X-box Binding Protein 1 Expression Research Articles

XBP1 Regulates the Transcription of HIF-1a in BALB/c Mice with Chronic Rhinosinusitis without Polyps.

X-box binding protein 1 (XBP1) is a transcription factor that recognizes the CRE-like element in enhancers of human T-cell leukemia virus and MHC class II gene and induces their transcription. This study was performed to characterize the function of XBP1, which was identified to be a differentially expressed gene via GEO database, in chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP). XBP1 expression was significantly elevated in both CRSsNP patients and mice who were accompanied with mucosal thickening, goblet cell hyperplasia and chemosis, glandular hyperplasia, and dense infiltration of inflammatory cells. Silencing of XBP1 suppressed the development of CRSsNP in mice. Mechanistically, knockdown of XBP1 downregulated the expression of hypoxia-inducible factor 1-alpha (HIF-1a), and overexpression of XBP1 led to the opposite result. Silencing of HIF-1a inhibited β-catenin expression and impaired the Wnt/β-catenin pathway. Further overexpression of HIF-1a in XBP1-silenced CRSsNP mice exacerbated pathological changes in mouse nasal mucosal tissues, promoted inflammation, and activated the Wnt/β-catenin pathway. Taken together, overexpression of XBP1 may be associated with increased expression of HIF-1a and possibly contribute to the Wnt/β-catenin pathway activation and the development of CRSsNP.

X-box binding protein 1 (XBP1): a potential role in chemotherapy response, clinical pathologic features, non-inflamed tumour microenvironment for breast cancer

X-box binding protein 1 (XBP1) is mainly expressed in breast cancer (BC) in human cancers. Its tumorigenesis and favourable prognosis are contradictory, and its essential role in chemotherapeutic response and immunosuppression is unknown in BC. The study firstly identified XBP1 who received neoadjuvant chemotherapy (NAC) from GSE25055 and GSE24460. Associations between XBP1 expression and clinicopathological characteristics was investigated using Oncomine, TCGA, UALCAN and bc-GenExMiner. The prognostic value of XBP1 was assessed using the Kaplan–Meier Plotter, bc-GenExMiner, GSE25055, and GSE25056. Furthermore, we systematically correlated XBP1 and immunological characteristics in the BC tumour microenvironment (TME) using TISIDB, TIMER, GSE25055, GSE25056 and TCGA dataset. Finally, an essential role of XBP1 in chemotherapy response was evaluated based on GSE25055, GSE25065, GSE24460, GSE5846, ROC Plotter and CELL databases. Furthermore, XBP1 mRNA expression levels were obviously highest in BC among human cancers and were significantly related to a good prognosis. In addition, XBP1 mRNA and protein levels were higher in the luminal subtype than in normal tissues and basal-like subtype, which might be attributed to membrane transport-related processes. Apart from BC, negative immunological correlations of XBP1 were not observed in other malignancies. XBP1 might shape the non-inflamed TME in BC. Finally, XBP1 expression was higher in chemo-resistive than chemo-sensitive cases, it had a predictive value and could independently predict chemotherapy response in BC patients receiving NAC. Our study suggests that the essential role of XBP1 in clinical pathologic features, non-inflamed TME, chemotherapy response in BC.

IL‐10 Attenuates ER‐Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes

Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox‐induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein‐kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X‐box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress‐initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase‐12 activation, phosphorylation of c‐JUN NH2‐terminal kinase (JNK) and pro‐apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning.In this study on isolated rat cardiomyocytes, we analyzed ER‐stress markers, spliced XBP1 mRNA, and ER‐apoptotic proteins. We also assessed protective effects of Interleukin‐10 (IL‐10) against Dox initiated ER‐induced apoptosis and overall ER‐stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL‐10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER‐stress markers as well as ER‐related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL‐10 treatment were recorded. IL‐10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox‐treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL‐10 in Dox treated cells. Dox also initiated activation of procaspase‐12, which is known to activate caspase‐3 and apoptosis. IL‐10 significantly reduced the activation of procaspase‐12 and cell death. These data suggest that IL‐10 provides protection against Dox‐induced cardiotoxicity by restoring ER homeostasis.

Oxidative Stress-Induced Growth Inhibitor (OSGIN1), a Target of X-Box-Binding Protein 1, Protects Palmitic Acid-Induced Vascular Lipotoxicity through Maintaining Autophagy.

Saturated free fatty acids (FFAs) strongly correlate with metabolic syndromes and are well-known risk factors for cardiovascular diseases (CVDs). The mechanism of palmitic acid (PA)-induced vascular lipotoxicity under endoplasmic reticulum (ER) stress is unknown. In the present paper, we investigate the roles of spliced form of X-box-binding protein 1 (XBP1s) target gene oxidative stress-induced growth inhibitor 1 (OSGIN1) in PA-induced vascular dysfunction. PA inhibited the tube formation assay of primary human umbilical vein endothelial cells (HUVECs). Simultaneously, PA treatment induced the XBP1s expression in HUVECs. Attenuate the induction of XBP1s by silencing the XBP1s retarded cell migration and diminished endothelial nitric oxide synthase (eNOS) expression. OSGIN1 is a target gene of XBP1s under PA treatment. The silencing of OSGIN1 inhibits cell migration by decreasing phospho-eNOS expression. PA activated autophagy in endothelial cells, inhibiting autophagy by 3-methyladenine (3-MA) decreased endothelial cell migration. Silencing XBP1s and OSGIN1 would reduce the induction of LC3 II; therefore, OSGIN1 could maintain autophagy to preserve endothelial cell migration. In conclusion, PA treatment induced ER stress and activated the inositol-requiring enzyme 1 alpha–spliced XBP1 (IRE1α–XBP1s) pathway. OSGIN1, a target gene of XBP1s, could protect endothelial cells from vascular lipotoxicity by regulating autophagy.

MiR-148a targets XBP1 to regulate trophoblast apoptosis induced by plasma reticulum stress in preeclampsia.

To study the relationship between miR-148a and preeclampsia (PE), and clarify that miR-148a can regulate the endoplasmic reticulum stress (ERS) of placental trophoblasts by targeting the ERS protein X box binding protein 1 (XBP1).Fifty patients with hypertension during pregnancy, patients with mild PE, patients with severe PE, and normal pregnant women were selected, and their placental tissues were collected. RT-polymerase chain reaction was used to detect the expression of miR-148a in placental tissues, and Western blot was used to detect XBP1 in placental tissues. Compare the expression differences of miR-148a and XBP1 in each group, and analyze the correlation between the expressions of the two.Compared with the Neg-miR group, MTT experiment result in pre-miR-148a group was decreased. MTT experiment result in anti-miR-148a group was increased. Cell cycle test result in pre-miR-148a group [G1 (%)] was increased. Cell cycle test result in anti-miR-148a group [S (%)] was increased. Apoptosis test result in pre-miR-148a group [early apoptotic cells (%), late apoptotic cells (%)] was increased. Apoptosis test result in anti-miR-148a group [early apoptotic cells (%), late apoptotic cells (%)] was decreased. XBP1 expression result in pre-miR-148a group was increased. XBP1 expression result in anti-miR-148a group was decreased. Compared with the normal population, XBP1 is expressed in hypertension, mild eclampsia, severe eclampsia increased. GRP78, CHOP, and caspase-12 expression result in pre-miR-148a group was increased. GRP78, CHOP, and caspase-12 expression result in anti-miR-148a group was decreased.miR-148a can regulate the ERS-mediated apoptosis by targeting XBP1, thereby intervening in the occurrence and development of PE.

XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and protects against diabetic beta cell failure during metabolic stress in mice

Aims/hypothesisPancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states.MethodsMice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate.ResultsDeletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response.Conclusions/interpretationThese findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.Graphical abstract

Role of XBP1 in regulating the progression of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is associated with the dysregulation of lipid metabolism and hepatic inflammation, though the underlying mechanisms remain unclear. We aimed to investigate the role of X-box binding protein-1 (XBP1) in the progression of NASH. Human liver tissues obtained from patients with NASH and controls were used to assess XBP1 expression. NASH models were developed in hepatocyte-specific Xbp1 knockout (Xbp1ΔHep), macrophage-specific Xbp1 knockout (Xbp1ΔMf), macrophage-specific Nlrp3 knockout, and wild-type (Xbp1FL/FL or Nlrp3FL/FL) mice fed with a high-fat diet for 26 weeks or a methionine/choline-deficient diet for 6 weeks. The expression of XBP1 was significantly upregulated in liver samples from patients with NASH. Hepatocyte-specific Xbp1 deficiency inhibited the development of steatohepatitis in mice fed the high-fat or methionine/choline-deficient diets. Meanwhile, macrophage-specific Xbp1 knockout mice developed less severe steatohepatitis and fibrosis than wild-type Xbp1FL/FL mice in response to the high-fat or methionine/choline-deficient diets. Macrophage-specific Xbp1 knockout mice showed M2 anti-inflammatory polarization. Xbp1-deleted macrophages reduced steatohepatitis by decreasing the expression of NLRP3 and secretion of pro-inflammatory cytokines, which mediate M2 macrophage polarization in macrophage-specific Xbp1 knockout mice. Steatohepatitis was less severe in macrophage-specific Nlrp3 knockout mice than in wild-type Nlrp3FL/FL mice. Xbp1-deleted macrophages prevented hepatic stellate cell activation by decreasing expression of TGF-β1. Less fibrotic changes were observed in macrophage-specific Xbp1 knockout mice than in wild-type Xbp1FL/FL mice. Inhibition of XBP1 suppressed the development of NASH. XBP1 regulates the development of NASH. XBP1 inhibitors protect against steatohepatitis. Thus, XBP1 is a potential target for the treatment of NASH. XBP1 is a transcription factor that is upregulated in liver tissues of patients with non-alcoholic steatohepatitis (NASH). Conditional knockout of Xbp1 in hepatocytes resulted in decreased lipid accumulation in mice, while genetic deletion of Xbp1 in macrophages ameliorated nutritional steatohepatitis and fibrosis in mice. Pharmacological inhibition of XBP1 protects against steatohepatitis and fibrosis, highlighting a promising therapeutic strategy for NASH.

ER Stress is Involved in Mast Cells Degranulation via IRE1α/miR-125/Lyn Pathway in an Experimental Intracerebral Hemorrhage Mouse Model.

The degranulation of mast cells accounts for the development of neuroinflammation following intracerebral hemorrhage (ICH). Inhibition of IRE1α, a sensor signaling protein related to endoplasmic reticulum stress, has been shown to exert anti-inflammatory effects in several neurological diseases. The objective of this study was to investigate the effects of IRE1α inhibition on mast cells degranulation in an ICH mouse model and to explore the contribution of miR-125/Lyn pathway in IRE1α-mediated mast cells degranulation. Male mice were subjected to ICH by intraparenchymal injection of autologous blood. STF083010, an inhibitor of IRE1α, was administered intranasally at 1h after ICH induction. AntimiR-125 was delivered by intracerebroventricular (i.c.v.) injection prior to ICH induction to elucidate the possible mechanisms. Western blot analysis, immunofluorescence staining, neurological test, hematoma volume, brain water content, toluidine blue staining and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), tryptase, interleukin-17A (IL-17A), tumor necrosis factor α (TNF-α) and tryptase mRNA were increased in time dependent manner while miR-125b-2-3p was decreased after ICH. Inhibition of IRE1α, with STF083010, remarkably reduced brain water content, improved neurological function, decreased hematoma volume, upregulated the expression of miR-125b-2-3p, decreased the number of mast cells, and downregulated the protein expression of Lyn kinase, XBP1s (spliced X-box binding protein-1), tryptase, IL-17A and TNF-α. The downregulation of Lyn kinase, tryptase, IL-17A, TNF-α, and decreased mast cells number were reversed by antimiR-125. The present findings demonstrate that IRE1α inhibition attenuates mast cells degranulation and neuroinflammation, at least partially, through IRE1α/miR-125/Lyn signaling pathway after ICH.

XBP1s promotes the development of lung adenocarcinoma via the p‑JNK MAPK pathway.

Spliced X-box binding protein 1 (XBP1s) has been reported to participate in the pathogenesis of numerous types of cancer; however, whether XBP1s plays a role in lung cancer remains to be elucidated. In the present study, bioinformatics analysis was performed to determine the mRNA expression level of XBP1 in lung cancer and adjacent normal tissues. Gene Ontology terms, pathway enrichment and Pearson's correlation analysis were performed to investigate the possible mechanism involved. Western blot and reverse transcription-quantitative PCR were performed to quantify the protein and mRNA expression level of target proteins, respectively. Small interfering RNA or overexpression plasmid were used to knockdown or overexpress the expression level of XBP1s. EdU staining, colony formation, Cell Counting Kit-8, Transwell and wound healing assays, and flow cytometry were performed to detect the proliferation, colony forming ability, cell viability, migration and invasion ability, and the apoptosis rate. The results showed that the mRNA and protein expression level of XBP1 was higher in tumor tissues compared with that in adjacent normal tissues using data from the TIMER2.0, ONCOMINE and UALCAN online databases. In addition, the mRNA expression level of XBP1 was also associated with clinical features, including age, smoking habit, individual cancer stage and nodal metastasis status. In the in vitro experiments, the mRNA and protein expression level of XBP1s was increased in the A549 cell line compared with that in the human bronchial epithelial (HBE), H1299, PC9 and H460 cell lines. Hypoxia further increased the protein expression level of XBP1s in the A549 cell line. Knockdown of XBP1s expression in the A549 cell line resulted in decreased proliferation, colony formation, cell viability, migration and invasion, and increased apoptosis. By contrast, overexpressing XBP1s in the HBE cell line led to the opposite results. To investigate the mechanism involved, proteins associated with XBP1 were analyzed using the LinkedOmics database. Pathway enrichment revealed the MAPK pathway to be the possible XBP1 downstream target. Furthermore, Pearson's correlation and western blot analyses verified that phosphorylated (p)-JNK rather than p-ERK or p-p38 was the downstream effector of XBP1s. Phosphorylation of JNK was decreased when XBP1s expression was knocked down in the A549 cell line under normoxic and hypoxic conditions. Inhibiting p-JNK with SP600125 reversed the increased prosurvival effects caused by XBP1s overexpression. The results from the present study suggest that XBP1s/p-JNK function as a prosurvival factors in the A549 cell line and could be a potential target for the treatment of lung adenocarcinoma.

Clinicopathologic and protein markers distinguishing the "polymerase epsilon exonuclease" from the "copy number low" subtype of endometrial cancer.

ObjectiveThe need to perform genetic sequencing to diagnose the polymerase epsilon exonuclease (POLE) subtype of endometrial cancer (EC) hinders the adoption of molecular classification. We investigated clinicopathologic and protein markers that distinguish the POLE from the copy number (CN)-low subtype in EC.MethodsNinety-one samples (15 POLE, 76 CN-low) were selected from The Cancer Genome Atlas EC dataset. Clinicopathologic and normalized reverse phase protein array expression data were analyzed for associations with the subtypes. A logistic model including selected markers was constructed by stepwise selection using area under the curve (AUC) from 5-fold cross-validation (CV). The selected markers were validated using immunohistochemistry (IHC) in a separate cohort.ResultsBody mass index (BMI) and tumor grade were significantly associated with the POLE subtype. With BMI and tumor grade as covariates, 5 proteins were associated with the EC subtypes. The stepwise selection method identified BMI, cyclin B1, caspase 8, and X-box binding protein 1 (XBP1) as markers distinguishing the POLE from the CN-low subtype. The mean of CV AUC, sensitivity, specificity, and balanced accuracy of the selected model were 0.97, 0.91, 0.87, and 0.89, respectively. IHC validation showed that cyclin B1 expression was significantly higher in the POLE than in the CN-low subtype and receiver operating characteristic curve of cyclin B1 expression in IHC revealed AUC of 0.683.ConclusionBMI and expression of cyclin B1, caspase 8, and XBP1 are candidate markers distinguishing the POLE from the CN-low subtype. Cyclin B1 IHC may replace POLE sequencing in molecular classification of EC.

XBP1 is required in Th2 polarization induction in airway allergy

Rationale: Th2 polarization plays a central role in the pathogenesis of allergic diseases such as airway allergy. The underlying mechanism is not fully understood yet. X-box-binding protein-1 (XBP1) can regulate immune cell activities upon exposing stressful events. The role of XBP1 in the development of Th2 polarization has not yet been explored.Methods: Mice carrying Xbp1-deficient CD4+ T cells were employed to observe the role of XBP1 in the induction of airway allergy. A cell culture model was established to evaluate the role of XBP1 in facilitating the Th2 lineage commitment.Results: We found that Xbp1 ablation in CD4+ T cells prevented induction of Th2 polarization in the mouse airway tract. XBP1 was indispensable in the Th2 lineage commitment. XBP1 mediated the effects of 3-methyl-4-nitrophenol (MNP) on facilitating inducing antigen-specific Th2 response in the airways. Exposure to MNP induced expression of XBP1 in CD4+ T cells. RhoA facilitated the binding between XBP1 and GATA3 in CD4+ T cells. XBP1 induced GATA3 phosphorylation to promote the Il4 gene transcription. Modulation of the RhoA/XBP1 axis mitigated experimental allergic response in the mouse airways.Conclusions: A potential therapeutic target, XBP1, was identified in this study. XBP1 was required in the development of skewed Th2 response in the airways. Inhibiting XBP1 alleviated Th2 polarization-related immune inflammation in the airways. The data suggest that inhibiting XBP1 has the translation potential for the treatment of airway allergy.

Tumor innervation is triggered by endoplasmic reticulum stress.

Nerve infiltration in the tumor microenvironment is emerging as a promoter of cancer progression that could be targeted in therapies, but the mechanisms initiating tumor innervation remain to be elucidated. Here we report that endoplasmic reticulum (ER) stress in cancer cells is transmitted to neuronal cells, resulting in neurite outgrowth and tumor innervation. In vitro, the induction of ER stress in various human cancer cells resulted in the synthesis and release of the precursor for brain-derived neurotrophic factor (proBDNF) through a mechanism dependent on the transcription factor X-box binding protein 1 (XBP1). Cancer cell-released proBDNF was found to mediate the transmission of ER stress to neurons, resulting in the stimulation of neurite outgrowth. Next-generation sequencing indicated the increased expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) that was mediated by c-MYC and necessary to neurite outgrowth induced by proBDNF. In orthotopic tumor xenograft, ER stress stimulated XBP1 and proBDNF expression as well as tumor innervation. Anti-proBDNF antibody inhibited both tumor innervation and cancer progression induced by ER stress. Interestingly, the chemotherapeutic drug 5-Fluorouracil (5-FU) was found to induce ER stress and tumor innervation, and this effect was inhibited by anti-proBDNF antibody. Finally, in human tumors, cancer tissues with nerve infiltration expressed high XBP1 and proBDNF while EGLN3 was upregulated in infiltrated nerves. This study reveals that ER stress participates in tumor innervation through the release of proBDNF and that targeting this pathway could be used in future therapies.

Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next‐generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis

Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested invitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. Invivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein1. Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Dihydroartemisinin triggers ferroptosis in primary liver cancer cells by promoting and unfolded protein response‑induced upregulation of CHAC1 expression.

Dihydroartemisinin (DHA), an artemisinin derivate, has been investigated as a potential antitumor drug in primary liver cancer (PLC). Ferroptosis is a form of iron-dependent cell death that can be driven by lipid peroxidation inducers. The present study aimed to determine whether and how DHA could promote the death of PLC cells by inducing ferroptosis. In total, four PLC cell lines with different p53 statuses, including Hep3B (p53 null), Huh7 (p53 mutant), PLC/PRF/5 (p53 mutant) and HepG2 (p53 wild-type), were treated with various concentrations of DHA. The effects of DHA on all three branches of the unfolded protein response (UPR) were evaluated. To deactivate the UPRs, small interfering RNA was used to knockdown the expression of activating transcription factor (ATF)4, X-box binding protein 1 (XBP1) or ATF6 in PLC cells. The effect of DHA on the promoter activity of Chac glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) was evaluated using a dual luciferase reporter assay. The results revealed that DHA-induced death in PLC cells was irrelevant of the p53 status. PLC cells exposed to DHA displayed classic features of ferroptosis, such as increased lipid reactive oxygen species and malondialdehyde levels, an iron overload, and decreased activity or expression of glutathione (GSH), glutathione peroxidase 4, solute carrier family (SLC) 7 member 11 and SLC family 3 member 2. The antitumor effects of DHA in PLC cells were significantly weakened by two typical ferroptosis inhibitors, ferrostatin-1 and deferoxamine mesylate salt, whereas the antitumor effects were augmented following iron overload. Furthermore, DHA activated all three branches of the UPR (eukaryotic translation initiation factor 2 α kinase 3/eukaryotic translation initiation factor 2A/ATF4, inositol-requiring transmembrane kinase/endoribonuclease 1α/XBP1 and ATF6 branches) in vitro. Notably, DHA-induced ferroptosis was significantly attenuated following the knockdown of ATF4, XBP1 or ATF6 expression. In addition, the promoter activity of CHAC1, a gene capable of degrading GSH, was enhanced by DHA, but weakened when the aforementioned three UPR transcription factors were knocked down. In conclusion, the findings of the present study suggested that DHA may effectively induce ferroptosis in PLC cells through the activation of anti-survival UPRs and the upregulation of CHAC1 expression.

Silencing circ‑BIRC6 inhibits the proliferation, invasion, migration and epithelial‑mesenchymal transition of bladder cancer cells by targeting the miR‑495‑3p/XBP1 signaling axis.

Circular RNAs (circRNAs) regulate gene expression by acting as a ‘sponge’ for microRNAs (miRs) and play crucial roles in tumorigenesis, including in bladder cancer (BC). circRNA-baculoviral IAP repeat-containing 6 (circ-BIRC6) has been reported to participate in the pathogenesis of several cancer types. The present study aimed to elucidate the roles and potential mechanisms of circ-BIRC6 in the progression of BC. circ-BIRC6 expression levels in BC cell lines were determined using reverse transcription-quantitative PCR. Following circ-BIRC6 knockdown, cell proliferation, invasion and migration were detected using Cell Counting Kit-8, colony formation, Transwell and wound healing assays, respectively. Western blotting was also conducted to evaluate the expression levels of X-box binding protein 1 (XBP1) and epithelial-mesenchymal transition (EMT)-associated proteins. In addition, rescue experiments were performed using by transfecting a miR-495-3p inhibitor into T24 cells following circ-BIRC6 knockdown. The interactions between circ-BIRC6, miR-495-3p and XBP1 was verified using dual luciferase reporter assays. Moreover, T24 cells with circ-BIRC6 knockdown and miR-495-3p inhibitor transfection were used for the tumor-bearing experiment. Tumor growth was observed and Ki-67 expression was determined using immunohistochemistry. The results demonstrated that circ-BIRC6 expression was upregulated in BC cell lines. Moreover, circ-BIRC6 knockdown notably attenuated the proliferation, invasion, migration and EMT of BC cells, which was blocked by the miR-495-3p inhibitor. It was also identified that circ-BIRC6 sponged miR-495-3p to regulate XBP1 expression. In addition, results from the xenograft experiments indicated that the knockdown of circ-BIRC6 and miR-495-3p expression significantly inhibited tumor growth. It was also found that the expression levels of XBP1, Ki-67 and EMT-associated proteins in tumor tissues of the co-transfection group were markedly restored compared with the circ-BIRC6 knockdown group. In conclusion, these findings demonstrated that circ-BIRC6 knockdown suppressed BC tumorigenesis and progression via regulation of the miR-495-3p/XBP1 signaling axis, offering a promising therapeutic target for the treatment of BC.

MicroRNA-64 Promotes Progression of Liver Cancer Through Targeting X-Box Binding Protein 1 (XBP1)

Liver cancer is still a horrible threat to people’s health worldwide. Herein, we aimed to explore the mechanism underlying microRNA (miR)-64’s role in liver cancer progression to provide novel insight into therapy. Expression of miR-64 in liver cancer cells was determined by RT-qPCR. Luciferase reporter gene assay detected the potential binding between miR-64 and X-box binding protein 1 (XBP1). Cancer cells were transfected with specific plasmids of XBP1, followed by MTT experiment and Ttranswell assay to assess invasion and proliferation. miR-64 was highly expressed in liver cancer cells and positively correlated with cell proliferation, invasion, and metastasis. miR-64 targeted and inhibited XBP1 expression. Overexpression of miR-64 significantly resulted in increased cell viability and invasion. Taken altogether, miR-64 promotes liver cancer through inhibiting XBP1, providing insight into miR-64-based targeted therapy against liver cancer.

Circ_0006168 Promotes the Migration, Invasion and Proliferation of Esophageal Squamous Cell Carcinoma Cells via miR-516b-5p-Dependent Regulation of XBP1.

BackgroundCircular RNAs (circRNAs) exert important roles in carcinogenesis. Here, we aimed to uncover the working mechanism of circ_0006168 in esophageal squamous cell carcinoma (ESCC) development.MethodsWestern blot assay and real-time quantitative polymerase chain reaction (RT-qPCR) were used to determine protein and RNA expression, respectively. Wound healing assay and transwell migration assay were performed to assess cell migration ability, whereas cell invasion ability was evaluated by transwell invasion assay. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay were utilized to analyze cell proliferation ability. Xenograft tumor model was utilized to assess the role of X-box binding protein 1 (XBP1) in xenograft tumor growth in vivo. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay were used to verify intermolecular interactions.ResultsXBP1 silencing suppressed the migration, invasion and proliferation of ESCC cells in vitro and restrained the xenograft tumor growth in vivo. MicroRNA-516b-5p (miR-516b-5p) interacted with the 3ʹ untranslated region (3ʹUTR) of XBP1 in ESCC cells. MiR-516b-5p overexpression inhibited the proliferation and motility of ESCC cells. MiR-516b-5p was a molecular target of circ_0006168 in ESCC cells. The interference of circ_0006168 restrained the motility and proliferation of ESCC cells. Circ_0006168 acted as miR-516b-5p sponge to up-regulate XBP1 expression in ESCC cells. MiR-516b-5p silencing or the accumulation of XBP1 largely rescued the proliferation ability and motility in circ_0006168-silenced ESCC cells.ConclusionIn conclusion, circ_0006168 contributed to ESCC development through promoting the proliferation and motility of ESCC cells via mediating miR-516b-5p/XBP1 axis.

Downregulation of XBP1 protects kidney against ischemia-reperfusion injury via suppressing HRD1-mediated NRF2 ubiquitylation

Ischemia-reperfusion (IR) injury to the renal epithelia is associated with endoplasmic reticulum stress (ERS) and mitochondria dysfunction, which lead to oxidative stress-induced acute kidney injury (AKI). X-box binding protein 1 (XBP1), an ERS response protein, could play a prominent role in IR-induced AKI. In this study, we revealed that XBP1 and its downstream target HRD1 participated in the crosstalk between ERS and mitochondrial dysfunction via regulation of NRF2/HO-1-mediated reactive oxidative stress (ROS) signaling. Mice with reduced expression of XBP1 (heterozygous Xbp1±) were resistant to IR-induced AKI due to the enhanced expression of NRF2/HO-1 and diminished ROS in the kidney. Downregulation of XBP1 in renal epithelial cells resulted in reduced HRD1 expression and increased NRF2/HO-1 function, accompanied with enhanced antioxidant response. Furthermore, HRD1 served as an E3-ligase to facilitate the downregulation of NRF2 through ubiquitination-degradation pathway, and the QSLVPDI motif on NRF2 constituted an active site for its interaction with HRD1. Thus, our findings unveil an important physiological role for XBP1/HRD1 in modulating the antioxidant function of NRF2/HO-1 in the kidney under stress conditions. Molecular therapeutic approaches that target XBP1-HRD1-NRF2 pathway may represent potential effective means to treat renal IR injury.

Therapeutic effects of sertraline on improvement of Ovariectomy-induced decreased spontaneous activity in mice.

We have already reported that ovariectomized (OVX) rats reduced the spontaneous activity during the dark period due to the decease of serotonin release in the amygdala. In this study, we examined the potential of sertraline, a selective serotonin reuptake inhibitor, on the recovery of less spontaneous activity seen in mice with OVX-induced despair-like behaviors. Female 9-week old ICR mice were underwent either OVX or sham surgery. Sertraline (10 mg/kg/day, s.c.) or saline were started to administer to each group for 8 weeks (6 times/week) from the 8th week after OVX. Each spontaneous activity of mouse was evaluated during the dark period (19:00-07:00) using an infrared sensor. Moreover, mRNA expression levels of tryptophan hydroxylase (TPH) and X-box binding protein 1 (XBP1) were measured in the hippocampus and prefrontal cortex using by a real-time PCR method. We found out that the OVX-induced despair-like behaviors were improved by the continuous administration of sertraline. After treatment of OVX, our real-time PCR data showed that sertraline significantly suppressed the upregulation of XBP1 expression levels in both hippocampus and prefrontal cortex, although this suppression of the downregulation of TPH expression levels was seen in only hippocampus. These results suggest that sertraline improves the decrease in spontaneous activity induced by OVX assessed by the hippocampus suppressing decreased serotonin synthesis in the serotonergic neuron.