Knockdown Of GRP78 Research Articles

Cannabidiol’s cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic’s such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.

Targeting GRP78 suppresses oncogenic KRAS protein expression and reduces viability of cancer cells bearing various KRAS mutations

KRAS is the most commonly mutated oncogene in human cancers with limited therapeutic options, thus there is a critical need to identify novel targets and inhibiting agents. The 78-kDa glucose-regulated protein GRP78, which is upregulated in KRAS cancers, is an essential chaperone and the master regulator of the unfolded protein response (UPR). Following up on our recent discoveries that GRP78 haploinsufficiency suppresses both KRASG12D-driven pancreatic and lung tumorigenesis, we seek to determine the underlying mechanisms. Here, we report that knockdown of GRP78 via siRNA reduced oncogenic KRAS protein level in human lung, colon, and pancreatic cancer cells bearing various KRAS mutations. This effect was at the post-transcriptional level and is independent of proteasomal degradation or autophagy. Moreover, targeting GRP78 via small molecule inhibitors such as HA15 and YUM70 with anti-cancer activities while sparing normal cells significantly suppressed oncogenic KRAS expression in vitro and in vivo, associating with onset of apoptosis and loss of viability in cancer cells bearing various KRAS mutations. Collectively, our studies reveal that GRP78 is a previously unidentified regulator of oncogenic KRAS expression, and, as such, augments the other anti-cancer activities of GRP78 small molecule inhibitors to potentially achieve general, long-term suppression of mutant KRAS-driven tumorigenesis.

Abstract 6372: Nanodrug delivery system for non-genotoxic p53 activator INZ-C

Abstract p53 is a known tumor suppressor protein whose gene is altered in 50% of all human cancers, while its function is inactivated in the many cancers harboring wild type p53, making it an attractive target for targeted cancer therapies. In this regard, our lab had previously discovered a small molecule, Inauhzin (INZ C), as a potent non-genotoxic p53 activator. In an effort to improve the bioavailability and efficacy, we have now encapsulated the INZ C small molecule in a chitosan/cyclodextrin nanoparticle delivery system. Evaluation of in vitro/in vivo activity and protien expression suggests nanoparticle encapsulation of INZ-C (n-INZ-C) improved p53 induction and inhibition of cell growth in lung and colorectal cancers, while maintaining a minimal effect for MEF and Wi38 normal cells. These results are consistent with the significantly reduced cellular uptake observed for normal cells as compared to the cancer cells. H/E staining of tissues for mice treated with n-INZ-C reveals no toxicity to other organs as well. Additionally, n-INZ-C demonstrates the ability to block migration of cells in vitro as demonstrated by both wound healing and trans-well migration assays. Our in vivo pharmacokinetic evaluation suggests nanoparticle encapsulation doubles the half-life of INZ-C from 2.5 to 5 h. These results suggest that nanoparticle encapsulation improved activity and bioavailability of INZ-C while maintaining the non-toxic nature of INZ-C in normal cells. In addition, we unveiled GRP78 as a potential new target of INZ-C and found out that knockdown of GRP78 can alleviate the inhibitory effect of INZ-C on lung and colorectal cancer proliferation. Further studies now focus on revealing the mechanism of enhanced cellular uptake for INZ-C in cancer cells as comapred to normal cells. Thus, these results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic and also identified a novel target of INZ-C. Citation Format: Nimisha Bhattarai, Jieqiong Wang, Daniel Nguyen, Xiaoxiao Yang, Linh Helmers, Jennifer Paruch, Li Li, Yiwei Zhang, Kun Meng, Alun Wang, Janarthanan Jayawickramarajah, Binghe Wang, Shelya Zeng, Hua Lu. Nanodrug delivery system for non-genotoxic p53 activator INZ-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6372.

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles.

Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Oxymatrine attenuates arsenic-induced endoplasmic reticulum stress and calcium dyshomeostasis in hepatic stellate cells.

BackgroundOxymatrine is the main bioactive component of Sophora flavescens. It exhibits various biological activities and has been used in various liver diseases, including hepatic fibrosis (HF). Hepatic stellate cells (HSCs) are the primary cell type involved during HF progression. Oxymatrine treatment could suppress the proliferation of HSCs and degrade the extracellular cell matrix (ECM), presumed to be associated with HF. However, the mechanism is still unknown.MethodsNaAsO2 induces HF in LX2 cells. Oxymatrine was used to treat NaAsO2- induced LX2 cells. Then, the LX2 cell proliferation, apoptosis, ECM secretion protein, oxidative stress index, and intracellular calcium concentration were respectively measured. Furthermore, after knocking down GRP78 [endoplasmic reticulum (ER) chaperone BiP] or overexpressing of SERCA2 (ATPase sarcoplasmic/ER Ca2+ transporting 2) in NaAsO2-induced LX2 cells, we detected the changes in ER stress and calcium homeostasis in LX2 cells.ResultsNaAsO2 exposure promoted apoptosis, increased ECM secretion, produced ER stress, and disrupted calcium homeostasis, which could be attenuated by oxymatrine treatment. Furthermore, knockdown of GRP78 to alleviate ER stress, or overexpression of SERCA2 to restore intracellular calcium homeostasis can inhibit the NaAsO2 effect.ConclusionsOxymatrine treatment could improve calcium homeostasis and attenuate ER stress to reverse NaAsO2-induced HSC activation and ECM secretion, which are the significant phenotypes of HF. The ER stress and calcium homeostasis may be the therapeutic targets for HF.

Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 μM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 μg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.

Endoplasmic Reticulum Stress in Subepithelial Myofibroblasts Increases the TGF-β1 Activity That Regulates Fibrosis in Crohn's Disease.

Endoplasmic reticulum (ER) stress is an essential response of epithelial and immune cells to inflammation in Crohn's disease. The presence and mechanisms that might regulate the ER stress response in subepithelial myofibroblasts (SEMFs) and its role in the development of fibrosis in patients with Crohn's disease have not been examined. Subepithelial myofibroblasts were isolated from the affected ileum and normal ileum of patients with each Montreal phenotype of Crohn's disease and from normal ileum in non-Crohn's subjects. Binding of GRP78 to latent TGF-β1 and its subcellular trafficking was examined using proximity ligation-hybridization assay (PLA). The effects of XBP1 and ATF6 on TGF-β1 expression were measured using DNA-ChIP and luciferase reporter assay. Endoplasmic reticulum stress components, TGF-β1, and collagen levels were analyzed in SEMF transfected with siRNA-mediated knockdown of DNMT1 and GRP78 or with DNMT1 inhibitor 5-Azacytidine or with overexpression of miR-199a-5p. In SEMF of strictured ileum from patients with B2 Crohn's disease, expression of ER stress sensors increased significantly. Tunicamycin elicited time-dependent increase in GRP78 protein levels, direct interaction with latent TGF-β1, and activated TGF-β1 signaling. The TGFB1 DNA-binding activity of ATF-6α and XBP1 were significantly increased and elicited increased TGFB1 transcription in SEMF-isolated from affected ileum. The levels of ER stress components, TGF-β1, and collagen expression in SEMF were significantly decreased following knockdown of DNMT1 or GRP78 by 5-Azacytidine treatment or overexpression of miR-199a-5p. Endoplasmic reticulum stress is present in SEMF of patients susceptible to fibrostenotic Crohn's disease and can contribute to development of fibrosis. Targeting ER stress may represent a novel therapeutic target to prevent fibrosis in patients with fibrostenotic Crohn's disease.

Spliced XBP1 Levels Determine Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitor Bortezomib Independent of the Unfolded Protein Response Mediator GRP78.

Background: Mechanisms mediating resistance against the proteasome inhibition by bortezomib (BTZ) in multiple myeloma (MM) cells are still unclear. We analyzed the activation of the unfolded protein response (UPR), induction of prosurvival, and apoptotic pathways after proteasome inhibition in BTZ-sensitive and -resistant cells. Thereafter, these findings from tissue culture were proofed on MM cells of BTZ-sensitive and BTZ-refractory patients.Methods: Proteasomal and ABC transporter activities were measured in sensitive and resistant cell lines by the use of the respective substrates. TP53 gene loss and mutations were determined by cytogenetics and targeted NGS. UPR pathways, proteasome subunit levels and protein secretion were studied by Western Blot analysis, and apoptosis was determined by flow cytometry. MM cell lines were stably transfected with inducible GRP78 expression to study unfolded protein expression. Transient knock-down of GRP78 was done by RNA interference. Splicing of XBP1 and expression of GRP78 was studied by real-time PCR in CD138-enriched MM primary cells of BTZ-refractory and -sensitive patients.Results: BTZ-sensitive cells displayed lower basal proteasomal activities. Similar activities of all three major ABC transporter proteins were detected in BTZ-sensitive and resistant cells. Sensitive cells showed deficiencies in triggering canonical prosurvival UPR provoked by endoplasmic reticulum (ER) stress induction. BTZ treatment did not increase unfolded protein levels or induced GRP78-mediated UPR. BTZ-resistant cells and BTZ-refractory patients exhibited lower sXBP1 levels. Apoptosis of BTZ-sensitive cells was correlating with induction of p53 and NOXA. Tumor cytogenetics and NGS analysis revealed more frequent TP53 deletions and mutations in BTZ-refractory MM patients.Conclusions: We identified low sXBP1 levels and TP53 abnormalities as factors correlating with bortezomib resistance in MM. Therefore, determination of sXBP1 levels and TP53 status prior to BTZ treatment in MM may be beneficial to predict BTZ resistance.

CSFV protein NS5A activates the unfolded protein response to promote viral replication

Classical swine fever is a world organization for animal health listed disease and is caused by classical swine fever virus (CSFV). CSFV can induced unfolded protein response (UPR) and whether NS5A protein plays a role in this process remains unknown. Here, we demonstrate that CSFV induced all the three signal pathways ATF6, IRE1 and PERK of UPR. Furthermore, this phenomenon may be mediated by the NS5A protein since expression of NS5A alone can achieve the same effect. In the current study, we show that NS5A can interact with GRP78 as measured by using the CO-IP and GST pulldown assays. This interaction plays a positive role in the promotion of CSFV replication. Overexpression or knockdown of GRP78 mediated by lentivirus can enhance or decrease viral replication, respectively. Our findings provide the evidence that CSFV infection can activate the cellular UPRs, in which NS5A and GRP78 play key roles in the process.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

CIGARETTE SMOKE EXTRACT PROMOTES HUMAN LUNG MYOFIBROBLAST DIFFERENTIATION BY THE INDUCTION OF ENDOPLASMIC RETICULUM STRESS

PURPOSE: IPF is a progressive and lethal fibrotic lung disease with an unknown aetiology. Cigarette smoking is a risk factor for IPF and an indicator of poor prognosis. Cigarette smoking induces endoplasmic reticulum (ER) stress, and it has been shown that ER stress promotes fibroblast-to-myofibroblast differentiation in lung fibrosis. In this study, we investigated whether cigarette smoke extract (CSE) promotes lung myofibroblast differentiation via the induction of ER stress. METHODS: Human embryonic lung fibroblasts (MRC-5 fibroblasts) were stimulated with various doses of CSE. Levels of α-smooth muscle actin (α-SMA) protein were evaluated by immunofluorescence and western blot analyses. ER stress was induced by thapsigargin (TG) and inhibited by 4-phenyl butyric acid (4-PBA). Protein levels of GRP78, IRE1, XBP-1 and ATF6 were determined by western blotting. GRP78 siRNA was transfected into MRC-5 cells using Lipofectamine RNAiMAX Reagent. RESULTS: CSE at a concentration of 1.0% significantly increased α-SMA expression in MRC-5 cells. There was no significant cell apoptosis after cells were exposed to CSE. CSE treatment significantly increased the expression of GRP78, IRE1, XBP-1 and ATF6 at the protein level at 48 h. Pretreatment with TG enhanced, whereas pretreatment with 4-PBA inhibited, the CSE-induced expression of α-SMA, GRP78 and XBP-1. Furthermore, knockdown of GRP78 blocked α-SMA expression in MRC-5 cells exposed to CSE. CONCLUSIONS: Our data suggested that CSE promotes lung fibroblast-to-myofibroblast differentiation by the induction of ER stress. CLINICAL IMPLICATIONS: Our studies offer a new strategy to treat smoking-related lung interstitial diseases, such as in IPF patients who smoke.

P118 TARGETING ENDOPLASMIC RETICULUM STRESS FOR THE TREATMENT OF FIBROSIS IN CROHN’S DISEASE

Progressive tissue remodeling characterized by extensive collagen production and excess extracellular matrix deposition leads to intestinal fibrosis and stricture formation in patients with fibrostenotic Crohn’s disease. Currently medical therapies can delay but not prevent the development of critical fibrostenosis necessitating surgery. Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are associated with intestinal epithelial cells damage and apoptosis in Crohn’s disease. However, the role of ER stress and UPR signaling in subepithelial myofibroblasts (SEMF) during the development of intestinal fibrosis awaits further clarification. Previously, we reported that increase in ER stress and UPR signaling in SEMF isolated from strictured ileum contributes to activation of TGF-β1 and fibrosis. Increased interaction between GRP78 and latent TGF-β complex on the cell membrane leads to αVβ3 integrin-mediated activation of latent TGF-β. The ER stress response was augmented in SEMF by silencing of inhibitory miR-199a-5p by DNA methyltransferase-1 (DNMT1). In current study we investigated the effects of selective targeting of activated ER stress sensors on intestinal fibrosis in vitro. SEMF were prepared from cells isolated from affected ileum and from normal ileum in the same patient undergoing resection for Crohn’s disease. SEMF were transfected with siRNA to the ER stress proteins: Grp78, ATF6, XBP1 and IRE1α, and to DNMT1 to knockdown these proteins and compared to scramble sense siRNA treatment. Some cells were co-transfected with a Smad-binding element (SBE) luciferase reporter and TGFB1 transcriptional activity measured. The protein levels of GRP78, DNMT1 and collagen I were measured by immunoblot and normalized to β-actin. Active and total TGF-β1 was measured by ELISA. Our results showed that knockdown of DNMT1 decreased GRP78 protein production, diminished activation of TGF-β1, and blocked ER stress-induced TGF-β1-dependent collagen production. Knockdown of Grp78, Atf6α, Xbp1, and Ire1α in normal SEMF decreased ER stress-induced TGFB1 transcriptional activity by 30 ± 2%, 33 ± 1.5%, 35 ± 1.8%, and 23 ± 1.2%, respectively. In 3D culture of normal SEMF with Grp78 knocked down, tunicamycin failed to induce ER stress, increase activation of TGF-β1 or increase collagen production. Taken together, our results indicate that ER stress in part mediates development of intestinal fibrosis in Crohn’s disease by where DNMT1-induced silencing of miR-199a-5p allows a heighted ER Stress response that results in greater TGF-β1 activation and collagen production in the SEMF.

Role of reciprocal interaction between autophagy and endoplasmic reticulum stress in apoptosis of human bronchial epithelial cells induced by cigarette smoke extract.

Endoplasmic reticulum stress (ERS)-induced apoptosis of airway epithelial cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Furthermore, autophagy is closely related to ERS under apoptosis. Here, this study aimed to investigate the role of the reciprocal interaction between autophagy and ERS in the cigarette smoke extract (CSE)-induced apoptosis of human bronchial epithelial (HBE) cells. Cell apoptosis was detected by flow cytometry analysis. Protein expression was examined by Western blot. The mRNA expression was detected using real-time quantitative reverse transcription PCR (qRT-PCR). The results showed that CSE treatment induced apoptosis, autophagy, and expression of ERS-related proteins in HBE cells. Furthermore, autophagy inhibition by 3-MA significantly decreased protein expression of GRP78, p-PERK, and p-eIF2α and increased CHOP, ATF4, and caspase-4, whereas ERS inhibition by 4-PBA led to autophagy suppression. Moreover, the CSE-induced autophagy was diminished by knockdown of GRP78, PERK, or eIF2α but enhanced by knockdown of ATF4 or CHOP; however, the CSE-induced HBE apoptosis was enhanced by knockdown of GRP78, PERK, or eIF2α but was attenuated by knockdown of ATF4 or CHOP. Additionally, both sodium hydrosulfide (NaHS) and melatonin attenuated the CSE-induced apoptosis, enhanced the CSE-induced autophagy, increased GRP78, p-PERK, and p-eIF2α, and decreased CHOP, ATF4, and caspase-4, via SIRT1/ORP150 pathway. Collectively, this study provided evidence about the role of the reciprocal interaction between autophagy and ERS in CSE-induced apoptosis of HBE cells. © 2018 IUBMB Life, 71(1):66-80, 2019.

Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.

Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response.

ABSTRACTMammalian Ecdysoneless (ECD) is a highly conserved ortholog of the Drosophila Ecd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

Abstract 3066: Inhibition of hsp90 by auy922 preferentially kills mutant KRAS colon cancer cells by activating Bim through ER stress

Abstract Colon cancer is one of the most common and deadly malignancies (1). Despite recent advances in early diagnosis and the development of molecularly targeted therapy, the overall survival of patients with metastatic colon cancers remains disappointing (1). This is often associated with resistance of colon cancer cells to systemic therapies resulting from oncogenic mutations of KRAS that drive activation of multiple downstream signalling pathways important for cell survival and proliferation. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the heat shock protein 90 (HSP90) inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with the comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with activating mutations of KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3 only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. In addition, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and that agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy. Citation Format: Chun Yan Wang, Su Tang Guo, Jia Yu Wang, Xu Guang Yan, Margaret Farrelly, Yuan Yuan Zhang, Fen Liu, Hamed Yari, Ting La, Fu Xi Lei, Lei Jin, Chen Chen Jiang, Xu Dong Zhang. Inhibition of hsp90 by auy922 preferentially kills mutant KRAS colon cancer cells by activating Bim through ER stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2017-3066

Abstract B08: ER chaperone GRP78 increases chemoresistance in pancreatic ductal adenocarcinoma

Abstract Purpose: We show that expression of GRP78, the master regulator of the endoplasmic reticulum (ER) stress response, leads to chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC) and utilize a small molecule inhibitor to block the activity of GRP78, increasing the efficacy of currently available therapies in vitro and in vivo. Background: Gemcitabine (GEM) is the standard chemotherapeutic agent for adjuvant therapy of PDAC and a commonly used agent in other treatment settings, including neoadjuvant treatment of borderline resectable PDAC and palliative treatment of metastatic PDAC. However, recurrent disease is observed in an alarming number of patients treated with GEM or GEM-based treatment regimens. A molecular-level understanding of the mechanisms that contribute to chemoresistance is essential for the development of more effective treatment strategies. ER chaperone proteins protect cells from apoptosis in response to cellular insults, including inflammation and pancreatitis. Elevated expression of chaperone proteins also causes the activation of many “pro-survival” pathways that promote cell survival and resistance to routinely utilized therapeutic regimens. We hypothesize that expression of ER chaperone protein GRP78, is responsible for innate and acquired resistant to GEM and that therapies targeting GRP78 by blocking its activity with the small molecule inhibitor, IT-139, could potentially improve patient response to GEM-based treatment regimens. Methods: We examined if GRP78 overexpression correlated with chemoresistance in PDAC through establishing a patient-derived orthotopic xenograft (PDX) model. Treating tumor-bearing mice with GEM continuously or releasing mice from GEM treatment after 3-4 weeks of exposure identified PDX lines that were either sensitive or resistant to GEM. Tumors from these cohorts were analyzed for GRP78 expression. To examine the role of GRP78 in chemoresistance in vitro, GRP78 expression was induced in GEM-sensitive cell lines prior to GEM treatment. Further, to investigate the effect of GRP78 loss on chemoresistance, siRNA knockdown of GRP78, followed by GEM treatment, was used in GEM-resistant cell lines. Finally, the efficacy of combination treatment with GEM and IT-139, a ruthenium-based anticancer drug and GRP78 inhibitor, was determined in vitro and in vivo. Results: Here, we report that PDX lines that result in GEM resistant PDAC have higher GRP78 expression than that of GEM sensitive PDX lines. In vitro, increased GRP78 induces chemoresistance in GEM-sensitive PDAC cell lines while knockdown of GRP78 reduces chemoresistance in GEM-resistant PDAC cell lines. Finally, we found that IT-139 can overcome GRP78 mediated chemoresistance. In vitro, IT-139 restores sensitivity to cytotoxic drugs in drug resistant PDAC cells and induces twice as much cell death in combination treatment compared with GEM alone. In vivo, a single weekly IT-139 treatment in combination with GEM caused a 35% increase in median survival and a 25% increase in overall survival compared to GEM alone. Conclusions: Collectively, our data show that GRP78 expression promotes chemoresistance in PDAC and therapeutic strategies blocking the activity of GRP78 increase the efficacy of currently available therapies. Citation Format: Jenifer B. Gifford, Wei Huang, Ann E. Zeleniak, Antreas Hindoyan, Hong Wu, Timothy R. Donahue, Reginald Hill.{Authors}. ER chaperone GRP78 increases chemoresistance in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B08.

Insulin enhanced leptin-induced STAT3 signaling by inducing GRP78.

Leptin, an adipocyte-derived hormone, centrally regulates energy homeostasis. Overlaps in the regulation of glucose and energy homeostasis have been reported between leptin and insulin. However, the effects of insulin on leptin’s actions in the central nervous system (CNS) have not yet been elucidated in detail. In the present study, we found that insulin potentiated leptin’s actions through GRP78 in the neuronal cell line, SH-SY5Y-ObRb. Since insulin induces GRP78, we speculated that it may also enhance leptin’s actions through this induction. We found that insulin enhanced leptin-induced STAT3 phosphorylation and this effect was ameliorated by the knockdown of GRP78. The role of GRP78 in leptin’s actions was also confirmed by impairments in leptin-induced STAT3 phosphorylation in HEK293-ObRb cells in which GRP78 was knocked down. Furthermore, we found that the overexpression of GRP78 enhanced leptin-induced STAT3 phosphorylation. These results suggest that GRP78 plays an important role in leptin’s actions. Furthermore, insulin may enhance the leptin-induced activation of STAT3 by inducing GRP78, which may provide an important connection between insulin and leptin in the CNS.

Abstract 291: MANF, a Structurally Unique ER Stress-inducible Protein, Restores ER-protein Folding in ER Stressed Cardiac Myocytes and in the Ischemic Heart

The sarco/endoplasmic reticulum (SR/ER) of cardiomyocytes is a critical site of protein synthesis and folding, as most secreted and membrane proteins including receptors, growth factors, ion channels, and calcium-handling proteins are made at this location. Myocardial ischemia induces ER stress, during which toxic, misfolded proteins accumulate in the SR/ER and contribute to cardiomyocyte death. The branch of the ER stress response mediated by the transcription factor, ATF6, induces ER chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a novel, ubiquitously expressed, ER-luminal protein of unknown function. MANF is structurally unique, thus its function cannot be inferred by structural analogy to known proteins. Since it is ATF6-inducible, and resides in the ER lumen, we hypothesized that MANF is an ER chaperone required for optimal viability of cardiac myocytes during ER stresses, including ischemia. The characteristics of MANF gene induction by ER stress, and the effects of MANF knockdown on the ER stress response and cell viability were determined in cultured neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to restore structure and function to model misfolded proteins was also examined. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo , were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. MANF induction and functional characteristics phenocopied those of a well-studied ER chaperone, glucose-regulated protein 78 (Grp78). Like Grp78, MANF was induced by ER stress in an ATF6-dependent manner. Like knockdown of Grp78, knockdown of MANF in NRVM increased myocyte death in response to ER stress. Like recombinant Grp78, recombinant MANF exhibited a robust ability to restore structure and function to model misfolded proteins, in vitro. Finally, MANF knockdown in the heart, in vivo , increased damage in a mouse model of myocardial infarction. These results suggest that MANF is an SR/ER-resident chaperone required for restoration of SR/ER protein folding during the adaptive ER stress response, and decreasing tissue damage in the ischemic heart.