusted P-value = 0.05. This effect was not observed in MAVERICC FOLFOX6 and FIRE-3 cet cohorts. A significant interaction was found between IRE1 rs16947383 and treatment on OS in both trials: P = 0.04 in MAVERICC (FOLFIRI vs FOLFOX6), and P = 0.03 in FIRE-3 (bev vs cet). In the MAVERICC FOLFOX6/bev arm, XBP1 rs2239815 and PERK rs6731022 SNPs were associated with shorter PFS and OS, while GADD34 rs610308 with longer OS, in multivariable analysis ( P &lt; 0.05). Conclusions: Our results provide evidence that germline polymorphisms in ER stress response genes may have a predictive value in mCRC pts receiving first-line bev-based treatment and contribute to modulate anti-VEGF efficacy.

Salt stress induces endoplasmic reticulum stress-responsive genes in a grapevine rootstock.

Grapevines, although adapted to occasional drought or salt stress, are relatively sensitive to growth- and yield-limiting salinity stress. To understand the molecular mechanisms of salt tolerance and endoplasmic reticulum (ER) stress and identify genes commonly regulated by both stresses in grapevine, we investigated transcript profiles in leaves of the salt-tolerant grapevine rootstock 1616C under salt- and ER-stress. Among 1643 differentially expressed transcripts at 6 h post-treatment in leaves, 29 were unique to ER stress, 378 were unique to salt stress, and 16 were common to both stresses. At 24 h post-treatment, 243 transcripts were unique to ER stress, 1150 were unique to salt stress, and 168 were common to both stresses. GO term analysis identified genes in categories including 'oxidative stress', 'protein folding', 'transmembrane transport', 'protein phosphorylation', 'lipid transport', 'proteolysis', 'photosynthesis', and 'regulation of transcription'. The expression of genes encoding transporters, transcription factors, and proteins involved in hormone biosynthesis increased in response to both ER and salt stresses. KEGG pathway analysis of differentially expressed genes for both ER and salt stress were divided into four main categories including; carbohydrate metabolism, amino acid metabolism, signal transduction and lipid metabolism. Differential expression of several genes was confirmed by qRT-PCR analysis, which validated our microarray results. We identified transcripts for genes that might be involved in salt tolerance and also many genes differentially expressed under both ER and salt stresses. Our results could provide new insights into the mechanisms of salt tolerance and ER stress in plants and should be useful for genetic improvement of salt tolerance in grapevine.

C 60 fullerene nanoparticle prevents β-amyloid peptide induced cytotoxicity in neuro 2A cells

Oxidative stress, which is an early determinant of Alzheimer's disease (AD), is involved in mediating neuronal apoptosis in Aβ-induced cell death. C 60 fullerenes are known to behave like a "radical sponge" as they can sponge up free radicals and act more effectively than other antioxidants. PEG-C 60-3, a C 60 fullerene derivative, was investigated against β-amyloid (Aβ) 25-35-induced toxicity toward Neuro-2A cells in this study. PEG-C 60-3 reduced Aβ 25-35-induced cytotoxicity, which showed an increasing cell viability with C 60 and Aβ 25-35 co-treated cells. Moreover, the intracellular reactive oxygen species (ROS) accumulation caused by Aβ-treated Neuro-2A cells was reduced by PEG-C 60-3 co-treatment. Microarray for the analysis of gene expressions was investigated. Endoplasmic reticulum (ER) stress responsive genes, ion-channel, cell-cycle and anti-oxidant related cell responses were found to be associated with C 60 protective mechanism against Aβ 25-35 treatment. The results offered new comprehension into the possible pathway of Aβ 25-35 gene expression and C 60 protective mechanism. With the understanding of the roles of Aβ and C 60 in cells, we can hopefully provide insight on therapeutic design using C 60 fullerene nanoparticles against Aβ-associated diseases.

Nrf2 Signaling in Sodium Azide-Treated Oligodendrocytes Restores Mitochondrial Functions.

Mitochondrial dysfunctions mark a critical step in many central nervous system (CNS) pathologies, including multiple sclerosis (MS). Such dysfunctions lead to depolarization of mitochondrial membranes and imbalanced redox homeostasis. In this context, reactive oxygen species (ROS) are potentially deleterious but can also act as an important signaling step for cellular maintenance. The transcription factor nuclearfactor (erythroid-derived2)-like 2 (Nrf2), the key regulator in the cellular oxidative stress-response, induces a battery of genes involved in repair and regeneration. Here, we investigated the relevance of Nrf2 signaling for the prevention of cellular damage caused by dysfunctional mitochondria. We employed sodium azide (SA) as mitochondrial inhibitor on oligodendroglial OliNeu cells in vitro, and the cuprizone model with wild type and GFAP-Cre+::Keap1loxP/loxP mice to induce mitochondrial defects. The importance of Nrf2 for cellular functions and survival after SA treatment was elucidated by in vitro knockdown experiments with shRNA directed against Nrf2 and its inhibitor Keap1 as well as by methysticin treatment. Metabolic activity, cytotoxicity, and depolarization of the mitochondrial membrane were analyzed after SA treatment. The expression of Nrf2 target genes as well as endoplasmic reticulum stress response genes was additionally measured by real-time PCR (in vitro) and PCR gene arrays (in vivo). Treatment of OliNeu cells with SA resulted in significant depolarization of the mitochondrial membrane, decreased metabolic activity, and increased cytotoxicity. This was partly counteracted in Nrf2-hyperactivated cells and intensified in Nrf2-knockdown cells. Our studies demonstrate a keyrole of Nrf2 in maintaining cellular functions and survival in the context of mitochondrial dysfunction.

Lysine demethylase inhibition protects pancreatic β cells from apoptosis and improves β-cell function

Transcriptional changes control β-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs) protects β cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate β-cell fate in response to inflammatory stress.Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting a possible role in inflammation-induced β-cell destruction. Inhibition of KDM6 demethylases using the selective inhibitor GSK-J4 protected insulin-producing cells and human and mouse islets from cytokine-induced apoptosis by blunting nuclear factor (NF)-κB signaling and endoplasmic reticulum (ER) stress response gene expression. GSK-J4 furthermore increased expression of insulin gene and glucose-stimulated insulin secretion. Expression of genes regulating purinergic and cytokine ligand-receptor interactions was downregulated following GSK-J4 exposure, while expression of genes involved in cell maintenance and survival was upregulated. These data suggest that KDMs are important regulators of inflammation-induced β-cell dysfunction and death.

Abstract 4501: CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival

Abstract CEBPD (C/EBP delta) is a member of the CCAAT-enhancer binding protein (C/EBP) family of transcription factors characterized by a b-Zip domain that mediates dimerization and DNA binding. CEBPD is induced in response to acute stressors such as cytokine stimulation, bacterial lipopolysaccharide (LPS), corticosteroids, radiation and hypoxia. We have previously reported that CEBPD has dual functions in breast cancer by both attenuating or enhancing oncogenic pathways depending on context (Balamurugan and Sterneck, 2013, Mendoza-Villanueva et al., 2016). Recent studies reveal that elevated Endoplasmic Reticulum (ER) stress is associated with the pathology of several diseases including cancer. Limiting supply of nutrients and oxygen in growing tumor cells disrupts the protein folding homeostasis resulting in activation of the unfolded protein response (UPR). The UPR includes pathways that support adaptation to stress, and that are also implicated in promoting malignant features and therapy resistance in breast cancer. Hence, we investigated a possible role for CEBPD in this stress response pathway. Using breast cancer cell lines as model systems, we found that CEBPD is dynamically regulated in response to chemical and physiological inducers of ER stress. Kinetic analyses indicate that CEBPD induction is concurrent with the activation of the UPR effectors XBP1s and ATF4 suggesting a potential role in stress adaptation. Examination of the global transcriptional prolife of the Thapsigargin-induced stress response in MDA MB-231 cells indicated that CEBPD activates genes involved in cytokine and chemokine production/signaling, immune cell recruitment as well as angiogenesis pathways. While XBP1s has been previously ascribed to activating these pathways, the levels of activated XBP1 were not affected by CEBPD silencing. This is the first report of a similar role for CEBPD in the ER stress response and warrants the investigation of possible co-operation of CEBPD and XBP1 in the UPR pathway. Further, we show that knock down of CEBPD in cancer cells increased PARP cleavage and susceptibility to ER stress induced cell death. CEBPD deficient cells also displayed reduced LC3II accumulation, suggesting impaired autophagy induction in response to ER stress. Taken together, our data suggests that CEBPD is a novel proximal effector of the ER stress response in breast cancer cells. Activation of this transcription factor regulates tumor promoting inflammatory and angiogenic signaling pathways. Additionally, CEBPD expression confers a survival advantage to cancer cells encountering ER stress. Therefore, we propose that targeting CEBPD may sensitize cancer cells to ER stress inducing therapeutics. Citation Format: Namratha Sheshadri, Shikha Sharan, Esta Sterneck. CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2017-4501

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the “Warburg effect”), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4-induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species

Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10μM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites.

Prenatal methyl-donor supplemented diet increases endoplasmic reticulum stress and inflammation in adipose tissue CD11b+ cells in F1 mice

Abstract The risk for development of metabolic and cardiovascular diseases in later life is increasing, as the population is becoming overweight and obese at earlier ages. Early-life nutritional programming influences late-life disease susceptibility across generations, in part through epigenetic mechanisms including DNA methylation. Using mouse models, it has been shown that prenatal diet rich in methyl donors (L-methionine, vitamin B-12, folic acid, choline, betaine, zinc) can increase airway reactivity but improves cardiovascular disease outcomes in F1 mice. Here, we used a prenatal diet enriched with methyl-donors to study the impact of the prenatal diet in the context of diet-induced obese F1 C57Bl/6 mice fed a 40% high-fat diet (HFD) for 16 weeks. F1 mice exposed to the prenatal methyl donor supplemented diet have increased number of adipose tissue ‘crown-like structures’ consisting of primarily CD11b+ cells. The prenatal diet was found to increase the expression of adipose tissue endoplasmic reticulum (ER) stress response genes Chop, Bip, and Atf4 in HFD F1 mice. Obesity-induced ER stress has been linked to adipose tissue chronic inflammation. We have previously established that ER stress contributes to aging adipose tissue macrophage inflammation. Interestingly, Mcp-1 and Tnfα gene expression were found to be increased in the CD11b+ cells of F1 mice adipose tissue. The results strongly support an important role of prenatal methyl-donors exposure in modulating diet-induced obesity ER stress response and inflammation in offspring.

SIRT3 Overexpression Attenuates Palmitate-Induced Pancreatic β-Cell Dysfunction.

Abnormally high levels of circulating free fatty acids can lead to pancreatic islet β-cell dysfunction and apoptosis, contributing to β-cell failure in Type 2 diabetes. The NAD+-dependent protein deacetylase Sirtuin-3 (SIRT3) has been implicated in Type 2 diabetes. In this study, we tested whether SIRT3 overexpression affects palmitate-induced β-cell dysfunction in cells of line NIT1, which are derived from mouse pancreatic β-cells. Two different lengths of SIRT3 were overexpressed: full length SIRT3 (SIRT3LF), which was preferentially targeted to mitochondria and partially to the nucleus, and its N-terminal truncated form (SIRT3SF), which was located in the nucleus and cytoplasm. Overexpression of SIRT3LF and SIRT3SF using an adenoviral system alleviated palmitate-induced lipotoxicity such as reduction of cell viability and mitogen-activated protein kinase (MAPK) activation. Chronic exposure to low concentrations of palmitate suppressed glucose-stimulated insulin secretion, but the suppression was effectively reversed by overexpression of SIRT3LF or SIRT3SF. The mRNA levels of the endoplasmic reticulum (ER) stress responsive genes ATF4, GRP94 and FKBP11 were increased by palmitate treatment, but the increases were completely inhibited by SIRT3LF overexpression and less effectively inhibited by SIRT3SF overexpression. This result suggests that overexpression of SIRT3 inhibits induction of ER stress by palmitate. Collectively, we conclude that overexpression of SIRT3 alleviates palmitate-induced β-cell dysfunction.