XBP1s Expression Research Articles

Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell’s viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

The inositol-requiring enzyme 1 (IRE1) endoplasmic reticulum stress pathway promotes MDA-MB-231 cell survival and renewal in response to the aryl-ureido fatty acid CTU

Current treatment options for triple-negative breast cancer (TNBC) are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity. However, there was a residual cell population that survived treatment. The present study evaluated the mechanisms that underlie survival and renewal in CTU-treated MDA-MB-231 TNBC cells. RNA-seq profiling identified several pro-inflammatory signaling pathways that were activated in treated cells. Increased expression of cyclooxygenase-2 and the cytokines IL-6, IL-8 and GM-CSF was confirmed by real-time RT-PCR, ELISA and Western blot analysis. Increased self-renewal was confirmed using the non-adherent, in vitro colony-forming mammosphere assay. Neutralizing antibodies to IL-6, IL-8 and GM-CSF, as well as cyclooxygenase-2 inhibition suppressed the self-renewal of MDA-MB-231 cells post-CTU treatment. IPA network analysis identified major NF-κB and XBP1 gene networks that were activated by CTU; chemical inhibitors of these pathways and esiRNA knock-down decreased the production of pro-inflammatory mediators. NF-κB and XBP1 signaling was in turn activated by the endoplasmic reticulum (ER)-stress sensor inositol-requiring enzyme 1 (IRE1), which mediates the unfolded protein response. Co-treatment with an inhibitor of IRE1 kinase and RNase activities, decreased phospho-NF-κB and XBP1s expression and the production of pro-inflammatory mediators. Further, IRE1 inhibition also enhanced apoptotic cell death and prevented the activation of self-renewal by CTU. Taken together, the present findings indicate that the IRE1 ER-stress pathway is activated by the anti-cancer lipid analogue CTU, which then activates secondary self-renewal in TNBC cells.

The gut microbe-derived metabolite trimethylamine-N-oxide induces aortic valve fibrosis via PERK/ATF-4 and IRE-1α/XBP-1s signaling in vitro and in vivo

Background and aimsThe gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) has been implicated in the development of cardiovascular fibrosis. Endoplasmic reticulum (ER) stress occurs after the dysfunction of ER and its structure. The three signals PERK/ATF-4, IRE-1α/XBP-1s and ATF6 are activated upon ER stress. Recent reports have suggested that the activation of PERK/ATF-4 and IRE-1α/XBP-1s signaling contributes to cardiovascular fibrosis. However, whether TMAO mediates aortic valve fibrosis by activating PERK/ATF-4 and IRE-1α/XBP-1s signaling remains unclear. MethodsHuman aortic valve interstitial cells (AVICs) were isolated from aortic valve leaflets. PERK IRE-1α, ATF-4, XBP-1s and CHOP expression, and production of collagen Ⅰ and TGF-β1 were analyzed following treatment with TMAO. The role of PERK/ATF-4 and IRE-1α/XBP-1s signaling pathways in TMAO-induced fibrotic formation was determined using inhibitors and small interfering RNA. ResultsDiseased valves produced greater levels of ATF-4, XBP-1, collagen Ⅰ and TGF-β1. Interestingly, diseased cells exhibited augmented PERK/ATF-4 and IRE-1α/XBP-1s activation after TMAO stimulation. Inhibition and silencing of PERK/ATF-4 and IRE-1α/XBP-1s each resulted in enhanced suppression of TMAO-induced fibrogenic activity in diseased cells. Mice treated with dietary choline supplementation had substantially increased TMAO levels and aortic valve fibrosis, which were reduced by 3,3-dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) treatment. Moreover, a high-choline and high-fat diet remodeled the gut microbiota in mice. ConclusionsTMAO promoted aortic valve fibrosis through activation of PERK/ATF-4 and IRE-1α/XBP-1s signaling pathways in vitro and in vivo. Modulation of diet, gut microbiota, TMAO, PERK/ATF-4 and IRE1-α/XBP-1s may be a promising approach to prevent aortic valve fibrosis.

IRE1-mediated degradation of pre-miR-301a promotes apoptosis through upregulation of GADD45A

The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here, we determine IRE1’s role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A’s 3’ UTR with a target protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these results indicate that IRE1’s endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis.9J7o_YRPucfgg8rDu5QLJgVideo

The endoplasmic reticulum acetyl-CoA transporter AT-1 plays a role in the protective unfolded protein response in the exocrine pancreas

Pancreatic acinar cells are responsible for the synthesis and secretion of digestive enzymes. In order to sustain high rates of secretory protein production, acinar cells rely on adaptive mechanisms to mitigate endoplasmic reticulum (ER) stress and maintain secretory homeostasis. The adaptive unfolded protein response (UPR), mediated by the IRE1-XBP1s axis, is essential for acinar function and protects against cellular injury. Recent work has demonstrated that the ER acetyl-CoA transporter, AT-1, is necessary for nascent secretory protein acetylation and export out of the ER and is regulated by IRE1-XBP1s. Furthermore, AT-1 is rapidly downregulated during experimental pancreatitis. To investigate the role of AT-1 in exocrine pancreas function, we generated inducible, acinar-specific AT-1 knockout mice (Ela-Cre AT-1-/-). Loss of AT-1 produced a chronic pancreatitis phenotype characterized by inflammation, immune cell infiltration, tissue fibrosis, and aberrant intracellular activation of the proteolytic digestive enzyme trypsin. Despite the histological and biochemical dysfunction observed, Ela-Cre AT-1-/- mice grow normally, have typical lifespans, and do not lose pancreatic mass. Interestingly, XBP1s expression is significantly upregulated in Ela-Cre AT-1-/- and may confer a protective effect with loss of AT-1. Our data suggests a role for AT-1 in the IRE1-XBP1s axis and highlights the previously undescribed role of protein acetylation in exocrine pancreas physiology. Given the numerous potential downstream effects of dysregulated acetyl-CoA transport, this model will advance our understanding of pancreatic function and disease. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The plasma cell proteome initiates early in B cell activation in advance of Blimp-1.

Abstract Plasma cell (PC) differentiation is a highly regulated process that radically changes the morphology and function of a B lymphocyte to that of a professional protein secretor. It is becoming clear that this process precedes increase in immunoglobulin synthesis rather than reacting to it. We have shown previously that large segments of the PC transcriptome are initiated in an mTORC1-dependent manner prior to both Blimp-1 and Xbp1s expression. Still, the kinetics of induction of the PC proteome remain largely unknown. We performed a whole proteome kinetic analysis of activated Blimp-1-reporter follicular B cells in PC-inductive conditions every 12 hours for three days and observed distinct patterns in induction of the PC proteome. To elucidate transcriptional networks, we performed single cell RNA-seq with the same conditions and correlated transcription factor expression with genes coding for these proteome modules within single cells. We found distinct subgroups within proteome modules corresponding to transcriptional patterns each with a highly correlated transcription factor set. These included robust, transient, Myc-associated factors required for translation initiation; rapid, sustained expression of protein synthesis machinery; and gradual, sustained increases in proteins associated with ER function and secretion. Each of these preceded the induction of Blimp-1 and increase in immunoglobulin protein. Finally, using inducible deletion of Blimp-1 in conjunction with the reporter allele we show that these unique transcriptional and proteomic programs are initiated independently of Blimp-1 expression. Thus, activated B cells prepare for protein secretion by initiating the PC proteome prior to antibody secretion. Supported by grants from the NIH: R01 AI113545, AI139123, AI154932, R21 AI133998 (PI:Allman) and BTG was supported by training grant T32 CA009140 (PI:Pear)

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.

Protein Aggregation Patterns Inform about Breast Cancer Response to Antiestrogens and Reveal the RNA Ligase RTCB as Mediator of Acquired Tamoxifen Resistance.

Simple SummaryAcquired resistance to antiestrogenic therapy remains the major obstacle to curing luminal subtype breast cancer. While current treatment in acquired endocrine-resistant settings includes combined therapy with receptor tyrosine kinase or cyclin-dependent kinase inhibitors, progression to incurable disease remains possible. In recent years, the antioxidant system and the protein quality control network have been associated with the enhanced resistance of breast cancer cells to hormonal therapy. In this work, we raise the hypothesis that antiestrogen treatment induces the accumulation of protein aggregates in sensitive cells, which in turn could hinder the activation of survival pathways. We present evidence concerning a novel way to identify antiestrogen response and disclose a novel protein, RTBC, that controls acquired antiestrogen resistance. This work opens a new avenue for research towards finding breast cancer prognostic markers and therapeutic targets, where the identification of proteins prone to aggregate could help to identify antiestrogen response and understand mechanisms of disease.The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.

Suppression of Cdc37 Induces BTZ Resistance through XBP1s Mediated Plasma Cell Immaturation in Multiple Myeloma

Backgrounds: Cell division cycle protein 37 (Cdc37) is considered as a key co-chaperone of heat shock protein 90 (Hsp90), guiding the stabilization and activation of client kinases with time specificity and substrate selectivity. Previous studies have demonstrated that Cdc37 is over-expressed in many kinds of cancers, contributing to the tumorigenesis. However, our previous study suggested that some MM cell population with low expression of Cdc37 was correlated with BTZ resistance in clinical MM samples and in vitro study. The purpose of this study is to investigate how Cdc37 contributes to drug resistance in MM.Materials and methods: Nineteen sequential MM samples with gene expression profiling (GSE19554) obtained from published studies were analyzed. The expression of Cdc37 was investigated in MM cell lines and primary CD138+ cells by qRT-PCR. Cdc37 function was then interrupted by Cdc37 shRNA or Hsp90-Cdc37 inhibitor Celastrol in MM cell line. Sensitivity of BTZ was evaluated. Bone marrow MM cells were enriched and gated into subpopulations by CD38 and CD138 status by FACS. CD38+CD138+ plasma cells, CD38+CD138- plasmablasts and CD38-CD138- B cells were examined for Xbp1s and Cdc37 expression by qRT-PCR and IF. Then cellular morphology, supernatant light chain immunoglobulin and makers of plasma cell maturation were checked when MM cells were exposed to Celastrol or underwent Xbp1s overexpression. Finally, the in vivo verification was carried out in 5TGM1 mouse model.Results: The GEP analysis showed that Cdc37 was down-regulated as the progression of MM (base line, pre-1st transplantation, pre-2nd transplantation, post-2nd transplantation) in 13 out of 19 sequential samples. Our results also showed that some relapsed MM patients had a down-regulated Cdc37 expression compared with newly diagnosed cases (0.78±1.13 vs 0.25±1.05, p <0.05). Moreover, Cdc37 was only suppressed in bortezomib (BTZ) resistant cell line ANBL-6. BR compared with their wild type ANBL-6.WT. While there were no significant differences between doxorubicin-, dexamethasone- resistant cell line. Indeed, suppression Cdc37 in MM cell line by Cdc37-shRNA or Celastrol resulted in a lower apoptosis rate after BTZ treatment. These results indicated that Cdc37 may participate in BTZ resistance.To explore the mechanism of Cdc37 mediating BTZ resistance, we tried to determine if Cdc37 suppression might cause immaturity of plasma cells, which is well established to cause MM cells resistant to BTZ. Cdc37 expression was first found to be positively correlated with Xbp1s in primary MM cells (r=0.61, p <0.001). Then CD38+CD138+ plasma cells, CD38+CD138- plasmablasts and CD38-CD138- B cells were examined for Xbp1s and Cdc37 and showed that Cdc37 and Xbp1s were gradually up-regulated during the maturation of plasma cells in 3/5 patients. The results indicated that Cdc37 may induce BTZ resistancethroughXBP1s mediated plasma cell immaturation.To certify our hypothesis, Cdc37-Hsp90 interaction were interrupted with Celastrol, NCI-H929 cells appeared smaller and rounder with larger nucleus, less cytoplasm, resembling plasmablast. ELISA analysis also demonstrated a decreased secretion of free light chain from MM cells. Partial repression of plasma cell maturation markers, such as Xbp1s, Chop, CD49e were also observed when Cdc37 was inhibited in NCI-H929 cells. However, the above changes were reversed when Xbp1s was overexpressed in MM cells. In vivo, the serum IgG2b level was (146.8±4.0) ng/ml and (166.0±13.0) ng/ml (p<0.05) in BTZ group and BTZ+Celastrol group, respectively. Although statistical significance were not reached, the BTZ+Celastrol group showed a shorter overall survival compared with BTZ group.Conclusion: Our results demonstrated that Cdc37 downregulation may result in BTZ resistance through Xbp1s mediated plasma cell immaturation. These findings provide a certain theoretical reference for the proper combination of BTZ and other therapy, forcing BTZ resistant cells differentiation, to fight against BTZ resistance. More importantly, Cdc37 may provide a diagnostic biomarker for predicting BTZ sensitivity prior to treatment. DisclosuresNo relevant conflicts of interest to declare.

Adar-Mediated Aberrant a-to-I RNA Editing Is Driven By 1q Amplification and Contributes to Proteasome Inhibitor Resistance in Multiple Myeloma

Multiple Myeloma (MM) is an incurable malignancy of plasma cells characterized by wide and remarkable genetic heterogeneity. The amplification of 1q identifies a class of patients characterized by a more aggressive disease course and poor prognosis. While previous research has identified several oncogenes with prognostic relevance located in 1q, the biological mechanisms underlying the poor clinical outcome associated with this alteration is still unclear.ADAR (Adenosine Deaminase Acting on RNA) is an enzyme responsible for A-to-I editing, a post-transcriptional process which contributes to transcriptome and proteome diversity by conversion of specific adenosines into inosines in both coding and non-coding transcripts. Such changes may affect RNA stability and regulation and introduce mutations in the corresponding proteins.Recent research has reported dysregulation of RNA editing in cancer, revealing its oncogenicity and clinical relevance. Since ADAR is located in 1q21, the critical amplified region in MM, we sought to investigate the implications of ADAR activity in MM.We have previously shown increased ADAR expression and A-to-I RNA editing in a small group of newly diagnosed patients with 1q21 amplification enrolled in the MMRF CoMMpass study (Lagana et al, ASH 2016), and that ADAR expression could differentiate prognosis. To further investigate and characterize the role of ADAR and RNA Editing in MM, we have analyzed 590 RNAseq samples from newly diagnosed patients in CoMMpass. ADAR copy number gain was observed in 111 samples (19%).ADAR expression was significantly higher in patients with 1q21 gain (p < 0.0001), supporting the role of copy number gain as a driver of ADAR expression. Moreover, patients with high expression of ADAR and no CN alterations, showed significant activation of Interferon pathways (Fig. 1A). This can be explained by the fact that one isoform of ADAR (p150) is regulated by an interferon-inducible promoter. Multivariate regression of ADAR expression against ADAR CN and STAT1 expression, as a measure of interferon signaling activation, showed that both ADAR CN and STAT1 expression were significant independent predictors of ADAR expression.To investigate the functional impact of ADAR activity, we implemented a computational pipeline to calculate sample-wise Alu Editing Index (AEI). A-to-I RNA editing targets predominantly Alu elements, repetitive sequences abundantly interspersed throughout the human genome, mostly within introns and untranslated regions (UTRs). AEI has been developed and validated as a global measure of RNA editing activity at the sample level (Bazak et al, Genome Res 2014). Our analysis in MM revealed that AEI could discriminate between good and poor prognosis in terms of both PFS and OS (p < 0.0001). Moreover, AEI could critically stratify patients with 1q21 gain in terms of PFS (p < 0.01) (Fig. 1B). This finding suggests that RNA editing can be an adverse prognostic factor independently of 1q21 gain.Patients with high AEI were characterized by down-regulation of immunoglobulin transcripts and of the Unfolded Protein Response (UPR) pathway. In particular, patients with high AEI exhibited down-regulation of key activators of UPR such as NFYC, ATF4 and XBP1, and of transcriptional targets of XBP1s, the active spliced form of XBP1. Decreased UPR and low XBP1s expression in pre-plasmablasts have been shown to induce resistance to proteasome inhibitors (PI) (e.g. Bortezomib) through de-differentiation of plasma cells (Leung-Hagesteijn et al, Cancer Cell 2013). Among patients who received Bortezomib-based first line therapy, we found that AEI was significantly higher (p<0.01) in patients whose best response was stable disease (SD) or progressive disease (PD) (23; 4.5%) compared to patients who had complete (CR) or stringent complete response (sCR) (118; 23%) (Fig. 1C). siRNA meditated knockdown of ADAR increased sensitivity to Bortezomib in MM cell lines with 1q amplification and high ADAR expression (OPM2), consistently with our in-silico findings.In conclusion, we propose a model of MM where ADAR-mediated RNA editing is driven by gain of 1q21 and interferon signaling and contributes to disease aggressiveness and resistance to PI-based therapy through down-regulation of UPR (Fig. 1D). [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Cho:Agenus, Inc.: Research Funding; Genentech: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Multiple Myeloma Research Foundation: Research Funding. Barlogie:Millenium Pharmaceuticals: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Jagannath:Merck: Consultancy; Medicom: Speakers Bureau; Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy; MMRF: Speakers Bureau. Dudley:Ecoeos, Inc.: Equity Ownership; Ayasdi, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy; NuMedii, Inc.: Equity Ownership, Patents & Royalties; Janssen Pharmaceuticals, Inc.: Consultancy; GlaxoSmithKline: Consultancy.

XBP1s Acts as a Tumor Suppressor to Inhibit the EMT Process and Metastasis of Papillary Thyroid Cancer.

PurposePapillary thyroid cancer (PTC) patients could obtain poor prognosis if they have lymph node metastasis, identification of informative and robust biomarkers for predicting cervical lymph node metastasis is critical for improving clinical decision-making and patient prognosis.Materials and MethodsIn this study, we analyzed the expression of X box binding protein 1 spliced-form (XBP1s) in 41 PTC tissue samples and extended our findings using public databases, then we investigated how XBP1s’ contributed to PTC progression in vitro.ResultsWe found that XBP1s’ expression was lower in PTC patients with cervical lymph node metastasis than non-metastasis patients by immunohistochemical analysis. With publicly accessible dataset, we showed that the XBP1 transcription was significantly decreased in thyroid cancer (TC) tissues with lymph node metastasis as compared to that without lymph node metastasis. Moreover, we also found that XBP1 expression was significantly correlated with patients’ gender, T classification, lymph node metastasis and PTC stages, and low XBP1 expression was associated with poor diseases free survival (DFS). In vitro, XBP1s overexpression could inhibit the invasion, migration, and wound healing capacity of PTC cells. Mechanistically, overexpression of XBP1s could enhance the expression of classical epithelial–mesenchymal transition (EMT) markers such as ZO-1 and E-cadherins, and downregulated N-cadherin in BCPAP cells.ConclusionThese findings suggest that XBP1s is a prognostic maker for thyroid carcinoma patients, and sustaining XBP1s expression might be a new strategy to control PTC progression.

Allicin alleviated acrylamide-induced NLRP3 inflammasome activation via oxidative stress and endoplasmic reticulum stress in Kupffer cells and SD rats liver

Acrylamide (AA) in heat-processed food leads to widespread concerns due to its hepatotoxicity. Allicin, a plant-derived antioxidant, possesses a significant protective effect on AA-induced hepatotoxicity, but the mechanism is still unclear. Herein, we investigated the mechanism in Kupffer cells and SD rats liver. Molecular docking, molecular dynamics simulation and LigPlus software speculated that allicin inhibited the activity of CYP2E1 expression by binding to its amino acid residues Phe116, Phe207, Leu210, Phe298, Ala299, Thr303, Val364 and Phe478 through hydrophobic interactions. Allicin decreased the reactive oxygen species (ROS) release and CYP2E1 protein expression and then alleviated the appearance of OS. Meanwhile, allicin significantly reduced ERS characteristic proteins GRP78, CHOP and UPR branch IRE1α pathway key proteins p-IRE, p-ASK, TRAF2 and XBP-1s expression. Simultaneously, allicin ameliorated OS and ERS activation, which inhibited the activation of the MAPK and NF-κB pathways, and down-regulated JNK, ERK, p38, p65 and IκBα phosphorylation. Allicin pre-treatment inhibited AA-induced inflammation as evidenced by reducing NLRP3 inflammasome activation, decreasing Cleaved-Caspase-1 expression as well as IL-1β, IL-18, IL-6 and TNF-α secretion. Taken together, our data provide new insights into possible signaling pathways involved in allicin attenuating AA-induced hepatotoxicity in vivo and in vitro.

Development of Tumor-Targeting IRE-1 Inhibitors for B-cell Cancer Therapy.

The IRE-1 kinase/RNase splices the mRNA of the XBP-1 gene, resulting in the spliced XBP-1 (XBP-1s) mRNA that encodes the functional XBP-1s transcription factor that is critically important for the growth and survival of B-cell leukemia, lymphoma, and multiple myeloma (MM). Several inhibitors targeting the expression of XBP-1s have been reported; however, the cytotoxicity exerted by each inhibitor against cancer cells is highly variable. To design better therapeutic strategies for B-cell cancer, we systematically compared the ability of these compounds to inhibit the RNase activity of IRE-1 in vitro and to suppress the expression of XBP-1s in mouse and human MM cell lines. Tricyclic chromenone-based inhibitors B-I09 and D-F07, prodrugs harboring an aldehyde-masking group, emerged as the most reliable inhibitors for potent suppression of XBP-1s expression in MM cells. The cytotoxicity of B-I09 and D-F07 against MM as well as chronic lymphocytic leukemia and mantle cell lymphoma could be further enhanced by combination with inhibitors of the PI3K/AKT pathway. Because chemical modifications of the salicylaldehyde hydroxy group could be used to tune 1,3-dioxane prodrug stability, we installed reactive oxygen species-sensitive structural cage groups onto these inhibitors to achieve stimuli-responsive activities and improve tumor-targeting efficiency.

HBEGF Induced XBP1s Expression, a Novel Form of Unfolded Protein Response

HBEGF Induced XBP1s Expression, a Novel Form of Unfolded Protein Response

Effects of fatty acids on inducing endoplasmic reticulum stress in bovine mammary epithelial cells

Fatty acids play important roles in the regulation of endoplasmic reticulum (ER) stress-induced apoptosis in different cells. Currently, the effects of fatty acids on bovine mammary epithelial cells (MEC) remain unknown. Our study examined bovine MEC viability and measured unfolded protein response (UPR)-related gene and protein expressions following fatty acid treatments. To evaluate the role of fatty acids, we treated MAC-T cells (a line of MEC) with 100 to 400 μM of saturated (palmitic and stearic acid) and unsaturated (palmitoleic, oleic, linoleic, and linolenic acid) fatty acids and 1 to 5 mM of short- and medium-chain fatty acids (acetic, propionic, butyric, and octanoic acid). Thereafter, we determined UPR-related gene expression using quantitative real-time PCR. Palmitic acid stimulated expression of XBP1s, ATF4, ATF6A, and C/EBP homologous protein (CHOP). Stearic acid increased expression of XBP1s and CHOP and decreased expression of ATF4 and ATF6A. Results of Western blot analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that palmitic and stearic acid reduced MAC-T cell viability and induced extreme ER stress by increasing the protein expression of ER stress markers, such as phospho-PKR-like endoplasmic reticulum kinase, phospho-eIF2α, cleaved CASP-3, and CHOP. Among unsaturated long-chain fatty acids, palmitoleic acid increased expression of ATF4 and ATF6A. Oleic acid increased expression of XBP1s, ATF4, and ATF6A. Linoleic and linolenic acids increased expression of XBP1s, ATF4, and ATF6A but decreased expression of XBP1s and ATF6A at the highest dose. Although palmitoleic, oleic, and linoleic acid decreased CHOP expression, only palmitoleic acid increased MAC-T cell viability. Therefore, unsaturated long-chain fatty acids did not induce severe ER stress. Acetic, propionic, and butyric acids decreased expression of ATF4, ATF6A, and CHOP and increased XBP1s expression. Although only octanoic acid increased ATF4 and ATF6A expressions, it lowered expression of XBP1s and CHOP. Although fatty acid treatment did not increase the levels of ER stress proteins, butyric and octanoic acids reduced cell viability, possibly because of early differentiation. These results suggest that saturated fatty acids play important roles in MEC viability by inducing severe ER stress compared with unsaturated fatty acids. In addition, acetic and propionic acids (short- and medium-chain fatty acids) reduced ER stress. Therefore, the present study reflects the new insight that serum fatty acid concentration plays an important role in maintaining the lactation physiology of dairy cows.

Rapid Death of Follicular B Cells and Burkitt Lymphoma Cells Effectuated by Xbp1s.

BCR-mediated tonic signaling is an indispensable requirement for the survival of follicular B (FOB) cells and Burkitt lymphoma (BL) cells. FOB cells of the I-A12% mutant mouse express unfolded protein response and are extremely short lived. Among the myriad molecules activated by unfolded protein response in I-A12% B cells, Xbp1s singularly "hijacked" p110 from p85:p110 heterodimeric PI3K, thereby abating BCR tonic signaling, resulting in their extremely short lifespan. Long-lived normal FOB cells became short lived upon ectopic Xbp1s expression. The proapoptotic Xbp1s role in FOB cells starkly contrasts with its antithetical prosurvival function in plasma cells. Also, tonic signaling and clonal expansion, two important functions mediated by the same BCR, operate in independent and distinct manners. Furthermore, concerning the development of new therapeutic treatment of drug-refractory BL patients, our finding of Xbp1s-mediated rapid death of BL cells brings forth a conceptual advancement based on blocking PI3K heterodimer formation rather than inhibition of PI3K enzyme activity.

LncRNA DANCR attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation through regulating of miR-33a-5p/XBP1s

Brain microvascular endothelial cell (BMEC) survival and angiogenesis after ischemic stroke has great significance for improving the prognosis of stroke. Abnormal variants of lncRNAs are closely associated with stroke. In this study, we examined the effects and molecular mechanisms of differentiation antagonizing non-protein coding RNA (DANCR) on apoptosis, migration, and angiogenesis of oxygen-glucose deprivation (OGD)-treated BMECs. We found that DANCR expression significantly increased at 2, 4, 6, 8, and 10 h after OGD. DANCR overexpression promoted cell viability, migration, and angiogenesis in OGD-treated BMECs. Additionally, we found that X-box binding protein l splicing (XBP1s) expression was positively correlated with DANCR expression. DANCR overexpression promoted XBP1s expression in OGD-treated BMECs. Silenced XBP1s reversed the effect of DANCR in OGD-treated BMECs. Furthermore, we found that microRNA (miR)-33a-5p bound to DANCR and the 3'-UTR of XBP1. miR-33a-5p overexpression inhibited proliferation, migration, angiogenesis, and XBP1s expression in OGD-treated DANCR-overexpressing BMECs, reversing the protective effect of DANCR. Finally, we found that XBP1s expression promoted proliferation, migration, and angiogenesis, reversing the damaging effect of miR-33a-5p. In conclusion, DANCR enhanced survival and angiogenesis in OGD-treated BMECs through the miR-33a-5p/XBP1s axis.

EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells

Targeting EGFR combined with chemotherapy is one of the most valuable therapeutic strategies in colorectal cancer. However, resistance remains a major obstacle to improve efficacy. IRE1α-XBP1s signaling pathway is activated in many malignant tumors, and plays important roles in chemoresistance. Therefore, IRE1α-XBP1s might be a potential target to overcome the chemoresistance in colorectal cancer. In this study, we detected the activation of IRE1α-XBP1s signaling in patient cancer tissues and colorectal cancer cell lines. The phosphorylation level of IRE1α and the spliced XBP1s were aberrantly elevated in colorectal cancer, and IRE1α-XBP1s signaling activation was correlated with high EGFR expression. By overexpression of EGFR protein or activation by EGF treatment, we found that EGFR activation could enhance the phosphorylation of IRE1α and spliced XBP1s expression. On the contrary, inhibition of EGFR decreased the IRE1α-XBP1s signaling. Further, we examined the downstream signaling pathways regulated by EGFR. Inhibition of ERK activity could reverse the EGFR induced IRE1α-XBP1s activation. Co-IP confirmed the physical interaction of ERK and IRE1α. Cell growth and colony formation assay showed that the inhibition of IRE1α activity could suppress EGFR driven colorectal cancer cell proliferation. Furthermore, we found that oxaliplatin could activate IRE1α-XBP1s signaling, and combination with cetuximab partially reversed the activation. Inhibition of EGFR signaling could enhance the efficacy of oxaliplatin in vitro and in vivo. Our results showed that IRE1α RNase activity is aberrantly elevated in colorectal cancer, and EGFR signaling could activate IRE1α/XBP1s possibly through EGFR-MEK-ERK pathway. IRE1α-XBP1s pathway might involve in EGFR driven tumor cell proliferation. Cetuximab could partially recover oxaliplatin-induced IRE1α-XBP1s activation, and therefore enhance the anti-tumor efficacy of oxaliplatin. Our findings declare a new mechanism that targeting EGFR could inhibit chemotherapy-induced IRE1α-XBP1s activation and therefore enhance the efficacy.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

Abstract A119: Inhibition of IRE1-mediated pro-tumorigenic responses enhances the sensitivity of MDA-MB-231 cells to the novel arylurea-fatty acid CTU

Abstract There is an unmet need for new agents with low toxicity for treatment of triple-negative breast cancer (TNBC) because current approaches are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity (Rawling et al., 2017). However, there was a residual cell population that survived CTU. The present study evaluated the mechanism underlying residual cell survival. From RNA-seq profiling, a major regulated process in CTU-treated cells was activation of the unfolded response and endoplasmic reticulum (ER)-stress. The ER transmembrane protein inositol-requiring enzyme 1 (IRE1) is a central mediator of the unfolded response and ER-stress that was activated by CTU. IRE1 is also implicated as a pro-tumorigenic factor that can activate the expansion of tumor-initiating cells. From RNA-seq several pro-tumorigenic factors, including cyclooxygenase-2 and the cytokines IL6, IL8 and GM-CSF, were strongly activated in CTU-treated cells; this was confirmed by real-time RT-PCR and Western blot analysis. Using the non-adherent, in vitro colony-forming mammosphere assay, that enables the quantification of cell self-renewal, it was found that CTU expanded tumor-initiating cells. Co-treatment with an inhibitor of IRE1 impaired IRE1 kinase and IRE1 RNase activities, as reflected by decreases in phospho-p65 and XBP1s expression. IRE1 inhibition also prevented the activation of pro-tumorigenic factors and self-renewal by CTU. Taken together, the present findings suggest that inhibition of IRE1 may be a novel approach to optimise the activity of CTU in TNBC. Citation Format: Md Khalilur Rahman, Tristan Rawling, Yassir Al-Zubaidi, Hassan Choucair, Bala Umashankar, Yongjuan Chen, Kirsi Bourget, Stanton Tam, Charlotte Smith, Michael Murray. Inhibition of IRE1-mediated pro-tumorigenic responses enhances the sensitivity of MDA-MB-231 cells to the novel arylurea-fatty acid CTU [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A119. doi:10.1158/1535-7163.TARG-19-A119