High GRP78 Expression Research Articles

Grp78 regulates NLRP3 inflammasome and participates in Sjogren’s syndrome

ObjectiveThe purpose of the present study was to potential effects of forsythiaside A (FA) on Sjogren’s syndrome (SS). MethodsEnzyme linked immunosorbent assay for detecting cytokines and Western blotting was used for detecting related protein expression. ResultsFA effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in SS. FA also effectively inhibited the high expression of Grp78 in SS. When Grp78 expression was silenced, it effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. FA effectively inhibit the secretion of inflammatory cytokines induced by overexpression of Grp78, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. ConclusionFA induces the degradation of Grp78 protein, regulates the NF-κB signaling pathway in SS and inhibited NLRP3 inflammasome activation and reduced the release of inflammatory cytokines to alleviate SS.

Synthesis and Evaluation of [18F]AlF-NOTA-c-DVAP: A Novel PET Probe for Imaging GRP78 in Cancer.

GRP78, a member of the HSP70 superfamily, is an endoplasmic reticulum chaperone protein overexpressed in various cancers, making it a promising target for cancer imaging and therapy. Positron emission tomography (PET) imaging offers unique advantages in real time, noninvasive tumor imaging, rendering it a suitable tool for targeting GRP78 in tumor imaging to guide targeted therapy. Several studies have reported successful tumor imaging using PET probes targeting GRP78. However, existing PET probes face challenges such as low tumor uptake, inadequate in vivo distribution, and high abdominal background signal. Therefore, this study introduces a novel peptide PET probe, [18F]AlF-NOTA-c-DVAP, for targeted tumor imaging of GRP78. [18F]AlF-NOTA-c-DVAP was radiolabeled with fluoride-18 using the aluminum-[18F]fluoride ([18F]AlF) method. The study assessed the partition coefficients, stability in vitro, and metabolic stability of [18F]AlF-NOTA-c-DVAP. Micro-PET imaging, pharmacokinetic analysis, and biodistribution studies were carried out in tumor-bearing mice to evaluate the probe's performance. Docking studies and pharmacokinetic analyses of [18F]AlF-NOTA-c-DVAP were also performed. Immunohistochemical and immunofluorescence analyses were conducted to confirm GRP78 expression in tumor tissues. The probe's binding affinity to GRP78 was analyzed by molecular docking simulation. [18F]AlF-NOTA-c-DVAP was radiolabeled in just 25 min with a high yield of 51 ± 16%, a radiochemical purity of 99%, and molar activity within the range of 20-50 GBq/μmol. [18F]AlF-NOTA-c-DVAP demonstrated high stability in vitro and in vivo, with a logD value of -3.41 ± 0.03. Dynamic PET imaging of [18F]AlF-NOTA-c-DVAP in tumors showed rapid uptake and sustained retention, with minimal background uptake. Biodistribution studies revealed rapid blood clearance and excretion through the kidneys following a single-compartment reversible metabolic model. In PET imaging, the T/M ratios for A549 tumors (high GRP78 expression), MDA-MB-231 tumors (medium expression), and HepG2 tumors (low expression) at 60 min postintravenous injection were 10.48 ± 1.39, 6.25 ± 0.47, and 3.15 ± 1.15% ID/g, respectively, indicating a positive correlation with GRP78 expression. This study demonstrates the feasibility of using [18F]AlF-NOTA-c-DVAP as a PET tracer for imaging GRP78 in tumors. The probe shows promising results in terms of stability, specificity, and tumor targeting. Further research may explore the clinical utility and potential therapeutic applications of this PET tracer for cancer diagnosis.

Lycium barbarum L. polysaccharide LBP3 exerts the anti-tumor effect through enhancing the function of tumor-associated dendritic cells via inhibiting IRE1α -XBP1 pathway of ER stress

Liver cancer is a huge global challenge. It has been exacerbated by tumor-associated dendritic cells (TDCs) due to Endoplasmic Reticulum (ER) stress. We planned to investigate the effects of LBP3 on TDCs immune function, and to explore whether the underlying mechanism was associated with IRE1α-XBP1 pathway of ER stress regulating lipid metabolism. In our early studies, LBP3 (40–350 kDa) with significant anti-tumor and immunomodulatory activities was isolated from the polysaccharide of Lycium barbarum L. (LBP). Here, we determined that LBP3 had a great anti-tumor efficacy in H22-tumor bearing mice by functional activation of TDCs. In vivo and in vitro results showed that LBP3 could up-regulate the expression of MHC Ⅱ, CD80 and CD86 on TDCs, increase the level of TNF-α secreted by TDCs, enhance TDCs’ ability to improve the proliferation and immunosuppression of lymphocytes. Furthermore, LBP3 could decrease the high expression of ER stress-related proteins IRE1α, GRP78, XBP1 and CHOP, reduce the lipid accumulation, raise the low expression of costimulatory molecules on TDCs induced by thapsigargin (TG). Taken together, LBP3 could reduce intracellular lipid accumulation by inhibiting IRE1α-XBP1 pathway of ER stress, improve the function of TDCs to stimulate T cells, thence play an anti-tumor role.

GRP78 improves the therapeutic effect of mesenchymal stem cells on hemorrhagic shock-induced liver injury: Involvement of the NF-кB and HO-1/Nrf-2 pathways.

Mesenchymal stem cells (MSCs) are a popular cell source for repairing the liver. Improving the survival rate and colonization time of MSCs may significantly improve the therapeutic outcomes of MSCs. Studies showed that 78-kDa glucose-regulated protein (GRP78) expression improves cell viability and migration. This study aims to examine whether GRP78 overexpression improves the efficacy of rat bone marrow-derived MSCs (rBMSCs) in HS-induced liver damage. Bone marrow was isolated from the femurs and tibias of rats. rBMSCs were transfected with a GFP-labeled GRP78 expression vector. Flow cytometry, transwell invasion assay, scratch assay immunoblotting, TUNEL assay, MTT assay, and ELISA were carried out. The results showed that GRP78 overexpression enhanced the migration and invasion of rBMSCs. Moreover, GRP78-overexpressing rBMSCs relieved liver damage, repressed liver oxidative stress, and inhibited apoptosis. We found that overexpression of GRP78 in rBMSCs inhibited activation of the NLRP3 inflammasome, significantly decreased the levels of inflammatory factors, and decreased the expression of CD68. Notably, GRP78 overexpression activated the Nrf-2/HO-1 pathway and inhibited the NF-κB pathway. High expression of GRP78 efficiently enhanced the effect of rBMSC therapy. GRP78 may be a potential target to improve the therapeutic efficacy of BMSCs.

Zinc attenuates arsenic overdose-induced brain damage via PERK/ATF6 and TLR/MyD88/NF-κB pathways

Exposure to arsenic (As), a widespread non-metallic toxicant in nature, often results in neurotoxicity, although the exact mechanism is unknown. Zinc (Zn) is a powerful nutrient often thought to be beneficial for growth, development and immunity. Whether Zn can rescue brain damage caused by As contamination remains to be demonstrated. Therefore, in this study, a 30-day model of As poisoning (2.83 mg/L) in carp was established and treated with Zn (1 mg/L) to investigate the detoxification mechanism involved. Histological observations showed that As induced the loosening of the molecular layer structure of the cerebellum and the dissolution or even disappearance of nuclei, accompanied by the occurrence of microthrombi in the granular layer, and the addition of Zn attenuated such As-induced damage. Further mechanistic studies indicated that Zn ameliorated As exposure-induced abnormalities in antioxidant capacity (decreased CAT and Cu/Zn-SOD), activation of the Nrf2/keap1 pathway and endoplasmic reticulum stress (ERs), which is a key factor in As-induced brain damage. ERs (high expression of PERK, ATF6, CHOP, eiF2α and GRP78) and inflammation (overexpression of TLR2, TLR4, MyD88, IKK, NF-κB, IL-1β and IL-6 and low expression of IκBα and IL-10). We suggest that Zn can alleviate excessive As-induced brain damage by attenuating As-induced oxidative stress, PERK/ATF6 and TLR/MyD88/NF-κB pathways. The present study fills in the preventive mechanism of As injury in fish and provides the possibility of prevention and control of As pollution-induced brain tissue injury by Zn rescue.

Novel NPR2 Gene Mutations Affect Chondrocytes Function via ER Stress in Short Stature.

Natriuretic peptide receptor 2 (NPR2) plays a key role in cartilage and bone morphogenesis. The NPR2 gene mutations result in acromesomelic dysplasia, Maroteaux type (AMDM), short stature with nonspecific skeletal abnormalities (SNSK), and epiphyseal chondrodysplasia, Miura type (ECDM). However, the pathogenic mechanism remains unclear. In our study, we identified one de novo (R557C) and six novel variants (G602W, V970F, R767*, R363*, F857S, and Y306S) in five independent Chinese families with familial short stature. Three patients with heterozygous mutations (G602W, V970F, and R767*) were diagnosed with SNSK (height SD score ranged from −2.25 to −5.60), while another two with compound heterozygous mutations (R363* and F857S, R557C and Y306S) were diagnosed with AMDM (height SD score ranged from −3.10 to −5.35). Among three patients with heterozygous status, two patients before puberty initiation with rhGH treatment significantly improved their growth (height velocity 7.2 cm/year, 6.0 cm/year), and one patient in puberty had a poor response to the rhGH treatment (height velocity 2.5 cm/year). Seven NPR2 gene variants were constructed and overexpressed in HEK293T and ATDC5 cells, and we found that ATDC5 cells with mutant NPR2 gene showed decreased differentiation, as evidenced by lower expression of ColII, ColX, and BMP4 and higher expression of Sox9. Moreover, the apoptosis rate was elevated in ATDC5 cells expressing the mutant NPR2 gene. N-glycosylation modification, plasma membrane localization, and ER stress resulted from the accumulation of mutant protein in ER, as shown by the higher expression of GRP78 and p-IRE1α. Overall, our results provide a novel insight into NPR2 loss of function, which could promote chondrocyte apoptosis and repress cell differentiation through ER stress and the unfolded protein response.

GRP78 expression and clinical outcomes in pancreatic cancer: A single institution review.

e16767 Background: Pancreatic Ductal Adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers. Poor prognosis can be attributed in part to late detection and chemoresistance. Perioperative chemotherapy with or without radiation is the standard treatment for resectable PDAC, however recurrence rates remain high. GRP78 (HSPA5/BiP) is an endoplasmic reticulum (ER) chaperone protein involved in regulation of the ER stress response that leads to pro-survival pathway activation and chemoresistance in tumor cells. Prior studies of suppression of GRP78 expression in PDAC cell lines led to increased pancreatic cell apoptosis in response to gemcitabine. In vivo studies of human PDAC xenografts showed that tumors with higher GRP78 expression were associated with increased chemoresistance. Methods: We conducted a single institution retrospective review of 53 patients who underwent resection of PDAC from 1/1/2009-7/1/2016. Archived tumor tissue expression of GRP78 was assessed by immunohistochemistry (IHC) and scored as 0-3+. GRP78 IHC scores of 0-1+ were categorized as low and scores of 2-3+ as high. Corresponding clinical data were collected. Adjusted effects of GRP78 expression on the hazard of treatment failure or death were estimated using Cox proportional hazards models. Type III Wald Chi-square p-values are reported for the overall effect in each model. Results: GRP78 expression level demonstrated a significant effect on disease free survival (DFS) in unadjusted analyses (HR 2.00, p = 0.05), as well as after adjusting for performance status (p = 0.03), initial overall stage (p = 0.05), pathological tumor stage (p = 0.02), pathological node stage (p < 0.01), resection margins (p = 0.05), and neoadjuvant therapy (p = 0.05) in separate models. In unadjusted analysis, high GRP78 expression showed a trend towards worsened overall survival (OS), but did not reach statistical significance (HR 1.8, p = 0.11). The effect on OS was significant when adjusting for pathological tumor stage (p = 0.04) and pathological node stage (p = 0.01). (575). Conclusions: Consistent with prior animal studies, our retrospective review revealed that high GRP78 expression was associated with worsened DFS in patients with resectable PDAC. When adjusting for pathological staging, high GRP78 expression had a similar impact on OS. These findings warrant further evaluation of the role of GRP78 in prognosis, response to therapy and as a potential treatment target.

Endoplasmic reticulum stress is involved in ventilator-induced lung injury in mice via the IRE1α-TRAF2-NF-κB pathway

Inflammation plays a criticalrole in the development of ventilator-induced lung injury (VILI). Endoplasmic reticulum (ER) stress is associated with a variety of diseases through the modulation of inflammatory responses. However, little is known about how ER stress is implicated in VILI. In this study, murine mechanical ventilation models were constructed. Total protein and inflammatory cytokines were measured in bronchoalveolar lavage fluid (BALF),and lung tissue injurywasassessedby histology. Our data revealed that mice subjected to high tidal ventilation (TV) for 4 h showed more severe pulmonary edema and inflammation than those of mice with spontaneous breathing and low TV-treatment. In addition, the high TV-treated animals upregulated the ER stress markers GRP78, CHOP, p-IRE1α, TRAF2, and p-NF-κB expression at both the mRNA and protein levels in lung tissue. Administration of thapsigargin exacerbated the histological changes, inflammation and expression of GRP78 and CHOP after high TV, but treatment with ER stress and IRE1α kinase inhibitors attenuated the pathological damage and downregulated the high expression of GRP78, CHOP, p-IRE1α, TRAF2, and p-NF-κB, suggesting that ER stress is involved in VILI though the IRE1α/TRAF2/NF-κB signaling pathway in mice.

The Gα-interacting vesicle-associated protein interacts with and promotes cell surface localization of GRP78 during endoplasmic reticulum stress.

Cell surface translocation of the chaperone glucose-regulated protein 78kDa (GRP78) is a key event that promotes cancer cell survival during endoplasmic reticulum (ER) stress. Here, we identify Gα-interacting vesicle-associated protein (GIV) - an enhancer of prosurvival signaling during ER stress - as a binding partner of GRP78. We show that GIV and GRP78 interact in an ER stress-dependent manner through their respective carboxyl terminal domains and that GIV aids in the localization of GRP78 to the plasma membrane. Kaplan-Meier analysis of disease-free survival in cancer patients shows poor prognosis for patients with high expression of both GIV and GRP78, further suggesting a vital role for these two proteins in enhancing cancer cell viability.

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture.

Cis‑diammine‑dichloro‑platinum II‑based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non‑small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin‑treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin‑treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post‑cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy‑related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15proteins [glucose‑regulated protein, 78kDa (GRP78), heat shock protein71, pre‑mRNA processing factor19, polypyrimidine tract binding protein1, translationally controlled tumor protein, CathepsinD, Cytochromec, thioredoxin domain containing5, MutS homolog (MSH)6, AnnexinA2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor1A interacting protein, MSH2, protein phosphatase2A 55kDa regulatory subunitBα, Rho glyceraldehyde‑3‑phosphate‑dissociation inhibitor1 and ANXA4] that were upregulated by >1.5‑fold in heavy(H)‑ and light(L)‑labeled A549R cells. In addition, 16 and 14proteins were downregulated by >1.5‑fold in the H‑ and L‑labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin‑resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin‑resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti‑apoptosis and/or autophagy promotion.

Proteomic Profile of Sorafenib Resistance in Hepatocellular Carcinoma; GRP78 Expression Is Associated With Inferior Response to Sorafenib.

The outcome of patients with advanced hepatocellular carcinoma (HCC) remains poor and therapeutic options, including sorafenib, the first anti-cancer drug proved to prolong survival in patients with advanced HCC, are limited. However, no clinically useful predictive biomarker for sorafenib has been reported. We exploited two-dimensional gel electrophoresis coupled with mass spectrometry to find de-regulated proteins by using conditioning of a sorafenib-resistant HCC cell line, Huh7. Tumor samples from 60 patients with HCC treated with sorafenib were analyzed and correlated with survival outcome. Comparative proteomics indicated three proteins including, 78 kDa glucose related protein (GRP78), 14-3-3ε, and heat shock protein 90β (HSP90β). The three proteins were over-expressed in sorafenib-resistant Huh7 cells. In HCC tumor samples from patients treated with sorafenib, 73% of tumor samples had a high expression of GRP78, 18% had high 14-3-3ε expression and 85% had high HSP90β expression. Among these, GRP78 was associated with the shortest progression-free survival of HCC patients treated with sorafenib. GRP78 can be a predictive biomarker in HCC patients treated with sorafenib. Strategies designed to inhibit the GRP78-related pathway may overcome sorafenib resistance.

Hexosamine pathway regulates StarD7 expression in JEG-3 cells.

StarD7 is a lipid binding protein involved in the delivery of phosphatidylcholine to the mitochondria whose promoter is activated by Wnt/β-catenin signaling. Although the majority of glucose enters glycolysis, ~ 2-5% of it can be metabolized via the hexosamine biosynthetic pathway (HBP). Considering that HBP has been implicated in the regulation of β-catenin we explored if changes in glucose levels modulate StarD7 expression by the HBP in trophoblast cells. We found an increase in StarD7 as well as in β-catenin expression following high-glucose (25mM) treatment in JEG-3 cells; these effects were abolished in the presence of HBP inhibitors. Moreover, since HBP is able to promote unfolded protein response (UPR) the protein levels of GRP78, Ire1α, calnexin, p-eIF2α and total eIF2α as well as XBP1 mRNA was measured. Our results indicate that a diminution in glucose concentration leads to a decrease in StarD7 expression and an increase in the UPR markers: GRP78 and Ire1α. Conversely, an increase in glucose is associated to high StarD7 levels and low GRP78 expression, phospho-eIF2α and XBP1 splicing, although Ire1α remains high when cells are restored to high glucose. Taken together these findings indicate that glucose modulates StarD7 and β-catenin expression through the HBP associated to UPR, suggesting the existence of a link between UPR and HBP in trophoblast cells. This is the first study reporting the effects of glucose on StarD7 in trophoblast cells. These data highlight the importance to explore the role of StarD7 in placenta disorders related to nutrient availability.

Prognostic role of beclin-1 in locally advanced non-small cell lung cancer in patients receiving docetaxel-platinum induction chemotherapy.

Background/AimsThe outcome of local treatment for advanced non-small cell lung cancer (NSCLC) remains poor, with therapies such as induction chemotherapy (IC) yielding conflicting results. This study aimed to assess the clinicopathologic and prognostic significance of the excision repair cross-complementation group 1 (ERCC1), beclin-1, and glucose-regulated protein of molecular mass 78 (GRP78) in patients with locally advanced NSCLC receiving docetaxel-platinum IC, along with efficacy and safety.MethodsThis is a retrospective observational cohort study. We reviewed medical records of 31 NSCLC patients receiving docetaxel-platinum IC, and conducted immunohistochemical staining of ERCC1, beclin-1, and GRP78.ResultsResponse rate was 67.8% with 10.7 months of median relapse-free survival (RFS) and 23.1 months of median overall survival (OS), and no treatment-related death was reported. High expression of ERCC1, beclin-1, and GRP78 was identified in 67.7%, 87.1%, and 67.7%, respectively. Expression of ERCC1 and GRP78 did not reveal statistical significance in survival, whereas high beclin-1 expression revealed longer OS (7.6 months vs. 23.2 months; log-rank p = 0.024). In multivariate analysis, histologic differentiation (hazard ratio [HR], 3.48; p < 0.001), stage (HR, 8.5; p = 0.024), and adjuvant treatment (HR, 16.1; p = 0.001) were related to RFS, and in OS, stage (HR, 5.4; p = 0.037), adjuvant treatment (HR, 8.6; p = 0.004), and beclin-1 expression (HR, 8.2; p = 0.011) were identified as significant prognostic factors.ConclusionsOur findings suggest that high beclin-1 expression predicts longer survival in locally advanced NSCLC and docetaxel-platinum IC is a treatment option that deserves consideration.

Overexpression of endoplasmic reticulum stress-related proteins, XBP1s and GRP78, predicts poor prognosis in pulmonary adenocarcinoma

ObjectivesEndoplasmic reticulum (ER) stress is associated with tumor development and progression via pro-tumorigenic and anti-tumorigenic effects. However, the clinicopathological implications of the ER stress pathway in non-small cell lung cancer remain unclear. Therefore, we sought to address these issues in this study. Materials and methodsExpression of two ER stress-related proteins, GRP78 and XBP1 spliced-form (XBP1s), was evaluated in pulmonary adenocarcinoma (pADC; n = 369) and squamous cell carcinoma (pSqCC; n = 246) using immunohistochemistry. ResultsExpression levels of GRP78 and XBP1s were significantly higher in pADCs and pSqCCs, respectively (both, P < 0.0001). In the pADC group, XBP1s expression was higher in patients with ALK translocation than in those with wild-type ALK, wild-type EGFR, or EGFR mutation (P < 0.005). No significant difference in GRP78 expression according to ALK or EGFR status was noted. pADC harboring high GRP78 expression exhibited an increased XBP1s expression (P = 0.0067). Higher XBP1s expression was associated with shorter disease-free survival (DFS) in patients with pADC (P = 0.026) and in those with ALK translocation (P = 0.001). Higher GRP78 expression was associated with shorter DFS in patients with pADC (P = 0.029) and those with EGFR mutation (P = 0.005). Multivariate survival analysis revealed that high XBP1s expression was an independent predictor of poor DFS in pADC (P = 0.004, hazard ratio [HR] = 3.115), and that high GRP78 expression was an independent predictor of poor DFS in EGFR-mutated pADC (P = 0.007, HR = 2.168). Taken together, high expression of XBP1s or GRP78 was an independent poor prognostic factor in pADC (P = 0.002, HR = 2.403). ConclusionGRP78 and XBP1s are expressed variably in pADC, but their overexpression is associated with poor patient prognosis. The ER stress pathway may be a prognostic biomarker and potential therapeutic target for pADC.

Neoisoliquiritigenin Inhibits Tumor Progression by Targeting GRP78-β- catenin Signaling in Breast Cancer.

Breast cancer mortality has been stable or decreasing in the world, its incidence and recurrence rates have sharply risen worldwide in the recent years. To investigate the clinicopathological significance and potential function of GRP78 in the development and progression of breast cancer. To explore the effects of neoisoliquiritigenin (NISL) in breast cancer and the underlying mechanism. GRP78 was detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs), and the association between GRP78 levels and clinicopathological factors and prognosis was analyzed. The functional effects of GRP78 on breast cancer were validated by an MTT assay, foci formation assay, Matrigel invasion assay and mouse xenograft assay. The effects of NISL were tested by an MTT assay, apoptosis assay and mouse xenograft assay. A LigandFit algorithm, ATPase activity assay, western blot and IHC assay were used to discover the underlying mechanism of the effects of NSIL. GRP78 was highly expressed in breast cancer cell lines and tissues. In addition, high expression of GRP78 was correlated to poor outcomes and distant metastasis. Functional experiments showed that GRP78 promoted breast cancer proliferation and invasion in vitro and in vivo. NISL inhibited cell proliferation and induced cell apoptosis in breast cancer by directly binding to GRP78 to regulate the β-catenin pathway. Taken together, these results highlighted the significance of GRP78 in breast cancer development and suggested NISL as a natural candidate to inhibit breast cancer by targeting GRP78 and β-catenin signaling.

Endoplasmic Reticulum (ER) Stress is Anti‐angiogenic and Anti‐tumorigenic

IntroductionThe functions of endoplasmic reticulum (ER) are significantly influenced by cellular microenvironment. The availability of oxygen (i.e., the hypoxia) or glucose (i.e., the hypoglycemia), hyperthermia, acidosis, calcium levels, the redox milieu, energy levels etc. influence and disturb proper functioning of the ER, resulting in ER stress and impacting protein folding in the lumen of the ER. The result is accumulation of unfolded or misfolded proteins. If the balance between accumulation and degradation of unfolded or misfolded proteins is shifted towards their accumulation, cells a suicidal pathway through unfolded protein response (upr) signaling. Our laboratory has recently shown that inhibiting asparagine‐linked (N‐linked) protein glycosylation with an Mr860Da glucosamine containing pyrimidine nucleoside tunicamycin [a natural product, a cytokine mimic, and a competitive inhibitor of N‐acetylglucosaminyl 1‐phosphate (GlcNAc‐1P) transferase activity] induces ER stress in capillary endothelial cells as well as in breast cancer cells. The treatment causes cell cycle arrest and induction of apoptosis in capillary endothelial cells. Since, angiogenesis is essential for normal growth and development as well as for the progression and metastasis of breast tumor, we have observed inhibition of ER−/PR−/Her2+ (double negative) and ER−/PR−/Her2− (triple negative) breast cancers in athymic balb/c (nu/nu) mice following tunicamycin treatment.HypothesisWe have therefore hypothesized that tumor microvessles are not only the unique target for ER stress in breast tumor; tumor epithelial cells are also equally vulnerable to tunicamycin‐induced ER stress.MethodsTo test the hypothesis, we have used human breast cancer cell lines as models and tunicamycin as a tool.ResultsWhen treated with tunicamycin, both MDA‐MB‐231(ER−/PR−/Her2−; triple negative) and MCF‐7 (ER+) breast cancer cells exhibited a dose and time dependent inhibition of cell proliferation. There were inhibition of surface expression of N‐glycans as well. Immunofluorescence microscopy of unfixed cells stained for FITC‐conjugated Concanavalin A (FITC‐Con A) and Texas Red‐conjugated Wheat germ agglutinin (Texas Red‐WGA). To measure the ER stress, we have monitored the expression of ER chaperone GRP78/Bip by immunofluorescence microscopy as well as by SDS‐PAGE followed by western blotting. Western blotting detected higher GRP78/Bip expression but none by immunofluorescence microscopy in unfixed tumor cells. Higher expression of GRP78 in tunicamycin treated cells is expected to support the presence of ER stress in breast cancer cells. So, the GRP78/Bip expression was tested by immunofluorescence microscopy after permeabilization of cells with ice‐cold methanol. There was a robust intracellular expression of GRP‐78/Bip. To evaluate if the induction of ER stress mediates its effect through upr signaling, we have analyzed the expression of IRE‐1, ATF4/ATF6 and PERK by western blotting.ConclusionInduction of ER stress both in endothelial cells and in tumor cells by targeting their asparagine‐linked (N‐linked) protein glycosylation machinery is unique and the concept could be used to develop new generation therapeutics for breast cancer..Support or Funding InformationSupported in part by the NIH‐NIDDK STEP‐UP program (AR) and NIH/NIMHD G12MD007583 (KB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Concomitant high expression of ERα36, GRP78 and GRP94 is associated with aggressive papillary thyroid cancer behavior.

Papillary thyroid cancer (PTC) is more common in women than in men. It has been suggested that estrogen may be involved in its development, as has previously been shown for breast, endometrial and ovarian cancer. The purpose of this study was to assess correlations between the expression of the estrogen receptor alpha36 (ERα36) and the glucose regulated proteins GRP78 and GRP94 (chaperones involved in glycoprotein folding) and various PTC clinicopathological features, as well as to evaluate the potential usefulness of these three potential oncogenic proteins in the prediction of aggressive PTC behavior. ERα36, GRP78 and GRP94 protein expression in 218 primary PTC tissues and PTC-derived BCPAP cells was examined using immunohistochemistry, Western blotting and immunocytochemistry. The proliferative, invasive and migrative capacities ofBCPAP cells in which the respective genes were either exogenously over-expressed or silenced were assessed using BrdU incorporation and Transwell assays, respectively. We found that ERα36, GRP78 and GRP94 protein expression was upregulated in the primary PTC tissues tested. We also found that ERα36, GRP78 and GRP94 expression modulation affected the proliferation, invasion and migration of PTC-derived BCPAP cells. A positive correlation and a positive feedback loop were noted between ERα36, GRP78 and GRP94 protein expression in the primary PTC tissues and in BCPAP cells, respectively. High ERα36 expression in combination with a high GRP78/ GRP94 expression was found to have a stronger correlation with extrathyroid extension (ETE), lymph node metastasis (LNM), distant metastasis (DM) and high TNM stage than high ERα36 expression in combination with either high GRP78 or high GRP94 expression (p = 0.028 for ETE, p = 0.002 for DM and p ≤ 0.001 for LNM and high TNM stage) or high ERα36 expression alone (p < 0.001 for ETE, LNM, DM and high TNM stage). From our data we conclude that a concomitant high expression of ERα36, GRP78 and GRP94 is strongly associated with aggressive PTC behavior and may be used as a predictor for ETE, LNM, DM and high TNM stage.

PGK1 and GRP78 overexpression correlates with clinical significance and poor prognosis in Chinese endometrial cancer patients.

The aim of this study was to measure the expression patterns of PGK1 and GRP78 in normal endometrial tissues and endometrial carcinoma, and associations between their combined effects and the pathological features of endometrial carcinoma. We used 30 normal endometrial tissue samples and 130 endometrial carcinoma samples, and separately evaluated PGK1 and GRP78 protein expression by immunohistochemistry. Scores ranging from 0 to 9 were obtained by multiplying the percentage of positive cells by the staining intensity (0–3). Immunohistochemical analysis revealed increased PGK1 and GRP78 expression in the cytoplasm of endometrial carcinoma cells compared with that in normal endometrial tissues. High PGK1 expression positively correlated with the FIGO stage (P < 0.001), histological grade (P = 0.002), and lymph node status (P < 0.001). High GRP78 expression positively correlated with the pathological type (P = 0.0125), FIGO stage (P < 0.001), and lymph node status (P < 0.001). In addition, PGK1 overexpression was positively correlated with GRP78 overexpression in endometrial carcinoma patients (P < 0.001), and the concurrent expression of both oncogenes in endometrial carcinoma patients correlated significantly with the lymph node status (P < 0.001) and FIGO stage (P < 0.001). Patients with high PGK1 and GRP78 expression levels had poorer overall survival rates than those with low expression levels of both proteins (P < 0.001). Our results suggested that the co-occurrence of PGK1 and GRP78 expression is potentially an unfavorable factor for endometrial carcinoma progression.

MiR-181a Inhibits Cervical Cancer Development via Downregulating GRP78.

Cervical cancer is among the most common cancers inflicting women worldwide. Understanding the pathological mechanisms of cervical cancer development is critical for identifying novel targets for cervical cancer treatment. MicroRNAs (miRs) have various roles in regulating cancer development. In this study, we investigated the potential role of miR-181a and its target in regulating cervical cancer development and chemotherapy resistance. The expression of miR-181a was evaluated and modulated in several human cervical cancer cell lines. The role of miR-181a in regulating cervical cancer growth and chemotherapy sensitivity was investigated in cell culture models and mouse tumor xenograft models. The target of miR-181a and its function were identified in cervical cancer models. We found a distinct expression profile for miR-181a in cervical cancer cell lines. Low expression of miR-181a was closely related to cervical cancer growth and oxaliplatin resistance. HSPA5/GRP78 was identified as a target of miR-181a in cervical cancer cells. Upregulation of GRP78 led to a high cell proliferation rate and oxaliplatin resistance in cervical cancer models. In a retrospective cervical cancer cohort, high GRP78 expression was correlated with poor survival. miR-181a suppressed cervical cancer development via downregulating GRP78. High expression of GRP78 is a tumor-promoting factor in cervical cancer and is thus a potential target for novel treatment.

Hepatocyte growth factor secreted by bone marrow stem cell reduce ER stress and improves repair in alveolar epithelial II cells

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis.