Key Unfolded Protein Response Research Articles

Exploring the mechanism of intestinal injury induced by Bisphenol S in freshwater crayfish (Procambarus clarkii): Molecular and biochemical approaches

Bisphenol S (BPS) is extensively utilized in various industries such as plastic manufacturing, food packaging, and electronics. The release of BPS into aquatic environments has been observed to have negative impacts on aquatic ecosystems. Research has shown that exposure to BPS can have adverse effects on the health of aquatic animals. This study aimed to explore the mechanism of oxidative stress and endoplasmic reticulum stress induced in freshwater crayfish (Procambarus clarkii) by exposure to BPS (0 µg/L, 1 µg/L, 10 µg/L, and 100 µg/L) for 14 days. The results showed that BPS exposure resulted in elevated levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and severe intestinal histological damage. In addition, oxidative stress can occur in the body by inhibiting the activity of antioxidant enzymes and the expression of related genes. BPS exposure induced a significant increase in the relative mRNA expression levels of inflammatory cytokines (NF-κB and TNF-α) and key unfolded protein response (UPR) related genes (Bip, Ire1, and Xbp1). At the same time, BPS exposure also induced up-regulation of apoptosis genes (Cytc and Casp3), suggesting that UPR and Nrf2-Keap1 signaling pathways may play a protective role in the process of apoptosis and oxidative stress. In conclusion, Our findings present the initial evidence that exposure to environmentally relevant levels of BPS can lead to intestinal injury through various pathways, highlighting concerns about the potential harm at a population level from BPS and other bisphenol analogs.

IRE1α regulates ROS and immune responses after UVB irradiation

Objective UV irradiation of the skin induces photo damage and generates cytotoxic intracellular reactive oxygen species (ROS), activating the unfolded protein response (UPR) to adapt or reduce these UVB-mediated damages. This study was designed to understand the role of the UPR mediator IRE1α in the antioxidant response following UVB irradiation of mouse skin and keratinocytes. Methods We used mice with an epidermal deletion of IRE1α and primary mouse keratinocytes to examine effects of UV on different parameters of the antioxidant response in the presence and absence of functional IRE1α. Results In the absence of IRE1α, PERK activity and protein levels are significantly compromised following UVB irradiation. Additionally, the loss of IRE1α suppressed phosphorylation of the PERK target, nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2-dependent antioxidant gene expression after UVB irradiation. Interestingly, IRE1α-deficient keratinocytes exhibit elevated basal ROS levels, while a robust ROS induction upon UVB exposure is abolished. Because UVB-induced ROS plays an essential role in regulating skin inflammation, we analyzed recruited immune cell populations and the expression of pro-inflammatory cytokines, Il-6 and Tnfα, in mice with epidermally targeted deletion of Ire1α. Following UVB irradiation, there was significantly less recruitment of neutrophils and leukocytes and reduced expression of pro-inflammatory cytokine genes in the skin of mice lacking IRE1α. Furthermore, keratinocyte proliferation was also significantly reduced after chronic UVB exposure in the skin of these mice. Conclusion Collectively, our findings indicate that IRE1α is essential for basal and UVB-induced oxidative stress response, UV-induced skin immune responses, and keratinocyte proliferation. Significance statement These findings shed new light on the protective function of IRE1α in the response to UV. IRE1α plays an important role in the regulation of ROS, PERK stability, and antioxidant gene expression in response to UVB in mouse keratinocytes and epidermis.

Expression of key unfolded protein response genes predicts patient survival and an immunosuppressive microenvironment in glioblastoma

Abstract Background Dysregulation of cellular processes related to protein folding and trafficking leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), triggering ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR), a signaling pathway that has been implicated in a variety of diseases, including cancer. However, the role of the UPR in cancer initiation and progression is still unclear. Methods Here we used bulk and single cell RNA sequencing data to investigate ER stress-related gene expression in glioblastoma, as well as the impact key UPR genes have on patient survival. Results ER stress-related genes are highly expressed in both cancer cells and tumor-associated macrophages, with evidence of high intra- and inter-tumor heterogeneity. High expression of the UPR-related genes HSPA5, P4HB, and PDIA4 was identified as risk factors while high MAPK8 (JNK1) expression was identified as a protective factor in glioblastoma patients, indicating UPR genes have prognostic potential in this cancer type. Finally, expression of XBP1 and MAPK8, two key downstream targets of the ER sentinel IRE1α, correlates with the presence of immune cell types associated with immunosuppression and a worse patient outcome. This suggests that the expression of these genes is associated with an immunosuppressive tumor microenvironment and uncover a potential link between stress response pathways, tumor microenvironment and glioblastoma patient survival. Conclusions We performed a comprehensive transcriptional characterization of the unfolded protein response in glioblastoma patients and identified UPR-related genes associated with glioblastoma patient survival, providing potential prognostic and predictive biomarkers as well as promising targets for developing new therapeutic interventions in glioblastoma treatment.

GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells

Lack of targetable antigens is a key limitation for developing successful Tcell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) Tcell target. We demonstrate that GRP78-CAR Tcells can recognize and kill GRP78+ brain and solid tumors invitro and invivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR Tcells upon Tcell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR Tcell therapeutic response.

Immunogenic landscape and risk score prediction based on unfolded protein response (UPR)-related molecular subtypes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of cancer and causes a significant number of cancer-related deaths worldwide. The molecular mechanisms underlying the development of HCC are complex, and the heterogeneity of HCC has led to a lack of effective prognostic indicators and drug targets for clinical treatment of HCC. Previous studies have indicated that the unfolded protein response (UPR), a fundamental pathway for maintaining endoplasmic reticulum homeostasis, is involved in the formation of malignant characteristics such as tumor cell invasiveness and treatment resistance. The aims of our study are to identify new prognostic indicators and provide drug treatment targets for HCC in clinical treatment based on UPR-related genes (URGs). Gene expression profiles and clinical information were downloaded from the TCGA, ICGC and GEO databases. Consensus cluster analysis was performed to classify the molecular subtypes of URGs in HCC patients. Univariate Cox regression and machine learning LASSO algorithm were used to establish a risk prognosis model. Kaplan-Meier and ROC analyses were used to evaluate the clinical prognosis of URGs. TIMER and XCell algorithms were applied to analyze the relationships between URGs and immune cell infiltration. Real time-PCR was performed to analyze the effect of sorafenib on the expression levels of four URGs. Most URGs were upregulated in HCC samples. According to the expression pattern of URGs, HCC patients were divided into two independent clusters. Cluster 1 had a higher expression level, worse prognosis, and higher expression of immunosuppressive factors than cluster 2. Patients in cluster 1 were more prone to immune escape during immunotherapy, and were more sensitive to chemotherapeutic drugs. Four key UPR genes (ATF4, GOSR2, PDIA6 and SRPRB) were established in the prognostic model and HCC patients with high risk score had a worse clinical prognosis. Additionally, patients with high expression of four URGs are more sensitive to sorafenib. Moreover, ATF4 was upregulated, while GOSR2, PDIA6 and SRPRB were downregulated in sorafenib-treated HCC cells. The UPR-related prognostic signature containing four URGs exhibits high potential application value and performs well in the evaluation of effects of chemotherapy/immunotherapy and clinical prognosis.

Effects of acute ammonia exposure on oxidative stress, endoplasmic reticulum stress and apoptosis in the kuruma shrimp (Marsupenaeus japonicus)

Ammonia is one of the main toxic substances in aquaculture water and causes serious physiological harm to aquatic animals. In the present study, we investigated the effects of ammonia exposure on antioxidant defense, endoplasmic reticulum stress and apoptosis in the Marsupenaeus japonicus hepatopancreas. Shrimp in three experimental groups (control, 15 mg/L and 45 mg/L ammonia) were sampled at 6, 12, 24, 48 and 96 h. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), and the corresponding relative mRNA expression levels in the hepatopancreas, showed slight or obvious increases during the early stage of ammonia exposure, and then significantly decreased after 48 h or 96 h of exposure, while the malondialdehyde (MDA) content increased continuously during ammonia exposure. In addition, the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) showed a similar regulation pattern to the antioxidant enzymes, whereas Kelch-like ECH associated protein 1 (Keap1), a negative regulator of Nrf2, showed the opposite pattern. Moreover, the relative mRNA expression levels of heat shock proteins (HSP70 and HSP90), endoplasmic reticulum stress marker (GRP78) gene and key unfolded protein response (UPR)-related genes (eIF2α, ATF4, IRE1 and XBP1) increased significantly during the early stage of ammonia exposure. Taken together, these results indicate that the antioxidant system, Nrf2-Keap1 signaling pathway, UPR and heat shock proteins play protective roles in ammonia-induced oxidative stress and apoptosis in M. japonicus. However, ammonia exposure also induced up-regulation of apoptosis genes (Caspase 3 and ASK1) levels, indicating that cell apoptotic may relate to oxidative stress. After 96 h of exposure to 45 mg/L ammonia, down-regulation of HSP70 and HSP90 gene transcription levels suggests that the protein synthesis system may be destroyed under severe ammonia-induced oxidative stress. Our results provide new insights into the mechanism through which crustaceans resist ammonia stress.

Spinal Stroke: Outcome Attenuation by Erythropoietin and Carbamylated Erythropoietin and Its Prediction by Sphingosine-1-Phosphate Serum Levels in Mice.

Spinal strokes may be associated with tremendous spinal cord injury. Erythropoietin (EPO) improves the neurological outcome of animals after spinal cord ischemia (SCI) and its effects on ischemia-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are considered possible molecular mechanisms. Furthermore, sphingosin-1-phosphate (S1P) is suggested to correlate with SCI. In this study, the effect of recombinant human EPO (rhEPO) and carbamylated EPO (cEPO-Fc) on the outcome of mice after SCI and a prognostic value of S1P were investigated. SCI was induced in 12-month-old male mice by thoracic aortal cross-clamping after administration of rhEPO, cEPO-Fc, or a control. The locomotory behavior of mice was evaluated by the Basso mouse scale and S1P serum levels were measured by liquid chromatography-tandem mass spectrometry. The spinal cord was examined histologically and the expressions of key UPR proteins (ATF6, PERK, and IRE1a, caspase-12) were analyzed utilizing immunohistochemistry and real-time quantitative polymerase chain reaction. RhEPO and cEPO-Fc significantly improved outcomes after SCI. The expression of caspase-12 significantly increased in the control group within the first 24 h of reperfusion. Animals with better locomotory behavior had significantly higher serum levels of S1P. Our data indicate that rhEPO and cEPO-Fc have protective effects on the clinical outcome and neuronal tissue of mice after SCI and that the ER is involved in the molecular mechanisms. Moreover, serum S1P may predict the severity of impairment after SCI.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.

Cyclosporine A but not tacrolimus induces pro‐apoptotic endoplasmic reticulum stress in renal tubular cells

Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first‐line immunosuppressive strategies in organ‐transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro‐apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro‐apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4‐PBA, alleviated the CsA‐induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9‐mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro‐apoptotic UPR in response to CsA.In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro‐apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity.

Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α-XBP1 pathway.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by survival of motor neuron (SMN) deficiency that induces motor neuron (MN) degeneration and severe muscular atrophy. Gene therapies that increase SMN have proven their efficacy but not for all patients. Here, we explored the unfolded protein response (UPR) status in SMA pathology and explored whether UPR modulation could be beneficial for SMA patients. We analysed the expression and activation of key UPR proteins by RT-qPCR and by western blots in SMA patient iPSC-derived MNs and one SMA cell line in which SMN expression was re-established (rescue). We complemented this approach by using myoblast and fibroblast SMA patient cells and SMA mouse models of varying severities. Finally, we tested in vitro and in vivo the effect of IRE1α/XBP1 pathway restoration on SMN expression and subsequent neuroprotection. We report that the IRE1α/XBP1 branch of the unfolded protein response is disrupted in SMA, with a depletion of XBP1s irrespective of IRE1α activation pattern. The overexpression of XBP1s in SMA fibroblasts proved to transcriptionally enhance SMN expression. Importantly, rebalancing XBP1s expression in severe SMA-like mice, induced SMN expression and spinal MN protection. We have identified XBP1s depletion as a contributing factor in SMA pathogenesis, and the modulation of this transcription factor proves to be a plausible therapeutic avenue in the context of pharmacological interventions for patients.

Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay

The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3′UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.

Canopy Homolog 2 contributes to liver oncogenesis by promoting unfolded protein response-dependent destabilization of tumor protein P53.

Abnormalities in the tumor protein P53(p53)gene and overexpression of mouse double minute 2 homolog (MDM2), a negative regulator of p53, are commonly observed in cancers. p53 destabilization is regulated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in cancer. However, the mechanisms remain enigmatic. Canopy homolog 2 (CNPY2) is a key UPR initiator that primarily involved in ER stress and is highly expressed in the liver, but its functional role in regulating liver carcinogenesis is poorly understood. Therefore, we aimed to investigate the role of CNPY2 in hepartocarcinogenesis through URP-dependent p53 destabilization. Here, we showed that CNPY2 expression is up-regulated in HCC and negatively correlated with survival rate in liver cancer patients. Deletion of Cnpy2 obliterates diethylnitrosamine (DEN)-induced HCC in mice. Mechanistic studies demonstrated that CNPY2 binds and prevents ribosome proteins from inhibiting MDM2 and enhances the UPR activity of protein kinase RNA-like endoplasmic reticulum kinase and inositol-requiring transmembrane kinase endoribonuclease-1α, leading to p53 destabilization and cell-cycle progression. In addition, transcriptome analyses uncovered that CNPY2 is also required for DEN-induced expression of oncogenes, including c-Jun and fibroblast growth factor 21. Intratumoral injection of nanoparticle-based CRISPR single-guide RNA/CRISPR-associated protein 9 mRNA against Cnpy2 has antitumor effects in HCC. These findings demonstrate that CNPY2 is crucial for liver oncogenesis through UPR-dependent repression of p53 and activation of oncogenes, providing insights into the design of a therapeutic target for HCC.

The PERK/ATF4/CHOP signaling branch of the unfolded protein response mediates cisplatin-induced ototoxicity in hair cells

Cisplatin is a widely used chemotherapeutic agent. However, its clinical application remains limited due to the high incidence of severe ototoxicity. It has been reported that the unfolded protein response (UPR) is involved in cisplatin-induced ototoxicity. However, the specific mechanism underlying its effect remains unclear. Therefore, the present study aimed to explore the sequential changes in the key UPR signaling branch and its potential pro-apoptotic role in cisplatin-induced ototoxicity. The hair cell-like OC-1 cells were treated with cisplatin for different periods and then the expression levels of the UPR- and apoptosis-related proteins were determined. The results showed that the apoptotic rate of cells was gradually increased with prolonged cisplatin treatment. Furthermore, the sequential changes in three UPR signaling branches were evaluated. The expression levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were gradually increased with up to 12 h of cisplatin treatment. The aforementioned expression profile was consistent with that observed for the apoptosis-related proteins. Subsequently, the proportion of apoptotic cells was notably decreased in CHOP-silenced hair cell-like OC-1 cells following treatment with cisplatin. Moreover, we found significant hair cells loss and a higher level of CHOP in cisplatin-treated cochlear explants in a time-dependent manner. Overall, the present study demonstrated that the protein kinase RNA‑like endoplasmic reticulum kinase (PERK)/ATF4/CHOP signaling branch could play an important role in cisplatin-induced cell apoptosis. Furthermore, the current study suggested that CHOP may be considered as a promising therapeutic target for cisplatin-induced ototoxicity.

BZIP17 regulates heat stress tolerance at reproductive stage in Arabidopsis

High temperature elicits a well-conserved response called the unfolded protein response (UPR) to bring protein homeostasis in the endoplasmic reticulum (ER). Two key UPR regulators bZIP28 and bZIP60 have been shown to be essential for maintaining fertility under heat stress conditions in Arabidopsis, however, the function of transcriptional activator bZIP17, a paralog of bZIP28, in heat stress response at reproductive stage is not reported. Here we found that bzip17 mutant plants were sensitive to heat stress in terms of silique length and fertility comparing to that of wildtype (WT) Arabidopsis plants, and transcriptomic analysis showed that 1380 genes were specifically up-regulated and 493 genes were specifically down-regulated by heat stress in the flowers of WT plants comparing to that in bzip17 mutant plants. These bZIP17-dependent up-regulated genes were enriched in responses to abiotic stresses such as water deprivation and salt stress. Further chromatin immuno-precipitation coupled with high-throughput sequencing (ChIP-Seq) uncovered 1645 genes that were direct targets of bZIP17 in MYC-bZIP17 expressing seedlings subjected to heat stress. Among these 1645 genes, ERSE-II cis-element was enriched in the binding peaks of their promoters, and the up-regulation of 113 genes by heat stress in flowers was dependent on bZIP17. Our results revealed direct targets of bZIP17 in flowers during heat stress responses and demonstrated the important role of bZIP17 in maintaining fertility upon heat stress in plants.

The Role of BiP and the IRE1α-XBP1 Axis in Rhabdomyosarcoma Pathology.

Simple SummaryThe expression of BiP (GRP78), spliced XBP1 (sXBP1, nuclear XBP1), and IRE1α, were significantly associated with Rhabdomyosarcoma (RMS) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS; (n = 192) compared to normal skeletal muscle tissues (n = 16). There was a significant correlation between BiP expression and the lymph node score, and between IRE1α, cytosolic XBP1 and sXBP1 expression and the stage score in all of the types of RMS. BiP and sXBP1 expression were significantly associated with all of the subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. There were correlations between BiP expression and the lymph node score in ARMS, and sXBP1 expression and the tumor score in PRMS.Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and is associated with a poor prognosis in patients presenting with recurrent or metastatic disease. The unfolded protein response (UPR) plays pivotal roles in tumor development and resistance to therapy, including RMS. Methods: In this study, we used immunohistochemistry and a tissue microarray (TMA) on human RMS and normal skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP, IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation of these proteins with the risk of RMS and several clinicopathological indices, such as lymph node involvement, distant metastasis, tumor stage and tumor scores. Results: Our results revealed that the expression of BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001) in all of the studied types of RMS tumors (n = 192) compared to normal skeletal muscle tissues (n = 16). In addition, significant correlations of BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004), cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage score were observed. At the subtype level, BiP and sXBP1 expression were significantly associated with all subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. Importantly, the expression levels of IRE1α and sXBP1 were more pronounced in ARMS than in any of the other subtypes. The results also showed correlations of BiP with the lymph node score in ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value = 0.002). Conclusions: In summary, this study demonstrates that the overall UPR is upregulated and, more specifically, that the IRE1/sXBP1 axis is active in RMS. The subtype and stage-specific dependency on the UPR machinery in RMS may open new avenues for the development of novel targeted therapeutic strategies and the identification of specific tumor markers in this rare but deadly childhood and young-adult disease.

Susceptibility factor RTP1 negatively regulates Phytophthora parasitica resistance via modulating UPR regulators bZIP60 and bZIP28.

The unfolded protein response (UPR) is a conserved stress adaptive signaling pathway in eukaryotic organisms activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). UPR can be elicited in the course of plant defense, playing important roles in plant-microbe interactions. The major signaling pathways of plant UPR rely on the transcriptional activity of activated forms of ER membrane-associated stress sensors bZIP60 and bZIP28, which are transcription factors that modulate expression of UPR genes. In this study, we report the plant susceptibility factor Resistance to Phytophthora parasitica 1 (RTP1) is involved in ER stress sensing and rtp1-mediated resistance against P. parasitica is synergistically regulated with UPR, as demonstrated by the simultaneous strong induction of UPR and ER stress-associated immune genes in Arabidopsis thaliana rtp1 mutant plants during the infection by P. parasitica. We further demonstrate RTP1 contributes to stabilization of the ER membrane-associated bZIP60 and bZIP28 through manipulating the bifunctional protein kinase/ribonuclease IRE1-mediated bZIP60 splicing activity and interacting with bZIP28. Consequently, we find rtp1bzip60 and rtp1bzip28 mutant plants exhibit compromised resistance accompanied with attenuated induction of ER stress-responsive immune genes and reduction of callose deposition in response to P. parasitica infection. Taken together, we demonstrate RTP1 may exert negative modulating roles in the activation of key UPR regulators bZIP60 and bZIP28, which are required for rtp1-mediated plant resistance to P. parasitica. This facilitates our understanding of the important roles of stress adaptive UPR and ER stress in plant immunity.

Abstract 4067: Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles

Abstract Recent advances in cancer therapy have focused on dysregulated local immunity, with paradigm-shifting discoveries like immune checkpoint inhibitors fundamentally altering the way we think about treating cancer. It is now well appreciated that local immune dysregulation is a viable target for cancer therapy, offering many advantages over traditional approaches. One mechanism at the origin of immune dysregulation is a sustained activation of the unfolded protein response (UPR) in the endoplasmic reticulum. The UPR is activated under conditions of stress, including hypoxia, DNA damage, and aneuploidy- stressors present within the tumor microenvironment. Unsurprisingly, the UPR is commonly dysregulated in cancer, acting to promote tumor growth. Multiple lines of evidence support this: TCGA data shows that UPR genes are upregulated in human cancer, and animal models show that tumor formation and growth rates are lower in mice lacking key UPR genes such as IRE1α and XBP1. The UPR promotes tumor growth not only cell-intrinsically, via cancer cell cytoprotection, but also cell-extrinsically, by polarizing tumor-infiltrating macrophages to a pro-tumor inhibitory/ immune stimulatory (IIS) phenotype. We recently found that the IRE1α-XBP1 branch of the UPR is responsible for the IIS signature that occurs in ER stressed macrophages. Here, we sought to develop a novel strategy to target the UPR in macrophages for the treatment of cancer. We focused on microRNAs (miRNA), since no approved small molecule inhibitors of the UPR exist at present for use in cancer patients due to toxicity. We used a proprietary technology developed by our lab that allows for the generation of “induced” extracellular vesicles (iEVs) from B cells that contain a microRNA of choice as cargo. Previously, we reported that transfection of B cells with plasmid DNA encoding for a pre-miRNA loop results in the secretion of large numbers of iEVs that are enriched in the corresponding mature miRNA. Using this technology, we engineered a plasmid DNA to code for miR-214, a miRNA known to target the 3'-UTR of XBP1 mRNA, and used this plasmid to transfected B cells, yielding iEV-214. iEV-214 were highly enriched in miR-214 relative to control iEVs, which include EVs from B cells that were not transfected (iEV-empty), transfected with a scrambled miRNA (iEV-scr), or transfected with an un-related miRNA (iEV-1291). Bone marrow-derived macrophages (BMDM) treated with iEV-214 were enriched in miR-214 relative to all of the controls, demonstrating that iEVs can transfer their miRNA payload to BMDM. When we induced ER stress in BMDM treated with iEV-214, XBP1 transcription was reduced over 50% compared to iEV-scr. Together, these data show that iEVs can transfer miR-214 into macrophages and reduce expression of XBP1 mRNA. We propose iEV-214 as a prototype of a new class of cancer therapeutics to correct UPR-mediated macrophage dysregulation. Citation Format: Stephen Searles, Gonzalo Alamanza, Maurizio Zanetti. Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4067.

Activation of Mitochondrial orf355 Gene Expression by a Nuclear-Encoded DREB Transcription Factor Causes Cytoplasmic Male Sterility in Maize

Coordination between mitochondria and the nucleus is crucial for fertility determination in plants with cytoplasmic male sterility (CMS). Using yeast one-hybrid screening, we identified a transcription factor, ZmDREB1.7, that is highly expressed in sterile microspores at the large vacuole stage and activates the expression of mitochondria-encoded CMS gene orf355. Δpro, a weak allele of ZmDREB1.7 with the loss of a key unfolded protein response (UPR) motif in the promoter, partially restores male fertility of CMS-S maize. ZmDREB1.7 expression increases rapidly in response to antimycin A treatment, but this response is attenuated in the Δpro allele. Furthermore, we found that expression of orf355 in mitochondria activates mitochondrial retrograde signaling, which in turn induces ZmDREB1.7 expression. Taken together, these findings demonstrate that positive-feedback transcriptional regulation between a nuclear regulator and a mitochondrial CMS gene determines male sterility in maize, providing new insights into nucleus–mitochondria communication in plants.

IRE1α Disruption in Triple-Negative Breast Cancer Cooperates with Antiangiogenic Therapy by Reversing ER Stress Adaptation and Remodeling the Tumor Microenvironment.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

MYC and the unfolded protein response in cancer: synthetic lethal partners in crime?

The transcription factors of the MYC family play pivotal roles in the initiation and progression of human cancers. High oncogenic level of MYC invades low‐affinity sites and enhancer sequences, which subsequently alters the transcriptome, causes metabolic imbalance, and induces stress response. The endoplasmic reticulum (ER) not only plays a central role in maintaining proteostasis, but also contributes to other key biological processes, including Ca2+ metabolism and the synthesis of lipids and glucose. Stress conditions, such as shortage in glucose or oxygen and disruption of Ca2+ homeostasis, may perturb proteostasis and induce the unfolded protein response (UPR), which either restores homeostasis or triggers cell death. Crucial roles of ER stress and UPR signaling have been implicated in various cancers, from oncogenesis to treatment response. Here, we summarize the current knowledge on the interaction between MYC and UPR signaling, and its contribution to cancer development. We also discuss the potential of targeting key UPR signaling nodes as novel synthetic lethal strategies in MYC‐driven cancers.