Induction Of Endoplasmic Reticulum Stress Research Articles

The Interplay Between Endoplasmic Reticulum Stress and Oxidative Stress in Chondrocyte Catabolism.

Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation. Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM). Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA in vivo. Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.

Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Tl(I) and Tl(III)-induce genotoxicity, reticulum stress and autophagy in PC12 Adh cells.

Thallium (Tl) and its two cationic species, Tl(I) and Tl(III), are toxic for most living beings. In this work, we investigated the effects of Tl (10-100µM) on the viability and proliferation capacity of the adherent variant of PC12 cells (PC12 Adh cells). While both Tl(I) and Tl(III) halted cell proliferation from 24h of incubation, their viability was ~ 90% even after 72h of treatment. At 24h, increased levels of γH2AX indicated the presence of DNA double-strand breaks. Simultaneously, increased expression of p53 and its phosphorylation at Ser15 were observed, which were associated with decreased levels of p-AKTSer473 and p-mTORSer2448. At 72h, the presence of large cytoplasmic vacuoles together with increased autophagy predictor values suggested that Tl may induce autophagy in these cells. This hypothesis was corroborated by images obtained by transmission electron microscopy (TEM) and from the decreased expression at 72h of incubation of SQSTM-1 and increased LC3β-II to LC3β-I ratio. TEM images also showed enlarged ER that, together with the increased expression of IRE1-α from 48h of incubation, indicated that Tl-induced ER stress preceded autophagy. The inhibition of autophagy flux with chloroquine increased cell mortality, suggesting that autophagy played a cytoprotective role in Tl toxicity in these cells. Together, results indicate that Tl(I) or Tl(III) are genotoxic to PC12 Adh cells which respond to the cations inducing ER stress and cytoprotective autophagy.

Identification of a novel RNA transcript TISPL upregulated by stressors that stimulate ATF4

Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5′ rapid amplification of cDNA ends (5′ RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.

Nr1h4 and Thrb ameliorate ER stress and provide protection in the MPTP mouse model of Parkinson's.

Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the differential expression of all the nuclear receptors (NRs) in the presence of classical ER stress inducers. Activation of Nr1h4 and Thrb by their cognate ligands (GW4064 and T3) ameliorates the tunicamycin (TM)-induced expression of ER stress genes. A combination of both ligands is effective in mitigating cell death induced by TM. Further exploration of their protective effects in the Parkinson's disease (PD) model shows that they reduce MPP+-induced dissipation of mitochondrial membrane potential and ROS generation in an in vitro PD model in neuronal cells. Furthermore, the generation of an experimental murine PD model reveals that simultaneous treatment of GW4064 and T3 protects mice from ER stress, dopaminergic cell death, and functional deficits in the MPTP mouse model of PD. Thus, activation of Nr1h4 and Thrb by their respective ligands plays an indispensable role in ER stress amelioration and mounts protective effects in the MPTP mouse model of PD.

Morroniside Protects C2C12 Myoblasts from Oxidative Damage Caused by ROS-Mediated Mitochondrial Damage and Induction of Endoplasmic Reticulum Stress.

Oxidative stress contributes to the onset of chronic diseases in various organs, including muscles. Morroniside, a type of iridoid glycoside contained in Cornus officinalis, is reported to have advantages as a natural compound that prevents various diseases. However, the question of whether this phytochemical exerts any inhibitory effect against oxidative stress in muscle cells has not been well reported. Therefore, the current study aimed to evaluate whether morroniside can protect against oxidative damage induced by hydrogen peroxide (H2O2) in murine C2C12 myoblasts. Our results demonstrate that morroniside pretreatment was able to inhibit cytotoxicity while suppressing H2O2-induced DNA damage and apoptosis. Morroniside also significantly improved the antioxidant capacity in H2O2-challenged C2C12 cells by blocking the production of cellular reactive oxygen species and mitochondrial superoxide and increasing glutathione production. In addition, H2O2-induced mitochondrial damage and endoplasmic reticulum (ER) stress were effectively attenuated by morroniside pretreatment, inhibiting cytoplasmic leakage of cytochrome c and expression of ER stress-related proteins. Furthermore, morroniside neutralized H2O2-mediated calcium (Ca2+) overload in mitochondria and mitigated the expression of calpains, cytosolic Ca2+-dependent proteases. Collectively, these findings demonstrate that morroniside protected against mitochondrial impairment and Ca2+-mediated ER stress by minimizing oxidative stress, thereby inhibiting H2O2-induced cytotoxicity in C2C12 myoblasts.

The proteasome inhibitor carfilzomib exerts anti-inflammatory and antithrombotic effects on the endothelium

BackgroundCarfilzomib (CFZ) is a second-generation proteasome inhibitor used to treat multiple myeloma. Potent inhibition of the proteasome results in chronic proteotoxic endoplasmic reticulum (ER) stress, leading to apoptosis. While CFZ has improved survival rates in multiple myeloma, it is associated with an increased risk of cardiovascular adverse effects. While this has been putatively linked to cardiotoxicity, CFZ could potentially also exhibit adverse effects on the endothelium. ObjectivesTo investigate the effects of CFZ on the endothelium. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with CFZ, and expression of relevant markers of ER stress, inflammation, and thrombosis was measured and functionally assessed. ResultsCFZ failed to induce ER stress in HUVECs but induced the expression of Kruppel-like factor 4, endothelial nitric oxide synthase, tissue plasminogen activator, and thrombomodulin and reduced tumor necrosis factor alpha (TNFα)-mediated intercellular adhesion molecule 1 and tissue factor expression, suggesting a potential protective effect on the endothelium. Consistent with these observations, CFZ reduced leukocyte adhesion under shear stress and reduced factor Xa generation and fibrin clot formation on the endothelium following TNFα treatment and inhibited von Willebrand factor (VWF) and angiopoietin-2 exocytosis from Weibel–Palade bodies. Subsequently, CFZ inhibited the formation of VWF-platelet strings, and moreover, media derived from myeloma cell lines induced VWF release, a process also inhibited by CFZ. ConclusionThese data demonstrate that CFZ is unable to induce ER stress in confluent resting endothelial cells and can conversely attenuate the prothrombotic effects of TNFα on the endothelium. This study suggests that CFZ does not negatively alter HUVECs, and proteasome inhibition of the endothelium may offer a potential way to prevent thrombosis.

Abstract LB303: Suppression of stress granule formation is a novel vulnerability imposed by mutant p53

Abstract Mutation of the tumor suppressor p53 is the most common cancer specific event. Most of these mutations are missense mutations in the DNA-binding domain of p53, causing loss of wild-type p53 (wtp53) transcriptional activity. Missense mutant p53 (mutp53) also enhances cancer malignancy in a manner independent of wtp53. Hence, mutp53 is an ideal therapeutic target due to its uniqueness and high expression levels in cancers; however, directly targeting mutp53 protein itself has been challenging. To better understand the mechanism of oncogenic function of mutp53, we performed in silico TCGA database analysis and found that patients with missense mutp53-carrying hepatocellular carcinoma (HCC), but not other types of cancer, had unexpectedly better overall survival than patients with p53-null HCC. Since sorafenib (SOR) has historically been used as a single agent chemotherapy for HCC, unlike other cancer types, we hypothesized that SOR treatment may have yet unappreciated advantages for treating mutp53-expressing tumors. Indeed, knockdown of mutp53 rendered HCC cells and xenografts more resistant to SOR. Consistent with a report showing that SOR induces ER stress-mediated pro-survival stress granule (SG) formation, mutp53-expressing HCC cells, treated with SOR or other ER stress inducers, formed significantly fewer SGs with increased apoptosis, as compared with mutp53-knockdown cells. This result was partly due to mutp53’s ability to interact with an eIF2α kinase PERK and a key SG core protein, G3BP1. Moreover, the oligomerization domain of mutp53 was required for the interaction of mutp53 with G3BP1 and subsequent suppression of ER-stress-mediated SG formation and survival. These results suggest that inhibition of SG formation is a novel vulnerability imposed by mutp53, leading to sensitization of cancer cells to ER stress. Our results also emphasize the use of ER stress inducers to treat mutp53-expressing tumors and mutp53 as a biomarker of SOR sensitivity. Citation Format: Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan Dixon, Sugako Oka, Tomoo Iwakuma. Suppression of stress granule formation is a novel vulnerability imposed by mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB303.

XBP1s activates METTL3/METTL14 for ER-phagy and paclitaxel sensitivity regulation in breast cancer

Cancer cells employ the unfolded protein response (UPR) or induce autophagy, especially selective removal of certain ER domains via reticulophagy (termed ER-phagy), to mitigate endoplasmic reticulum (ER) stress for ER homeostasis when encountering microenvironmental stress. N6-methyladenosine (m6A) is one of the most abundant epitranscriptional modifications and plays important roles in various biological processes. However, the molecular mechanism of m6A modification in the ER stress response is poorly understood. In this study, we first found that ER stress could dramatically elevate m6A methylation levels through XBP1s-dependent transcriptional upregulation of METTL3/METTL14 in breast cancer (BC) cells. Further MeRIP sequencing and relevant validation results confirmed that ER stress caused m6A methylation enrichment on target genes for ER-phagy. Mechanistically, METTL3/METTL14 increased ER-phagy machinery formation by promoting m6A modification of the ER-phagy regulators CALCOCO1 and p62, thus enhancing their mRNA stability. Of note, we further confirmed that the chemotherapeutic drug paclitaxel (PTX) could induce ER stress and increase m6A methylation for ER-phagy. Furthermore, the combination of METTL3/METTL14 inhibitors with PTX demonstrated a significant synergistic therapeutic effect in both BC cells and xenograft mice. Thus, our data built a novel bridge on the crosstalk between ER stress, m6A methylation and ER-phagy. Most importantly, our work provides novel evidence of METTL3 and METTL14 as potential therapeutic targets for PTX sensitization in breast cancer.

HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus

Aspergillus flavus is a fungus notorious for contaminating food and feed with aflatoxins. As a saprophytic fungus, it secretes large amounts of enzymes to access nutrients, making endoplasmic reticulum (ER) homeostasis important for protein folding and secretion. The role of HacA, a key transcription factor in the unfolded protein response pathway, remains poorly understood in A. flavus. In this study, the hacA gene in A. flavus was knockout. Results showed that the absence of hacA led to a decreased pathogenicity of the strain, as it failed to colonize intact maize kernels. This may be due to retarded vegetable growth, especially the abnormal development of swollen tips and shorter hyphal septa. Deletion of hacA also hindered conidiogenesis and sclerotial development. Notably, the mutant strain failed to produce aflatoxin B1. Moreover, compared to the wild type, the mutant strain showed increased sensitivity to ER stress inducer such as Dithiothreitol (DTT), and heat stress. It also displayed heightened sensitivity to other environmental stresses, including cell wall, osmotic, and pH stresses. Further transcriptomic analysis revealed the involvement of the hacA in numerous biological processes, including filamentous growth, asexual reproduction, mycotoxin biosynthetic process, signal transduction, budding cell apical bud growth, invasive filamentous growth, response to stimulus, and so on. Taken together, HacA plays a vital role in fungal development, pathogenicity and aflatoxins biosynthesis. This highlights the potential of targeting hacA as a novel approach for early prevention of A. flavus contamination.

Photodynamic Therapy of Head and Neck Cancers with Ruthenium-based Photosensitizers

SignificanceThe management of patients with head and neck cancers relies mainly on platinum-based chemotherapies. However, severe side effects and tumor resistance impair treatment efficacy, notably due to frequent mutations of the TP53 tumor suppressor. Here we propose a ruthenium-based photodynamic therapy as a p53-independent therapeutic strategy, and show that its phototoxicity relies on the induction of endoplasmic reticulum stress and immunogenic cell death. ApproachSQ20B cells were treated with the Ru I ruthenium-based photosensitizer prior to illumination. Ru I cytotoxicity and mode of action were studied using molecular and cellular read-outs. ResultsRu I showed an interesting phototoxicity and triggered the expression of key markers of endoplasmic reticulum stress and early stages of immunogenic cell death. ConclusionsRu I-PDT appears to be efficient in vitro on head and neck cancer models. Its phototoxicity seems to be associated to two p53-independent pathways, whose functional links remain to be clarified.

381 BiP knockdown decreases antibody production in malignant and non-malignant plasma cells

OBJECTIVES/GOALS: Numerous diseases, including AL amyloidosis, are due to expression of aberrant antibodies. Significant effort has gone into plasma cell toxic therapies with varying degrees of success, but no therapies preventing antibody synthesis have been developed. The goal of this study is to assess BiP targeting to prevent antibody secretion in plasma cells. METHODS/STUDY POPULATION: Using 4 multiple myeloma cell lines (KMS11, RPMI8226, ANBL-6, U266), we knocked down BiP expression with RnaseH dependent siRNA or subA toxin, a bacterial toxin that specifically cleaves BiP, and measured changes in unfolded protein and intracellular light chains by flow cytometry during drug induced ER stress created by the intracellular calcium depleting agent thapsigargin. BiP is the master regulator of the unfolded protein response (UPR), an ER stress pathway important for protein folding. BiP is also an ER resident protein folding chaperone important for proper antibody folding. We hypothesized that BiP downregulation will lead to decreased folded antibody in the cell, increased unfolded antibody and constitutive activation of the UPR. RESULTS/ANTICIPATED RESULTS: 1 to 4 hours after treatment with thapsigargin plus siRNA against BiP, levels of BiP are significantly decreased. The levels of intracellular light chains decrease, and the level of unfolded protein within the cells increases dramatically. Interestingly, in alignment with the UPR literature, 24 hours post treatment, these levels have normalized again in surviving cells. SubA treatment increased BiP expression by 4 hours, contrary to our hypothesis, and minimally increased unfolded proteins and minimally decreased intracellular light chains. We expect that further functional testing of antibody secretion by ELIspot assays will show decreased secretion of antibody with BiP siRNA treatment. Combination therapies with other UPR stressing agents may act synergistically to affect antibody production. DISCUSSION/SIGNIFICANCE: BiP knockdown reduces antibodies and boosts unfolded proteins. SubA toxin ineffectiveness likely stems from increased BiP due to feedback loops. Combining anti-BiP treatments with UPR stressing drugs like bortezomib may halt antibody synthesis and induce cell death. These findings support BiP as a viable drug target for antibody-related diseases.

Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

Social defeat stress induces liver injury by modulating endoplasmic reticulum stress in C57BL/6J mice

Social defeat stress is associated with endoplasmic reticulum (ER) stress, inflammation and apoptosis. ER stress is thought to contribute to many lifestyle diseases such as liver injury, cardiovascular dysfunction and depression. We investigated the expression of the ER stress markers RNA-dependent protein kinase-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP), as well as inflammatory and apoptotic factors, to assess how social defeat stress induces liver injury. Furthermore, we evaluated the effects of the ER stress inhibitor phenylbutyric acid (PBA) and ER stress inducer thapsigargin (TG) on liver injury. Adult mice were divided into the control, social defeat, social defeat + PBA, TG, PBA and TG + PBA groups. The social defeat and social defeat + PBA groups were simultaneously exposed to social defeat stress for 10 days. The social defeat + PBA, TG, PBA and TG + PBA groups were treated with PBA or TG via intraperitoneal injections. PBA was injected 1 h before the TG injection into the TG + PBA group. Liver samples from six groups of mice were analyzed by histological analysis and western blotting. Social defeat stress promoted ER stress, increased the expression of inflammatory factors and induced apoptosis in the liver of socially defeated mice, which was reversed by PBA. Moreover, ER stress induces TG-induced liver injury by initiating ER stress. Social defeat stress initiates ER stress, promotes the expression of inflammatory and apoptotic factors, and induces liver injury. PBA suppresses liver injury caused by social defeat stress and TG treatment.

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1β production in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

XAF1 overexpression inhibits the malignant progression and cisplatin resistance of NSCLC by activating endoplasmic reticulum stress.

XIAP-associated factor 1 (XAF1) has been found to participate in the progression of multiple human cancers. Nevertheless, its role as well as the reaction mechanism in non-small cell lung cancer (NSCLC) still remains obscure. In this study, the protein expression of XAF1 in NSCLC cell lines was evaluated using western blot. With the employment of CCK-8 assay, EdU staining, wound healing and transwell, capabilities of NSCLC cells to proliferate, migrate and invade were assessed. Cell apoptotic level and cell cycle were resolved utilizing flow cytometry. Western blot was applied for the estimation of apoptosis- and endoplasmic reticulum (ER) stress-related proteins. It was discovered that XAF1 expression was conspicuously reduced in NSCLC cell lines. XAF1 overexpression suppressed H1299 cell proliferative, invasive and migrative capabilities, but exhibited promotive effects on cell cycle arrest. Meanwhile, XAF1 overexpression inhibited cisplatin resistance in H1299 and H1299/DDP cells by promoting cell apoptosis and enhanced the expression levels of ER stress-related proteins CHOP, GRP78 and ATF4. What's more, 4-PBA treatment reversed the impacts of XAF1 overexpression on the proliferative, invasive, migrative and apoptotic capabilities of H1299 cells, as well as cell cycle and cisplatin resistance. In conclusion, XAF1 overexpression impeded the advancement of NSCLC and repressed cisplatin resistance of NSCLC cells through inducing ER stress, which indicated that XAF1 might be a novel targeted-therapy for NSCLC.

Transcriptomic changes behind Sparus aurata hepatic response to different aquaculture challenges: An RNA-seq study and multiomics integration.

Gilthead seabream (Sparus aurata) is an important species in Mediterranean aquaculture. Rapid intensification of its production and sub-optimal husbandry practices can cause stress, impairing overall fish performance and raising issues related to sustainability, animal welfare, and food safety. The advent of next-generation sequencing technologies has greatly revolutionized the study of fish stress biology, allowing a deeper understanding of the molecular stress responses. Here, we characterized for the first time, using RNA-seq, the different hepatic transcriptome responses of gilthead seabream to common aquaculture challenges, namely overcrowding, net handling, and hypoxia, further integrating them with the liver proteome and metabolome responses. After reference-guided transcriptome assembly, annotation, and differential gene expression analysis, 7, 343, and 654 genes were differentially expressed (adjusted p-value < 0.01, log2|fold-change| >1) in the fish from the overcrowding, net handling, and hypoxia challenged groups, respectively. Gene set enrichment analysis (FDR < 0.05) suggested a scenario of challenge-specific responses, that is, net handling induced ribosomal assembly stress, whereas hypoxia induced DNA replication stress in gilthead seabream hepatocytes, consistent with proteomics and metabolomics' results. However, both responses converged upon the downregulation of insulin growth factor signalling and induction of endoplasmic reticulum stress. These results demonstrate the high phenotypic plasticity of this species and its differential responses to distinct challenging environments at the transcriptomic level. Furthermore, it provides significant resources for characterizing and identifying potentially novel genes that are important for gilthead seabream resilience and aquaculture production efficiency with regard to fish welfare.

Abstract 3335: A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers

Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network called the unfolded protein response (UPR). Inositol-requiring enzyme 1α (IRE1α) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved way of the UPR. The IRE1α-XBP1 pathway, one of the most essential branches of the UPR, is activated in various types of cancer. When IRE1α is activated, the spliced form of XBP1 (XBP1s) is generated to activate the transcription of many pro-survival genes to prevent cellular death and relieve cellular stress. IRE1α plays an instrumental pro-tumoral role in various cancers, such as glioblastoma, myeloma, lung adenocarcinoma, and breast cancers, as well as high IRE1α activity is associated with poor prognoses. Moreover, IRE1-XBP1 plays an essential role in the UPR and ER stress responses, which have been implicated in the development of drug resistance. Thereby, blockade of the IRE1α-XBP1 pathway is crucial as it is considered as a potential anti-cancer strategy. Here, we developed a potent and selective IRE1α RNase inhibitor, which have a hydroxy-aryl-aldehydes (HAA) moiety that binds to IRE1α within the RNase catalytic site. Our lead compound, HM100168, potently inhibited the RNase activity of human IRE1α at nanomolar concentrations without affecting its kinase activity. Experiments were performed on MCF7 breast cancer cells to confirm the inhibitory effect in the mRNA and protein levels of XBP1s, which was increased by tunicamycin (TM), a ER stress inducer. HM100168 strongly inhibited ​​XBP1s levels in a dose-dependent manner, displaying the potent IC50 values. We tested cell growth inhibition activity in 3D as well as 2D cell culture condition of various cancer cell lines. Interestingly, HM100168 effectively inhibited cell proliferation of diverse cancer cell lines in both 2D and 3D culture condition. In addition, we explored the promising anti-cancer effects of HM100168 and its synergistic potential when combined with anti-tumorigenic agents. As a result, HM100168 in combination with anti-tumorigenic agents significantly enhanced suppression of tumor growth in human cancer cell transplanted mice. In conclusion, we have identified a novel compound with unique properties that specifically blocked the endonuclease activity of IRE1α. HM100168 inhibited IRE1α endonuclease activity, without affecting its kinase activity after endoplasmic reticulum stress in vitro. The identification of this novel IRE1α inhibitor supports the hypothesis that the IRE1α-XBP1 axis is a promising target for anticancer therapy. Also, co-targeting of IRE1α-XBP1 in combination with anti-tumorigenic agents can be considered a novel approach to overcome cancer resistance. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Citation Format: Jisook Kim, Seung Hyun Jung, Minjeong Kim, Wongi Park, Jooyun Byun, Soonki Park, Miyoung Lee, Yu-Yon Kim, Junghwa Park, Seokhyun Hong, Minhwa Kim, Young Gil Ahn. A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3335.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy

BackgroundThe aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear.Methods and resultsRNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson’s analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM.ConclusionsThe findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.