Endoplasmic Reticulum Stress Signaling Research Articles

Jintiange proteins promote osteogenesis and inhibit apoptosis of osteoblasts by enhancing autophagy via PI3K/AKT and ER stress pathways.

Tiger bone, which had long been used in traditional Chinese medicine, had the action of removing wind and alleviating pain, strengthening the sinews and bones, and often used to treat bone impediment, and atrophic debility of bones in TCM clinical practice. As a substitute of natural bone tiger, artificial tiger bone Jintiange (JTG), has been approved by the State Food and Drug Administration of China for relief the symptom of osteoporosis, such as lumbago and back pain, lassitude in loin and legs, flaccidity and weakness legs, and walk with difficulty based on TCM theory. JTG has similar chemical profile to natural tiger bone, and contains mineral substance, peptides and proteins, and has been shown to protect bone loss in ovariectomized mice and exert the regulatory effects on osteoblast and osteoclast activities. But how the peptides and proteins in JTG modulate bone formation remains unclear. To investigate the stimulating effects of JTG proteins on osteogenesis and explore the possible underlying mechanisms. JTG proteins were prepared from JTG Capsules by extracting calcium, phosphorus and other inorganic elements using SEP-PaktC18 desalting column. MC3T3-E1 cells were treated with JTG proteins to evaluate their effects and explore the underlying mechanisms. Osteoblast proliferation was detected by CCK-8 method. ALP activity was detected using a relevant assay kit, and bone mineralized nodules were stained with alizarin red-Tris-HCl solution. Cell apoptosis was analyzed by flow cytometry. Autophagy was observed by MDC staining, and autophagosomes were observed by TEM. Nuclear translocations of LC3 and CHOP were detected by immunofluorescence and observed under a laser confocal microscope. The expression of key proteins related to osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways was analyzed by Western Blot analysis. JTG proteins improved osteogenesis as evidenced by the alteration of proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, inhibited their apoptosis, and enhanced autophagosome formation and autophagy. They also regulated the expression of key proteins of PI3K/AKT and ER stress pathways. In addition, PI3K/AKT and ER stress pathway inhibitors could reverse the regulatory effects of JTG proteins on osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways. JTG proteins increased the osteogenesis and inhibited osteoblast apoptosis by enhancing autophagy via PI3K/AKT and ER stress signaling pathways.

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to osteocyte death induced by orthodontic compressive force.

During orthodontic tooth movement (OTM), osteocytes, the most mechanosensitive cells in alveolar bone, suffer the heavy orthodontic force and initiate alveolar bone resorption on the compression side. However, the inherent mechanisms of compressive force-induced osteocyte death are not fully understood. In this study, we established an OTM model on Sprague-Dawley rats by inserting coil springs to investigate osteocyte damage on the compression side of alveolar bone. We then applied compressive force on the MLO-Y4 osteocyte-like cell line in vitro to explore whether the reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ERS) pathway is involved in compressive force-induced osteocyte death. We found that the orthodontic force caused apparent alveolar bone loss, osteocyte death, and elevated serum sclerostin and receptor activator of NF-κB ligand (RANKL) levels in rats. In vitro, compressive force inhibited cell viability but increased the LDH leakage and loss of mitochondrial membrane potential in MLO-Y4 cells. Simultaneously, it activated protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 (eIF2α), and their downstream pro-apoptotic ERS signaling proteins and caused significant osteocyte apoptosis, which can be blocked by ERS inhibitor salubrinal. Moreover, the compressive force elevated intracellular ROS levels, while the ROS scavenger N-acetyl-L-cysteine (NAC) alleviated ERS and apoptosis in loaded osteocytes. These results suggest that the orthodontic compressive force induced osteocyte apoptosis via the ROS-mediated ERS pathway. This study first proposes the ERS pathway as a new potential pathway for regulating the rate of OTM based on osteocyte death. RESEARCH HIGHLIGHTS: The orthodontic force increases osteocyte death in rat alveolar bone. The compressive force causes osteocyte apoptosis by the endoplasmic reticulum stress (ERS) pathway in vitro. The ROS scavenger NAC blocked compressive force-induced ERS and osteocyte apoptosis.

The plasma membrane-associated transcription factor NAC091 regulates unfolded protein response in Arabidopsis thaliana

Adverse environmental stresses may cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), and the unfolded protein response (UPR) pathway is initiated to mitigate the ER stress. Previous studies demonstrate that NAC062, a plasma membrane-associated transcription factor, plays important roles in promoting cell survival under ER stress conditions in Arabidopsis thaliana. In this study, we identified another plasma membrane-associated transcription factor, NAC091 (also known as ANAC091/TIP), as an important UPR mediator. ER stress induces the expression of NAC091, which is mainly dependent on the ER stress regulators bZIP60 and bZIP28. In addition, NAC091 has transcriptional activation activity, and the truncated form of NAC091 devoid of the C-terminal transmembrane domain (TMD) forms a homodimer in the nucleus. Under ER stress conditions, NAC091 relocates from the plasma membrane to the nucleus and regulates the expression of canonical UPR genes involved in cell survival. Further, the loss-of-function mutant of NAC091 confers impaired ER stress tolerance. Together, these results reveal the important role of NAC091 in ER stress response in Arabidopsis, and demonstrate that NAC091 relays the ER stress signal from the plasma membrane to the nucleus to alleviate ER stress and promote cell survival in plants.

Protective effect of dexmedetomidine pre treatment against myocardial ischemia reperfusion injury in rats

Purpose: To determine the protective effect of dexmedetomidine pre-treatment against myocardial ischemia-reperfusion injury (MRI), and the involvement of ER stress and protein kinase R-like ER enzyme (PERK) signaling pathway in the process.
 Methods: Sixty male Sprague-Dawley (SD) rats were assigned to three groups: sham, model (ischemia-reperfusion), and dexmedetomidine pre-treatment groups. Myocardial infarction area was measured using 2,3,5-triphenyl tetrazolium chloride (TTC) method. Changes in heart structure were examined using H & E, while myocardial cell apoptosis was determined with TUNEL assay. The protein expression levels of CHOP, glucose regulatory protein 78, ATF4, eukaryotic translation initiation factor 2-α (elf2-α), and PERK were assayed with Western blotting.
 Results: RPP, myocardial infarction area, myocardial cell apoptosis rate, and the expression levels of CHOP, GRP78, ATF4, elf2-α, and PERK were significantly raised, relative to sham. The structure of myocardial cells in sham-operated rats was intact and orderly. In contrast, the arrangement of myocardial cells in the model group was disordered, and the myofibrils were broken or wavy. However, the arrangement of myocardial cells in the dexmedetomidine group was significantly improved, when compared with the model group.
 Conclusion: Pretreatment with dexmedetomidine may exert a protective effect against MRI through suppression of ER stress and PERK signaling pathway. Further preclinical studies would be required to validate these findings.

GLP-1 receptor agonists as promising disease-modifying agents in WFS1 spectrum disorder.

WFS1 spectrum disorder (WFS1-SD) is a rare monogenic neurodegenerative disorder whose cardinal symptoms are childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus, and neurological signs ranging from mild to severe. The prognosis is poor as most patients die prematurely with severe neurological disabilities such as bulbar dysfunction and organic brain syndrome. Mutation of the WFS1 gene is recognized as the prime mover of the disease and responsible for a dysregulated ER stress signaling, which leads to neuron and pancreatic β-cell death. There is no currently cure and no treatment that definitively arrests the progression of the disease. GLP-1 receptor agonists appear to be an efficient way to reduce elevated ER stress in vitro and in vivo, and increasing findings suggest they could be effective in delaying the progression of WFS1-SD. Here, we summarize the characteristics of GLP-1 receptor agonists and preclinical and clinical data obtained by testing them in WFS1-SD as a feasible strategy for managing this disease.

Fisetin derivatives exhibit enhanced anti-inflammatory activity and modulation of endoplasmic reticulum stress.

Inflammation and endoplasmic reticulum (ER) stress are often hand in hand in the context of chronic disease. Both are activated upon perceived disturbances in homeostasis, being deleterious when intensely or chronically activated. Fisetin (FST) is a dietary flavonol that is known to possess multiple relevant bioactivities, raising the question of its potential health benefits and even its use in novel pharmacological approaches against ER stress and inflammation. To attain this prospect, some limitations to this molecule, namely its poor bioavailability and solubility, must be addressed. In an attempt to improve the biological properties of the parent molecule, we have synthesized a set of FST derivatives. These new molecules were tested along with the original compound for their ability to mitigate the activation of the signaling pathways underlying inflammation and ER stress. By reducing LPS-induced nuclear factor-kappa B (NF-κB) activation, cytokine release, inflammasome activation and reactive oxygen species (ROS) generation, FST has proven to be effective against the onset of inflammation. The molecule also decreases the activation of the unfolded protein response (UPR), as evidenced by the reduced expression of relevant UPR-related genes upon ER stress induction. Some of the tested derivatives are novel inhibitors of targets associated to inflammation and ER stress signaling, in some cases more potent than the parent compound. Furthermore, the reduced cytotoxicity of some of these molecules enabled the use of higher concentrations than that of FST, resulting in the observation of enhanced bioactivities.

WED-541 - The role of chronic ER stress and calcium signalling in NASH associated carcinogenesis

WED-541 - The role of chronic ER stress and calcium signalling in NASH associated carcinogenesis

Notch3/Hes5 Induces Vascular Dysfunction in Hypoxia-Induced Pulmonary Hypertension Through ER Stress and Redox-Sensitive Pathways.

Notch3 (neurogenic locus notch homolog protein 3) is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development. Studies were performed in TgNotch3R169C mice (harboring gain-of-function [GOF] Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca2+ transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor). Hypoxia induced a more severe PH phenotype in TgNotch3R169C mice versus controls. TgNotch3R169C mice exhibited enhanced Notch3 activation and expression of Notch3 targets Hes Family BHLH Transcription Factor 5 (Hes5), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC (soluble guanylyl cyclase)/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice. Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.

Salivary proteins offer insights into keratinocyte death during aphthous stomatitis. A case-crossover study

BackgroundThe death of oral keratinocytes is a crucial step in the emergence of recurrent aphthous stomatitis (RAS, also known as aphthae or aphthous ulcers). Since there are no experimental models available to research aphthous ulcers, little is understood about this process. We hypothesize that saliva can be a data bank of information that offers insights on epithelial damage.MethodsIn this case-crossover study, we assessed the salivary proteome of patients with RAS (n = 36) in the presence and absence of ulcers using discovery proteomics and bioinformatics. Additionally, we contrasted these patterns with those of healthy individuals (n = 31) who had no prior aphthous ulceration.ResultsSalivary proteome showed that during the ulcerative phase, controlled cell death was downregulated. Due to its ability to distinguish between individuals with and without ulcers, the ATF6B protein raises the possibility that endoplasmic reticulum (ER) stress is responsible for the damage that leads to the death of oral keratinocytes. The high abundance of TRAP1 and ERN1 matches with this biological discovery. The type of death is immunogenic, according to the functional data found in a cell death database.ConclusionWe identified a cellular process that can lead to the death of oral keratinocytes in the etiopathogenesis process of RAS. Future studies should be conducted to identify what is responsible for the increase in ER stress signaling that would lead to an anti-cell death response.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress.

Cryptotanshinone (CPT) has wide biological functions, including anti-oxidative, antifibrosis, and anti-inflammatory properties. However, the effect of CPT on hepatic fibrosis is unknown. To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action. Hepatic stellate cells (HSCs) and normal hepatocytes were treated with different concentrations of CPT and salubrinal. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to measure apoptosis and cell cycle arrest. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress (ERS) signaling pathway related molecules, respectively. Carbon tetrachloride (CCL4) was used to induce in vivo hepatic fibrosis in mice. Mice were treated with CPT and salubrinal, and blood and liver samples were collected for histopathological examination. We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro. CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs. Furthermore, we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers (CHOP and GRP78) and activating ERS pathway molecules (PERK, IRE1α, and ATF4), which were inhibited by salubrinal. Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model. CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway, which represents a promising strategy for treating hepatic fibrosis.

Free fatty acid exacerbates diabetic kidney injury by suppressing autophagy-lysosomal function via ER stress

Diabetic kidney disease (DKD), the leading cause of chronic kidney disease, is characterized by accumulation of renal tubule free fatty acids (FFAs) and triglyceride (TG)-enriched lipid droplets (LDs) and cell death. The present study aimed to investigate how FFA or TG induces renal tubular cell apoptosis, thereby contributing to the progression of DKD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated ER stress and organelle dysfunction in tubular cell injury. Six-week-old wild-type (WT) and eNOS-deficient C57BL/6J mice were placed on normal diet (ND, 5% fat calories) or high-fat diet (HFD, 42% fat calories) for two weeks, and then rendered diabetic with alloxan. Alloxan injection resulted in equivalent hyperglycemia in ND and HFD-fed WT and eNOS-deficient mice. However, a massive increase in urinary albumin was observed in HFD plus alloxan-treated eNOS-deficient mice, which was accompanied by excessive accumulation of neutral lipids in damaged proximal tubules. Moreover, in the diabetic eNOS-deficient kidneys, HFD feeding resulted in suppression of autophagy-lysosomal function and increase in tubular cell apoptosis. Cell studies revealed that saturated FFA palmitate but not unsaturated oleate induced eukaryotic initiation factor (eIF)2α phosphorylation, and then stimulated the activating transcription factor (ATF)4 and C/EBP homologous protein (CHOP), which, in turn, impaired autophagy-lysosomal function, leading to cell apoptosis. In addition, we observed a dramatic upregulation of LD-associated protein ADRP expression and TG-enriched LD formation in oleate-treated renal tubular cells, which was not apparent in palmitate-treated ones. Together, this study demonstrates that accumulation of renal FFA, rather than TG, plays a crucial role in the pathogenesis of diabetic kidney injury by suppressing autophagy-lysosomal function through ER stress signaling. This work was supported by the National Institutes of Health Grants SC1DK112151 and S21MD000101. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Palmitate alters miR-2137 and miR-503-5p to induce orexigenic Npy in hypothalamic neuronal cell models: Rescue by oleate and docosahexaenoic acid.

MicroRNAs (miRNAs) are short noncoding RNA implicated in the pathogenesis of obesity. One cause of obesity is excess exposure to the saturated fatty acid palmitate that can alter miRNA levels in the periphery. Palmitate also promotes obesity by acting on the hypothalamus, the central coordinator of energy homeostasis, to dysregulate hypothalamic feeding neuropeptides and induce ER stress and inflammatory signaling. We hypothesized that palmitate would alter hypothalamic miRNAs that control genes involved in energy homeostasis thereby contributing to the obesity-promoting effects of palmitate. We found that palmitate upregulated 20 miRNAs and downregulated six miRNAs in the orexigenic NPY/AgRP-expressing mHypoE-46 cell line. We focused on delineating the roles of miR-2137 and miR-503-5p, as they were strongly up- and downregulated by palmitate, respectively. Overexpression of miR-2137 increased Npy mRNA levels and downregulated Esr1 levels, while increasing C/ebpβ and Atf3 mRNA. Inhibiting miR-2137 had the opposite effect, except on Npy, which was unchanged. The most downregulated miRNA by palmitate, miR-503-5p, negatively regulated Npy mRNA levels. Exposure to the unsaturated fatty acids oleate or docosahexaenoic acid completely or partially blocked the effects of palmitate on miR-2137 and miR-503-5p as well as Npy, Agrp, Esr1, C/ebpβ and Atf3. MicroRNAs may therefore contribute to palmitate actions in dysregulating NPY/AgRP neurons. Effectively combating the deleterious effects of palmitate is crucial to help prevent or reduce the impact of obesity.

The key mediators involved in myocardial endoplasmic reticulum stress induced by ischaemia reperfusion injury in rats

Objective: Endoplasmic reticulum stress (ERS) occurs throughout the pathological process of myocardial ischaemia reperfusion injury (MIRI); however, the key mediators involved in the biological processes of ERS or that can influence the outcomes of ischaemia reperfusion injury (IRI) are unclear. To identify the differentially expressed genes (DEGs) encoding promising mediators in rat hearts in the context of MIRI, gene profiles were mined through transcriptome analysis. Methods: By searching the Gene Expression Omnibus (GEO) database, a suitable GEO dataset was identified in the context of MIRI with 24 h reperfusion after 45 min ischaemia in rats. DEGs were extracted and analysed with R studio, and the properties of those DEGs were elucidated by utilizing protein‒protein-interaction (PPI), Cytoscape, GO and KEGG bioinformatics techniques. The potential hub genes in the first cluster of the DEG dataset were retrieved and verified by basal experiments. Results: By searching the target dataset GSE122020, 1647 mRNAs, including 790 upregulated and 857 downregulated genes, were found in the datasets with criteria of | fold change ≥ 1.2 and p value <0.05. The upregulated and downregulated genes were primarily involved in the “positive regulation of cytokine production”, “regulation of inflammatory response” and “response to pheromone” biological processes. Most of them were mainly correlated with the MAPK, Ras and calcium signalling pathways. In particular, 15 DEGs were found to be involved in the ERS signalling pathways, including Bfar, Itpr1, Srpx, Hspa1b, Usp25, Jun, Casp12, Eif2ak2, Casp4, Trim25, Ifng, Atf3, Hspa1a, Ppp1r15a, and Creb3. Through analysis with the MCODE plugin in Cytoscape, six genes, Atf3, Ppp1r15a, Casp12, Eif2ak2, Ifng, and Hspa1a, were identified as hub genes by the K-means algorithm from Cluster 1. In the in vivo MIRI model, the six genes mentioned above exhibited significant mRNA overexpression in the left anterior descending coronary artery ligation (LAD) model in rats. Conclusions: Altogether, the present study identified six potential DEGs involved in ERS induced by MIRI, which could be therapeutic targets in dealing with IRI-related ERS.

Unfolding the Interactions between Endoplasmic Reticulum Stress and Oxidative Stress.

Oxidative stress is caused by an imbalance in cellular redox state due to the accumulation of reactive oxygen species (ROS). While homeostatic levels of ROS are important for cell physiology and signaling, excess ROS can induce a variety of negative effects ranging from damage to biological macromolecules to cell death. Additionally, oxidative stress can disrupt the function of redox-sensitive organelles including the mitochondria and endoplasmic reticulum (ER). In the case of the ER, the accumulation of misfolded proteins can arise due to oxidative stress, leading to the onset of ER stress. To combat ER stress, cells initiate a highly conserved stress response called the unfolded protein response (UPR). While UPR signaling, within the context of resolving ER stress, is well characterised, how UPR mediators respond to and influence oxidative stress is less defined. In this review, we evaluate the interplay between oxidative stress, ER stress and UPR signaling networks. Specifically, we assess how UPR signaling mediators can influence antioxidant responses.

Abstract LB165: A novel PDX-based 2-stage screening platform can identify active drug combinations against KRAS-mutated colorectal cancer

Abstract Introduction: Targeting RASmut colorectal cancers (CRC) remains challenging and targeting has focused on the RAS-MAPK cascade. MEK inhibitors for KRASmut tumors were not successful in vivo or in clinic as they slowed tumor growth, but did not cause regression at clinically achievable doses, however, potential for synergy has been shown. In vivo modeling commonly guides decisions to advance to the clinic but these studies are heterogeneously conducted, with rare use of statistically justified sample sizes or prespecified alternate hypotheses. The purpose of this study was to develop a robust PDX screening platform for pre-clinical evaluation of combination strategies, and pilot it with MEK inhibitor combinations. Methods: The screening model was based on Simon's two-stage design, with alternate hypothesis of 30% response rate (null hypothesis of 5% response rate). Stage 1 involved testing drug combinations against 5 KRASmut PDX models. Drug combinations that demonstrated efficacy in Stage 1 (≥10% decrease in tumor volume at Day 22 post-treatment) were tested in Stage 2 against an additional 13 PDX models. The null hypothesis was rejected if ≥3/18 PDX models showed response. This design yields a type I error rate of 0.05 and power of 0.8. Twenty RAS-MAPK cascade inhibitor-based combinations were tested. RP2D drug doses were used. Results: In Stage 1, MEKi plus CDK4/6i combination demonstrated efficacy in 3/5 models. This effect was sustained in Stage 2 (9/13 models with ≥10% decrease in tumor volume) for a total of 12/18. MEKi/BETi was next best (total 4/18 models). MEKi/PARPi and MEKi/ERKi - 3/18. Two combinations (MEKi/TKi and MEKi/AKTi) passed Stage 1, but failed in Stage 2 (2/18 total). Other combinations did not meet efficacy threshold in Stage 1 and were deemed inactive. In an expansion study of various RASmut PDX models (6 codon 12/13 KRASmut, 5 codon 61/146 RASmut and 4 RASwt), trametinib/palbociclib differentially attenuated tumor growth relative to untreated controls and caused tumor regression in all tested PDX models. Protein expression analysis (RPPA) demonstrated increases in cell cycle regulators (Cyclin-B1, FOXM1, PLK1) in non-responders, and higher pMEK and FOXO3 protein levels in responders. Combination treatment resulted in decreased expression of MAPK/ERK and CDK4/6 pathway-related proteins. mRNA expression analysis (GSEA) revealed that MEKi-resistant PDX models were enriched in cell cycle, ERK, mTORC1, ER stress and DDR signaling pathways. Primary drug combination toxicity (weight loss) was most pronounced with trametinib/palbociclib compared to tram or palbo alone. Using in vivo dose/response matrix screening, we maintained >90% efficacy while significantly reducing toxicity (decreasing weight loss from >15% to <10%) by decreasing palbociclib dose and maintaining tram dose intensity. Conclusions: The two-stage screening design demonstrated efficacy, time and cost-efficiency in drug combination screening. Efficacy of MEKi/CDK4/6i, MEKi/PARPi and MEKi/BETi combinations in RASmut CRCs was confirmed. These combinations are now being explored in prospective clinical trials. Citation Format: Alexey Sorokin, Preeti Kanikarla Marie, Fengqin Gao, Zhensheng Liu, David Menter, Scott Kopetz. A novel PDX-based 2-stage screening platform can identify active drug combinations against KRAS-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB165.

The apoptotic effects of soybean agglutinin were induced through three different signal pathways by down-regulating cytoskeleton proteins in IPEC-J2 cells

Soybean agglutinin (SBA) is a main anti-nutritional factor in soybean. SBA exhibits its anti-nutritional functions by binding to intestinal epithelial cells. Keratin8 (KRT8), Keratin18 (KRT18) and Actin (ACTA) are the representative SBA-specific binding proteins. Such cytoskeletal proteins act a crucial role in different cell activities. However, limited reports reveal what the signal transduction pathway of apoptosis caused by SBA when binding to KRT8, KRT18 and ACTA. We aimed to evaluate the effects of SBA on cell apoptosis and the expression of the cytoskeletal protein (KRT8, KRT18 and ACTA), reveal the roles of these cytoskeletal proteins or their combinations on SBA-induced cell apoptosis in IPEC-J2 cell line, evaluate the influences of SBA on the mitochondria, endoplasmic reticulum stress and death receptor-mediated apoptosis signal pathway and to show the roles of KRT8, KRT18 and ACTA in different apoptosis signal pathways induced by SBA. The results showed that SBA induced the IPEC-J2 cell apoptosis and decreased the mRNA expression of KRT8, KRT18 and ACTA (p < 0.05). The degree of effect of three cytoskeleton proteins on cell apoptosis was ACTA > KRT8 > KRT18. The roles of these three cytoskeletal proteins on IPEC-J2 apoptotic rates had a certain accumulation effect. SBA up-regulated mitochondrial fission variant protein (FIS1) and fusion protein (Mfn2) promoted CytC and AIF in mitochondria to enter the cytoplasm, activated caspase-9 and caspase-3, damaged or declined mitochondrial function and reduced ATP synthesis (p < 0.05). Also, SBA up-regulated the expression of GRP78, XBP-1, eIF2α, p-eIF2α and CHOP (p < 0.05), down-regulated the expression level of ASK1 protein (p < 0.05). SBA led to the recruitment of FADD to the cytoplasmic membrane and increased the expression of FasL, resulting in caspase-8 processing. SBA up-regulated the expression level of Bax protein and decreased cytosolic Bcl-2 and Bid (p < 0.05). In addition, there was a significant negative correlation between the gene expression of cytoskeleton proteins and apoptosis, as well as the expression of key proteins of apoptosis-related signal transduction pathways. In conclusion, SBA induced the activation of the mitochondria, endoplasmic reticulum stress and the death receptor-mediated apoptosis signal pathway and the crosstalk between them. The effect of SBA on these three pathways was mainly exhibited via down-regulation of the mRNA expression of the three cytoskeletal expressions. This study elucidates the molecular mechanism and signaling pathway of SBA that lead to apoptosis from the perspective of cell biology and molecular biology and provides a new perspective on the toxicity mechanism of other food-derived anti-nutrients, medical gastrointestinal health and related cancer treatment.

Abstract 3247: Combination therapy using PERK and PD1/PD-L1 inhibitors reprograms tumor associated macrophages and reduces tumor burden

Abstract Background: The role of endoplasmic reticulum (ER) stress is profound in increasing malignant activity of neoplastic tumor cells and has been recently demonstrated to also increase immunosuppressive activity of tumor-associated macrophages (TAMs). Immunosuppressive TAM activity is thought to underly resistance to PD-1/PD-L1-targeted immune checkpoint inhibitors used for cancer therapy. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is a rate-limiting enzyme central to the ER-stress response mechanism that transduces ER-stress signals in cells to regulate protein synthesis, cell death, and cell survival. We hypothesized that inhibition of PERK would improve responses to ICI therapy by reprogramming TAM from immunosuppressive to immunoactivating cells. Methods: To investigate if ER-stress underlies resistance to PD-1/PD-L1 targeted therapies, we utilized ex vivo assays to investigate ER-stress regulation of macrophage phenotype, and in vivo syngeneic murine models of melanoma growth, with HC-5404 (PERKi), a selective and potent first-in-human small molecule PERK inhibitor currently in a phase 1 clinical trial for solid tumors (NCT04834778). Results: Treatment with PERKi sensitized αPD-1/PD-L1 mAb-resistant melanoma tumors (Y1.7/YR1.7) to PD-1/PD-L1 blockade with concomitant increase in tumor infiltrating leukocytes, TH1- reprogrammed TAM, and cytotoxic CD8+ T cells. In addition to tumor microenvironment changes by PERKi, gene expression analysis confirmed reprograming of the tumor microenvironment, marked by a relative increase in TH1-associated genes and downregulation of TH2-associated genes when compared with untreated tumors. We utilized an ex vivo macrophage-splenocyte co-culture assay to depict macrophage dependent T cell suppression prevalent in tumors. This revealed that treating macrophages with PERKi prior to co-culturing with T cells or adding PERKi to the macrophage-T cell co-culture relieved macrophage dependent T cell suppression, illustrating a potential mechanism of therapeutic efficacy. We are now studying how ER-stress is regulated in TAMs and are focusing on identifying components of PERK-dependent cell signaling that increases the tumor promoting behavior of TAMs. Conclusions: The combination therapy targeting PERK and PD-1/PD-L1 signaling increased adaptive immune responses, reprogramed TAMs and reduced tumor growth kinetics. Results from these studies highlight a role for ER-stress signaling in TAMs to maintain an immunosuppressive tumor microenvironment and demonstrate the potential therapeutic strategy of PERK inhibition in αPD-1/PD-L1 resistant tumors. Citation Format: Sushil Kumar, Michael McCane, Jeremy Drees, Nandita Bose, Lisa Coussens. Combination therapy using PERK and PD1/PD-L1 inhibitors reprograms tumor associated macrophages and reduces tumor burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3247.

Abstract 5489: Synergistic activity of SCD1 blockade in combination with tyrosine kinase inhibitors lenvatinib and cabozantinib in hepatocellular carcinoma (HCC)

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Tyrosine kinase inhibitors (TKIs) are approved as a first-line treatment for unresectable HCC. Lenvatinib and cabozantinib are two of the most used TKIs, but the therapeutic duration is limited due to the development of drug resistance. Therefore, understanding the mechanisms of resistance and combining TKIs with other drugs antagonizing resistance should lead to antitumor synergy, eliminating drug resistance. It turns out that the simultaneous use of TKIs together with an inhibitor of stearoyl-CoA desaturase 1 (SCD1) prevents the development of drug resistance, leading to a durable response. SCD1 is the enzyme responsible for de novo fatty acid (FAs) synthesis, converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). MUFAs are an alternative energy source to glucose, integral to cellular membranes, and prevent endoplasmic reticulum (ER) stress and other signaling pathways. In our laboratory, we developed a novel, highly specific SCD1 inhibitor - SSI-4. We have tested the biological activity of SSI-4 against different HCC cell lines and patient-derived xenografts (PDX) mouse models. Of the twelve tested HCC cell lines, four were highly sensitive to SSI-4 (IC50 1-50 nM). Other cell lines showed moderate or no sensitivity to SSI-4. We tested the concomitant use of SSI-4 with lenvatinib and cabozantinib, tyrosine kinase inhibitors (TKIs) FDA-approved for HCC, in HCC cell lines in vitro and using HCC PDX in vivo mouse models. Our studies showed that the combination of the SCD1 inhibitor with both lenvatinib and cabozantinib showed a highly synergistic effect and no development of drug resistance i.e. durable response versus single TKI therapy. Ongoing mechanistic studies are examining whether known molecular targets of tested TKI’s such as VEGFR1, 2, and 3, PDGFRα, FGFR, KIT, and RET dominate in HCC drug resistance to lenvatinib and cabozantinib, and how the use of SSI-4 overcomes the phenomenon of resistance. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew L. Pawlush, Ahmet Bilgili, Peyton G. Hickman, Kabir Mody, Lewis R. Roberts, Steven R. Alberts, Mark J. Truty, Tushar C. Patel, Han W. Tun, John A. Copland. Synergistic activity of SCD1 blockade in combination with tyrosine kinase inhibitors lenvatinib and cabozantinib in hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5489.

Apigenin Alleviates Endoplasmic Reticulum Stress-Mediated Apoptosis in INS-1 β-Cells.

The improvement of type 2 diabetes mellitus induced by naturally occurring polyphenols, known as flavonoids, has received considerable attention. However, there is a dearth of information regarding the effect of the trihydroxyflavone apigenin on pancreatic β-cell function. In the present study, the anti-diabetic effect of apigenin on pancreatic β-cell insulin secretion, apoptosis, and the mechanism underlying its anti-diabetic effects, were investigated in the INS-ID β-cell line. The results showed that apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion, which peaked at 30 µM. Apigenin also concentration-dependently inhibited the expression of endoplasmic reticulum (ER) stress signaling proteins, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3, which was elevated by thapsigargin in INS-1D cells, with peak suppression at 30 µM. This was strongly correlated with the results of flow cytometric analysis of annexin V/propidium iodide (PI) staining and DNA fragmentation analysis. Moreover, the increased expression of thioredoxin-interacting protein (TXNIP) induced by thapsigargin was remarkably reduced by apigenin in a concentration-dependent manner. These results suggest that apigenin is an attractive candidate with remarkable and potent anti-diabetic effects on β-cells, which are mediated by facilitating glucose-stimulated insulin secretion and preventing ER stress-mediated β-cell apoptosis, the latter of which may be possibly mediated by reduced expression of CHOP and TXNIP, thereby promoting β-cell survival and function.

Rt269L-Type hepatitis B virus (HBV) in genotype C infection leads to improved mitochondrial dynamics via the PERK–eIF2α–ATF4 axis in an HBx protein-dependent manner

BackgroundIn our previous report, the rt269I type versus the rt269L type in genotype C2 infection led to poor clinical outcomes and enhanced mitochondrial stress in infected hepatocytes. Here, we sought to investigate differences between the rt269L and rt269I types in mitochondrial functionality in hepatitis B virus (HBV) genotype C2 infection, mainly focusing on endoplasmic reticulum (ER) stress-mediated autophagy induction as an upstream signal.MethodsMitochondrial functionality, ER stress signaling, autophagy induction, and apoptotic cell death between rt269L-type and rt269I-type groups were investigated via in vitro and in vivo experiments. Serum samples were collected from 187 chronic hepatitis patients who visited Konkuk or Seoul National University Hospital.ResultsOur data revealed that genotype C rt269L versus rt269I infection led to improved mitochondrial dynamics and enhanced autophagic flux, mainly due to the activation of the PERK–eIF2α–ATF4 axis. Furthermore, we demonstrated that the traits found in genotype C rt269L infection were mainly due to increased stability of the HBx protein after deubiquitination. In addition, clinical data using patient sera from two independent Korean cohorts showed that, compared with rt269I, rt269L in infection led to lower 8-OHdG levels, further supporting its improved mitochondrial quality control.ConclusionOur data showed that, compared with the rt269I type, the rt269L type, which presented exclusively in HBV genotype C infection, leads to improved mitochondrial dynamics or bioenergetics, mainly due to autophagy induction via activation of the PERK–eIF2α–ATF4 axis in an HBx protein-dependent manner. This suggests that HBx stability and cellular quality control in the rt269L type predominating in genotype C endemic areas could at least partly contribute to some distinctive traits of genotype C infection, such as higher infectivity or longer duration of the hepatitis B e antigen (HBeAg) positive stage.Graphical