anti-GRP78 Autoantibodies Research Articles

Small molecules targeting GRP78 mitigate anti-GRP78 autoantibody–mediated tissue factor procoagulant activity in cultured endothelial cells

BackgroundThe 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor (TF) procoagulant activity (PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. ObjectivesDetermine the effects of the engagement of the anti-GRP78 autoantibody to csGRP78 on ECs and the underlying mechanisms that impact TF PCA. MethodsImmunofluorescent staining was used to determine the presence of csGRP78 in tumor necrosis factor α–treated ECs. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay (Abcam) was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind GRP78 were identified using artificial intelligence. Enzyme-linked immunosorbent assays were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. ResultsIn tumor necrosis factor α–treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by endoplasmic reticulum stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin attenuated anti-GRP78 autoantibody–induced TF PCA. We identified B07∗, as a GRP78 binder that diminished anti-GRP78 autoantibody–induced TF PCA on ECs. ConclusionThese findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

Scratching the Surface-An Overview of the Roles of Cell Surface GRP78 in Cancer.

The 78 kDa glucose-regulated protein (GRP78) is considered an endoplasmic reticulum (ER)-resident molecular chaperone that plays a crucial role in protein folding homeostasis by regulating the unfolded protein response (UPR) and inducing numerous proapoptotic and autophagic pathways within the eukaryotic cell. However, in cancer cells, GRP78 has also been shown to migrate from the ER lumen to the cell surface, playing a role in several cellular pathways that promote tumor growth and cancer cell progression. There is another insidious consequence elicited by cell surface GRP78 (csGRP78) on cancer cells: the accumulation of csGRP78 represents a novel neoantigen leading to the production of anti-GRP78 autoantibodies that can bind csGRP78 and further amplify these cellular pathways to enhance cell growth and mitigate apoptotic cell death. This review examines the current body of literature that delineates the mechanisms by which ER-resident GRP78 localizes to the cell surface and its consequences, as well as potential therapeutics that target csGRP78 and block its interaction with anti-GRP78 autoantibodies, thereby inhibiting further amplification of cancer cell progression.

Glucose-Regulated Protein 78 Autoantibodies Are Associated with Carotid Atherosclerosis in Chronic Obstructive Pulmonary Disease Patients.

Atherosclerosis prevalence is increased in chronic obstructive pulmonary disease (COPD) patients, independent of other risk factors. The etiology of the excess vascular disease in COPD is unknown, although it is presumably related to an underlying (if cryptic) systemic immune response. Autoantibodies with specificity for glucose-regulated protein 78 (GRP78), a multifunctional component of the unfolded protein response, are common in COPD patients and linked to comorbidities of this lung disease. We hypothesized anti-GRP78 autoreactivity might also be a risk factor for atherosclerosis in COPD patients. Carotid intima-medial thickness (cIMT) was measured in 144 current and former smokers by ultrasound. Concentrations of circulating IgG autoantibodies against full-length GRP78, determined by ELISA, were greater among subjects with abnormally increased cIMT (p <, 0.01). Plasma levels of autoantibodies against a singular GRP78 peptide segment, amino acids 246–260 (anti-GRP78aa 246–260), were even more highly correlated with cIMT, especially among males with greater than or equal to moderate COPD (rs = 0.62, p = 0.001). Anti-GRP78aa 246–260 concentrations were independent of CRP, IL-6, and TNF-α levels. GRP78 autoantigen expression was upregulated among human aortic endothelial cells (HAECs) stressed by incubation with tunicamycin (an unfolded protein response inducer) or exposure to culture media flow disturbances. Autoantibodies against GRP78aa 246–260, isolated from patient plasma by immunoprecipitation, induced HAEC production of proatherosclerotic mediators, including IL-8. In conclusion, anti-GRP78 autoantibodies are highly associated with carotid atherosclerosis in COPD patients and exert atherogenic effects on HAECs. These data implicate Ag-specific autoimmunity in the pathogenesis of atherosclerosis among COPD patients and raise possibilities that directed autoantibody reduction might ameliorate vascular disease in this high-risk population.

Prostate cancer-derived anti-GRP78 autoantibodies compromise the blood-brain barrier and accelerate atherosclerosis progression in vivo.

205 Background: Pathological conditions of prostate cancer (PCa) drive the translocation of the endoplasmic reticulum-resident chaperone, GRP78, to the cell surface (cs) where it acts as an antigenic protein with signaling properties. In PCa, csGRP78 drives the production of anti-GRP78 autoantibodies (AutoAbs) that engage csGRP78 and promote PCa survival/progression. New studies now demonstrate csGRP78 expression on endothelial cells (EC) that line the arterial vasculature and the blood-brain barrier (BBB) suggesting that these AutoAbs can affect other systems in the body. Based on this, we investigated how the engagement of anti-GRP78 AutoAbs to csGRP78 on EC can contribute to EC-dysfunction that can promote atherosclerosis and compromise the integrity of the BBB. Methods: Anti-GRP78 AutoAbs were purified from PCa patients (St. Joseph’s Healthcare Hamilton); human aortic EC and the ApoE -/- mouse model were used for in vitro and in vivo investigations, respectively. EC or mice were treated with anti-GRP78 AutoAbs or IgG control (60µg/mL); EC-dysfunction was investigated by measuring attachment protein expression, in vitro. In vivo evaluation was carried out by studying atherosclerotic plaque progression (immunohistochemistry; aorta); the BBB integrity was examined using the Evans Blue dye. Results: Mice injected with anti-GRP78 AutoAbs, and not human IgG, demonstrated larger atherosclerotic plaque volume and hallmarks of a leaky BBB. In terms of a mechanism, in vitro studies demonstrated that treating EC with anti-GRP78 AutoAbs resulted in activation of the NFκB pathway that led to increased expression of attachment proteins. All these effects were reversed by using a recombinant molecule that interfere with the binding of the AutoAb to csGRP78. Conclusions: We have identified anti-GRP78 AutoAb as a driver of EC-dysfunction that promote atherosclerotic plaque progression and damage to the BBB. Our results indicate that interfering with anti-GRP78 AutoAb:csGRP78 complex can reverse the pathological effects of the AutoAbs. This novel data suggests that patient-derived anti-GRP78 autoantibodies systemically drive pathologies, other than cancer, in vivo.

855 - Prostate cancer-derived anti-GRP78 autoantibodies compromise the blood-brain barrier and accelerate atherosclerosis progression in vivo

855 - Prostate cancer-derived anti-GRP78 autoantibodies compromise the blood-brain barrier and accelerate atherosclerosis progression in vivo

Anti-GRP78 autoantibodies induce endothelial cell activation and accelerate the development of atherosclerotic lesions.

The 78-kDa glucose-regulated protein (GRP78) is an ER molecular chaperone that aids in protein folding and secretion. However, pathological conditions that cause ER stress can promote the relocalization of GRP78 to the cell surface (csGRP78), where it acts as a signaling receptor to promote cancer progression. csGRP78 also possesses antigenic properties, leading to the production of anti-GRP78 autoantibodies, which contribute to tumor growth. In contrast, the presence and role of anti-GRP78 autoantibodies in atherosclerosis is unknown. Here, we show that atherosclerotic-prone ApoE-/- mice develop circulating anti-GRP78 autoantibodies that bind to csGRP78 on lesion-resident endothelial cells. Moreover, GRP78-immunized ApoE-/- mice exhibit a marked increase in circulating anti-GRP78 autoantibody titers that correlated with accelerated lesion growth. Mechanistically, engagement of anti-GRP78 autoantibodies with csGRP78 on human endothelial cells activated NF-κB, thereby inducing the expression of ICAM-1 and VCAM-1, a process blocked by NF-κB inhibitors. Disrupting the autoantibody/csGRP78 complex with enoxaparin, a low-molecular-weight heparin, reduced the expression of adhesion molecules and attenuated lesion growth. In conclusion, anti-GRP78 autoantibodies play a crucial role in atherosclerosis development, and disruption of the interaction between anti-GRP78 autoantibodies and csGRP78 represents a therapeutic strategy.

Cell surface GRP78 activation by anti‐GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.

BackgroundProstate cancer (PC) presentation is associated with the production of anti‐GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an endoplasmic reticulum resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules. We hypothesize that the disruption of the autoantibody/cell surface GRP78 complex represent a viable surrogate treatment of PC.MethodsWild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti‐GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT‐PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph's Hamilton.Results ‐Anti‐GRP78 autoantibodies correlate with PC stage: PC patients demonstrated high levels of anti‐GRP78 autoantibodies (60–100 μg/ml, pre‐prostatectomy stage) vs. healthy individuals (5–10 μg/ml). These titers were significantly reduced 24‐weeks post‐prostatectomy. ‐Anti‐GRP78 autoantibodies drive tumour progression: We show here that anti‐GRP78 autoantibodies activate the unfolded protein response, a pro‐survival cellular pathway in cancer cells. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model. ‐Heparin and low molecular weight heparin abolished the effect of anti‐GRP78 autoantiboides on PC progression. In vitro: We show that heparin and low molecular weight heparin inhibit the binding of anti‐GRP78 autoantibodies. In vivo: a low molecular weight heparin was used as an intervention treatment for anti‐GRP78 autoantibodies and was shown to reverse the effect of anti‐GRP78 autoantibodies on increased tumour growth. ConclusionsWe have identified the function of an agent in patients' blood, anti‐GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.Support or Funding InformationThis work was supported in part by Prostate Cancer Canada (PCC) Discovery Grant (Discovery Grants # 2010‐590, and D2017‐1949), and McMaster Surgical Associate Grant 176725.

Cell surface GRP78 activation by anti-GRP78 autoantibodies confers prostate tumour growth via tissue factor activation

Introduction: Prostate cancer (PC) presentation is associated with the production of anti-GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an ER-resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules.

Disrupting the interaction between anti-GRP78 autoantibodies and cell surface GRP78 with low molecular weight heparin as a treatment for prostate cancer

Disrupting the interaction between anti-GRP78 autoantibodies and cell surface GRP78 with low molecular weight heparin as a treatment for prostate cancer

Cell surface GRP78 activation by anti-GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.

31 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increases PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titers were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Endoplasmic reticulum stress responses in placentation - A true balancing act

The unfolded protein response (UPR) is recognized as a key mechanism to promote protein folding and processing in eukaryotes when endoplasmic reticulum stress (ERS) occurs. Some conditions such as hypoxia or glucose deprivation are factors that may elicit ERS response. Recent literature collectively proposes that ERS response is crucial for mammalian reproduction by allowing decidualization and placentation to occur. However, prolonged ERS and activation of UPR pathways can lead to apoptosis and autophagy, which in turn could pose adverse effects on pregnancy outcomes and placentation. ERS associated pregnancy pathologies include intrauterine growth restriction and early-onset preeclampsia. Given these findings, evidence suggests that overactivation of UPR may lead to harmful reproductive circumstances, whereas physiological regulation of ERS response is essential for mammalian reproduction and placental function.In this review, we discuss the dual role of UPR activation with respect to its contribution to placental development as well as pathologies caused by pathway overactivation. In addition, we suggest potential protein markers associated with the UPR, as circulating C-terminal GRP78 or anti-GRP78 autoantibodies which may prove to be of clinical interest.

93 - Cell surface GRP78 activation by anti-GRP78 autoantibodies confers prostate tumour growth via tissue factor activation

93 - Cell surface GRP78 activation by anti-GRP78 autoantibodies confers prostate tumour growth via tissue factor activation

Effect of anti-GRP78 autoantibodies on prostate cancer progression through tissue factor procoagulant activity.

258 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increase PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumour growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titres were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumour growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Autoreactivity to glucose regulated protein 78 links emphysema and osteoporosis in smokers.

RationaleEmphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders.ObjectivesTo discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers.MethodsReplicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays.Measurements and Main ResultsCirculating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7–5.7, p = 0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7–13.3, p = 0.002), and increased circulating bone metabolites (p = 0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002) and IFN-gamma production (p = 0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005) and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively).ConclusionsHumoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance for the pathogenesis of smoking-associated diseases, and development of biomarker immunoassays and/or novel treatments for these disorders.

Activation of cell surface grp78 by anti-grp78 autoantibodies augments endothelial cell activation and accelerates atherosclerotic lesion development

Activation of cell surface grp78 by anti-grp78 autoantibodies augments endothelial cell activation and accelerates atherosclerotic lesion development

Abstract 256: Activation of Cell Surface GRP78 by Anti-GRP78 Autoantibodies Accelerates Lesion Development by Promoting Endothelial Cell Activation

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is a molecular chaperone localized to the endoplasmic reticulum (ER) in healthy cells that functions to assist in the correct folding of newly synthesized proteins and to prevent aggregation of folding intermediates. In addition, GRP78 is also present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a membrane signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies isolated from the serum of cancer patients. We have demonstrated previously that high levels of anti-GRP78 autoantibodies accelerate lesion development in apoE -/- mice. However, the role of anti-GRP78 autoantibody activation of cell surface GRP78 on endothelial cells and how this contributes to atherogenesis is unknown. The objective of this study is to identify factors that mediate surface GRP78 expression on endothelial cells and to determine the mechanism by which surface GRP78 activation on endothelial cells contributes to atherogenesis. Here we demonstrate that induction of ER stress by tunicamycin increased surface GRP78 expression in cultured human aortic endothelial cells. We also show that activation of surface GRP78 on endothelial cells by anti-GRP78 autoantibodies significantly increases gene expression of the adhesion molecules ICAM-1 and VCAM-1. Pretreatment of these cells with a calcium chelator or an inhibitor of NF-B activation attenuated the anti-GRP78 autoantibody-induced promotion of ICAM-1 and VCAM-1. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation, an important initiating event in atherogenesis. These findings further our understanding of the role of anti-GRP78 autoantibodies and the activation of surface GRP78 in endothelial cell function and lesion development. Furthermore, inhibiting the interaction of anti-GRP78 autoantibodies with surface GRP78 could present a novel therapeutic strategy to modulate lesion growth and thereby reduce the risk for atherosclerosis and cardiovascular disease.

Autoantibodies against glucose-regulated protein 78 as serological diagnostic biomarkers in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a type of cancer with a very poor prognosis. Although α-fetoprotein (AFP) is the most effective marker available to detect HCC, the sensitivity and specificity are not optimal. Therefore, there is a need for the development of more sensitive and specific methods that can supplement AFP in the early detection of this cancer. In this study, autoantibody responses to glucose-regulated protein 78 (GRP78) were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting and indirect immunofluorescence assay in sera from patients with HCC, liver cirrhosis (LC) and chronic hepatitis (CH), as well as from normal human individuals. Immunohistochemistry (IHC) with tissue array slides was also preformed to analyze protein expression profiles of GRP78 in HCC and control tissues. The prevalence of autoantibodies against GRP78 was 35.5% (27/76) in HCC, which was significantly higher than that in LC, CH and normal human sera (NHS; P<0.01). The average titer of autoantibodies against GRP78 in HCC sera was higher compared to that in LC, CH and NHS(P<0.01). When both autoantibodies against GRP78 and AFP were used simultaneously as diagnostic markers, sensitivity reached 71.4%. Our data indicate that anti-GRP78 autoantibodies may be potential diagnostic markers for HCC, especially in conjunction with AFP.

OC-07 Binding of anti-GRP78 autoantibodies to cell surface GRP78 promotes cancer progression by activating tissue factor procoagulant activity

OC-07 Binding of anti-GRP78 autoantibodies to cell surface GRP78 promotes cancer progression by activating tissue factor procoagulant activity

Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival

A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.

Binding of Anti-GRP78 Autoantibodies to Cell Surface GRP78 Increases Tissue Factor Procoagulant Activity via the Release of Calcium from Endoplasmic Reticulum Stores

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.