Cell Surface GRP78 Research Articles

Abstract 4506: UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy

Abstract Much attention has been directed to the study of triple-negative breast cancer (TNBC) because of its short disease-free interval from diagnosis and more aggressive course in the metastatic setting. TNBC also lacks specific target receptors for treatment. Third-generation metronomic chemotherapy using available agents that target DNA repair complexes (i.e., taxanes), and cell proliferation (i.e., anthracycline) have shown promise results. However, specific regimens for TNBC at the different disease stages have not been clearly defined. Therefore, researchers are seeking molecular biomarkers to predict response to current chemotherapeutic agents. In a previous study of TNBC, we demonstrated that low doses of anthracyclines and taxanes, which are known to increase endoplasmic reticulum stress, increased tumor cell expression of glucose-regulated protein 78 (GRP78), a key regulator of the unfolding protein response (UPR), resulting in tumor cell apoptosis. GRP78 gene expression is upregulated via CREB2L1, an endoplasmic reticulum transmembrane transcription factor and member of the CREB/ATF family of transcription factors. CREB3L1 is a metastatic suppressor and functions as a transducer of UPR. The aim of the present study was to investigate the effect of low doses of doxorubicin and paclitaxel on UPR activation in metastatic TNBC cells by determining CREB3L1 protein expression in correlation to cell-surface GRP78 expression. Furthermore, we related CREB3L1 and cell-surface GRP78 expression with the migration potential of the cells in response to treatment. We found that metronomic doses of doxorubicin significantly induced CREB3L1 and cell surface GRP78 expression in TNBC cells. CREB3L1 increased by 2.5-fold in MDAMB231 and by 3-fold in MDAMB468 (p<0.04); GRP78 increased by 3.7-fold and 6.1-fold, respectively (p<0.01). Similar results were obtained with low doses of paclitaxel. A strong correlation was observed between CREB3L1 and cell-surface GRP78 protein expression. The increased expression of both proteins in MDAMB231 (highly metastatic) and MDAMB468 (moderated metastatic) was associated with significant inhibition of the migration capacity of the treated TNBC cells. In contrast, the migration capacity of treated estrogen-positive MCF7 cells (non-metastatic) and Her2- positive BT474 cells (highly metastatic) was poorly inhibited, coinciding with a non-significant increase in CREB3L1 and GRP78 expression. The results demonstrated that the drug effect via the UPR was specific for TNBC and unrelated to the metastatic profile of the cells. This study is the first stage in the identification of UPR-related biomarkers of beneficial outcome of metronomic chemotherapy for TNBC. Future mechanistic studies should focus on the possible use of cell surface GRP78 and CREB3L1 as targets for combination therapies. Citation Format: Annat Raiter, Julia Lipovetsky, Britta Hardy, Rinat Yerushalmi. UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy [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 4506. doi:10.1158/1538-7445.AM2017-4506

Inhibitory effect of Par-4 combined with cisplatin on human Wilms’ tumor cells

Wilms' tumor is associated with a high treatment success rate, but there is still a risk of recurrence. Cisplatin, which is one of the chemotherapeutic agents used for its treatment, is associated with a very high rate of resistance. Par-4 (prostate apoptosis response 4) is a tumor suppressor, which is capable of sensitizing tumor cells to chemotherapy. Therefore, the aim of this study was to determine whether combined treatment with Par-4 and cisplatin is effective for inhibiting growth of Wilms' tumor. Wilms' tumor and control cell samples were collected and analyzed by immunofluorescence assay and immunohistochemistry. Total proteins extracted from cultured cells were analyzed using western blotting and flow cytometry. In addition, a mouse xenograft model was established. We discovered significantly low expression of Par-4 in the tumor tissue, which was positively correlated with high expression of GRP78 (glucose-regulated protein 78). In addition, we found that ectopic Par-4 co-localized with cell surface GRP78 and induced high expression of the endoplasmic reticulum proteins ATF4 and BAX, which activated the endoplasmic reticulum apoptosis pathway. Moreover, treatment with ectopic Par-4 and cisplatin suppressed xenograft growth in nude mice. In conclusion, our results showed that Par-4 overexpression and cisplatin had a synergistic effect on SK-NEP-1 cells, as a result of which cell growth was inhibited and cellular apoptosis was induced. Thus, in vitro and in vivo upregulation of Par-4 expression is indispensable for the trafficking of GRP78 to the cell membrane and subsequent apoptosis of cancer cells.

Activation of cell surface GRP78 decreases endoplasmic reticulum stress and neuronal death.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER) -related stress conserved pathway that aims to protect cells from being overwhelmed. However, when prolonged, UPR activation converts to a death signal, which relies on its PERK-eIF2α branch. Overactivation of the UPR has been implicated in many neurological diseases, including cerebral ischaemia. Here, by using an in vivo thromboembolic model of stroke on transgenic ER stress-reporter mice and neuronal in vitro models of ischaemia, we demonstrate that ischaemic stress leads to the deleterious activation of the PERK branch of the UPR. Moreover, we show that the serine protease tissue-type plasminogen activator (tPA) can bind to cell surface Grp78 (78 kD glucose-regulated protein), leading to a decrease of the PERK pathway activation, thus a decrease of the deleterious factor CHOP, and finally promotes neuroprotection. Altogether, this work highlights a new role and a therapeutic potential of the chaperone protein Grp78 as a membrane receptor of tPA capable to prevent from ER stress overactivation.

Cell surface GRP78 expression on T and NK cell sub-populations of breast cancer patients.

11561 Background: The targeting of unfolded protein response (UPR) in tumor cells has received much attention. However, data are sparse on the impact of UPR on T and NK cells. The master regulator of UPR is the glucose-regulated protein 78 (GRP78) that is expressed in some tumor cells or normal stressed cells. There are few studies concerning GRP78 expression on T and NK cells in cancer and its relationship to stress induction by chemotherapy. We aimed to reveal the effect of UPR activation on the peripheral T and NK cells of breast cancer patients by the evaluation of cell surface GRP78 expression on T and NK cells before and after neoadjuvant chemotherapy. Methods: Forty-seven patients with triple negative, ER positive/Her2 negative and Her2 positive breast cancer were included. FACS analysis of their blood specimens before and after neoadjuvant treatment was performed. For multicolor FACS analysis, anti-CD3, CD4, CD8, CD56, CD16, NKG2D, CD45RA, CD45RO, CCR7 CD62L and anti-GRP78 antibody (AF488) were added to one of the tubes. A second tube was incubated with IgG-AF488 as isotype control. Analysis of the different T and NK subpopulations that expressed cell surface GRP78 were analyzed with the Gallios Flow cytometer and Kaluza Flow Analysis Software (Beckman Coulter, Inc.). Results: The percentage of cell surface GRP78 baseline expression in CD3 (1.8±0.9), CD8 (2.9±1.5), NKG2D (4.8±2) and CD45RO/CD62L/CCR7 active T memory cells (2.1±0.5) in Her2 positive patients were significantly higher than in triple negative and ER positive/Her2 negative patients. GRP78 expression on CD56, CD16 and NKG2D cells measured after neoadjuvant treatment was significantly higher in patients with complete response (CR) compared to patients without CR. 89% of the CR patients presented with Her2+ subtype. The non-CR patients include triple negative and Her2 negative subtypes. Conclusions: GRP78 was found to be expressed in the different T and NK sub-populations. The level of expression changed with each breast cancer subtype and response to chemotherapy. These novel findings suggest that GRP78 may be used as a new predictive biomarker. It sets the stage for understanding the mechanism of UPR activation on the immune system in breast cancer.

Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR

The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (−)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells.

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

Cell surface GRP78 as a biomarker and target for suppressing glioma cells.

High-grade glioma is a highly malignant and metastatic brain cancer, resistant to many existing anticancer treatments. In such glioma cancer cells, the glucose-regulated protein 78 kDa (GRP78) is particularly highly up-regulated. Former studies have thus targeted mutation-free GRP78 not only to detect glioma cancer cells specifically but also to enhance cytotoxic effect. We focus on cell surface-expressed GRP78 as a target for suppressing high-grade glioma cell lines. Glioblastoma multiforme (GBM) cell line, highly malignant glioma cells, was first injected into 5-week-old athymic mice to confirm and compare GRP78 expression in vivo in xenografted and normal brain tissue. Immunofluorescence and immunoblotting were utilized to detect surface-localized GRP78 in diverse high-grade glioma cell lines. By treating glioma cell lines with the polyclonal N-20 antibody against surface-localized GRP78, we subsequently studied the significance of surface GRP78 to the survival and growth of the glioma cell lines. We found that inhibiting the function of surface GRP78 suppressed cancer cell survival and growth proving that the surface-expressed GRP78 is a vital receptor involved in the proliferation of high-grade glioma. Our findings provide opportunities to target surface GRP78 as a biomarker for high-grade glioma and to develop effective cell-specific anticancer therapy.

Activated α2-Macroglobulin Regulates Transcriptional Activation of c-MYC Target Genes through Cell Surface GRP78 Protein

Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression.

Peptide Binding Glucose Regulated Protein 78 Improves Type 1 Diabetes by Preventing Pancreatic β Cell Apoptosis.

Diabetes Type 1 is characterized by hyperglycemia due to reduced insulin secretion that results from the death of pancreatic β cells. It was suggested that endoplasmic reticulum (ER) stress is associated with the autoimmune-mediated β cell destruction. Glucose regulated protein 78 (GRP78) functions as a key regulator to maintain the ER function. Under stress conditions GRP78 is up-regulated and expressed on the cell surface serving as a signaling receptor. Our first objective was to examine the effects of peptide binding cell surface GRP78 to reduce the deleterious effects of diabetes induced by streptozotocin. The second objective was to demonstrate the ability of the peptide to protect the pancreatic β cells from apoptosis. The effect of ADoPep on weight loss, HbA1c levels and anti GRP78 antibody titers was evaluated in a diabetes mouse model. The effect of ADoPep on the pancreatic β Ins1E cell apoptosis was determined by FACS analysis. The administration of ADoPep to diabetic mice retained the weight loss and reduced HbA1c significantly in 60% of mice. Titers of anti GRP78 antibodies increased in 70% of the treated mice. Apoptosis was significantly inhibited in stressed pancreatic β Ins 1E cells. We demonstrate that administration of the peptide ADoPep to diabetic mice improved type 1 diabetes by preventing pancreatic β cell apoptosis.

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.

Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line.

Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1-17, which possesses the most typical characteristics of PDLSCs. Confocal co-localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1-17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration-associated genes in 1-17 cells. Moreover, Grp78 overexpression enhanced rM180-induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin-induced migration in 1-17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin-induced periodontal tissue regeneration.

Abstract POSTER-THER-1420: Ascites drives ovarian cancer stem-like cell growth: therapeutic opportunities

Abstract Background: A significant literature suggests that cancer stem-like cells drive the aggressive behavior of ovarian cancer. Cancer stem-like cells are distinct from bulk tumor cells in possessing self-renewing/tumor-initiating activity and exhibiting therapy resistance. Accordingly, targeting cancer stem-like cells is a logical approach to developing an effective ovarian cancer therapy. The presence of ascites in ovarian cancer patients is associated with decreased five-year survival. Ascites promotes anti-apoptotic signaling in ovarian cancer cells. However, an ability of ascites to drive cancer stem-like behavior has not been tested. Glucose-regulated protein of 78 kDa (GRP78) is expressed on the surface of a variety of tumor types. This receptor is an ideal therapeutic target because it promotes tumor cell survival(Li and Lee, 2006) and is not detected in normal tissues. In head and neck cancer, cell surface GRP78-positive tumor cells exhibit cancer stem-like behaviors (Wu et al., 2010. Mol Cancer. 9:283.). However, it remains unknown if targeting cell surface GRP78 is an efficient strategy for killing cancer stem-like cells. We have produced and characterized a unique set of monoclonal antibodies specific for the carboxy-terminus (C-terminus) of GRP78. These antibodies inhibit Akt signaling and suppress melanoma growth (Misra et al. , 2010. Cancer biology & therapy. 9:142-152; de Ridder et al. 2012. Melanoma Res. 22:225-235). Neither the ability of these antibodies to target cancer stem-like cells, nor their therapeutic potential for ovarian cancer has been previously assessed. Purpose of the current studies: 1) Address the hypothesis that ascites fluid enriches for therapy-resistant ovarian cancer stem-like cells, 2) Determine efficacy of targeting cell surface GRP78 to eliminate ascites-enriched ovarian cancer stem-like cells. Experimental Procedures: Ovarian cancer cells (murine and human) were incubated with acellular ascites fluid obtained from an ovarian cancer mouse model or from ovarian cancer patients. Ability of these cells to grow as self-renewing spheres in vitro and to initiate tumor growth in a mouse model were measured. Stem cell marker (CD133, Oct4) and cell surface GRP78 expression were assessed by flow cytometry. Effects of anti-GRP78 antibodies on cancer stem-like cell behaviors in vitro and on tumor growth in vivo were measured. Data Summary: Incubation of ovarian cancer cells with ascites enriched for tumor cells with increased self-renewing/tumor initiating activity and increased cell surface GRP78 expression. A C-terminal anti-GRP78 antibody suppressed ascites-enriched ovarian cancer stem-like cells by inhibiting Akt signaling. Ovarian cancer bearing mice receiving the C-terminal anti-GRP78 antibody survived significantly longer than those receiving an IgG control. Conclusions: Acellular ascites fluid enriches for therapy-resistant cancer stem-like cells that express cell surface GRP78. An antibody specific for the C-terminus of GRP78 suppresses ascites-enriched ovarian cancer stem-like cells. We are currently testing the ability of this antibody to increase chemotherapy sensitivity of ovarian cancer by eliminating therapy-resistant cancer stem-like cells. These studies are expected to lay the groundwork for future clinical trials investigating if a humanized form of this anti- GRP78 antibody +/- chemotherapy reduces incidence of ovarian cancer mortality. Citation Format: Lihong Mo, Margaret Kennedy, Andrew Berchuck, George Cianciolo, Robin E. Bachelder, Salvatore V. Pizzo. Ascites drives ovarian cancer stem-like cell growth: therapeutic opportunities [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1420.

Cell surface GRP78: A potential marker of good prognosis and response to chemotherapy in breast cancer.

The 78-kDa glucose-regulated protein (GRP78) is a stress induced heat shock protein which, under limiting conditions, functions as a cell surface signaling receptor. Tumor cells are considered to be subjected to a physiologically stressful microenvironment due to their excessive growth. The role of GRP78 in tumor survival has been of notable interest. The present study aimed to assess the potential prognostic and predictive value of cell surface GRP78 expression in breast cancer tumor cells. Cell surface and cytoplasmic expression of GRP78 was examined by immunohistochemical staining of GRP78 in breast cancer archival paraffin-embedded tumor specimens. The cohort studied included breast cancer patients with operable T1,2, estrogen receptor-positive, node-negative cancer who were assessed using the Oncotype DX gene profile, as well as patients with locally advanced disease prior to and following neoadjuvant systemic treatment. GRP78 values were compared between the 2 groups, and prior to and following systemic treatment. Association analyses between GRP78 expression and prognostic markers were also performed. Cox regression analysis was used to examine the impact of these variables on disease-free survival (DFS). No differences in cytoplasmic GRP78 expression were observed. By contrast, the rates of cell surface GRP78 expression were 74.1% in the early stage operable patients, 36% in neoadjuvant systemic treatment patients prior to treatment and 62.5% in patients following systemic treatment (P<0.039). Positive cell surface GRP78 expression was associated with increased expression of the progesterone receptor (P=0.024), p53 expression (P=0.022) and improved DFS (P=0.047). In the case of GRP78 positivity, a trend for a superior response to chemotherapy was observed (P=0.19). The results of the present study indicated that cell surface GRP78 may be used as a marker for good prognosis in breast cancer and a potential marker for response to chemotherapy.

Cell Surface GRP78 Accelerated Breast Cancer Cell Proliferation and Migration by Activating STAT3.

High levels of cell surface glucose regulated protein 78 (sGRP78) have been implicated in cancer growth, survival, metastasis, and chemotherapy resistance. However, the underlying mechanism remains largely unknown. Here we report that the level of sGRP78 expression in human breast tumors gradually increases during cancer progression. Overexpression of GRP78 significantly enhanced its membrane distribution in human MCF-7 breast cancer cells, but had no effect on endoplasmic reticulum (ER) stress. High levels of sGRP78 facilitated cell proliferation and migration, as well as suppressed cell apoptosis. Neutralization of sGRP78 by a specific antibody against GRP78 alleviated sGRP78-induced cell growth and migration. Importantly, high phosphorylation levels of the signal transducer and activator of transcription 3 (STAT3) were found in human breast tumors that express sGRP78 and MCF-7 cells infected with adenovirus encoding human GRP78. Pretreatment with a GRP78 antibody suppressed STAT3 phosphorylation. Furthermore, genetic and pharmacological inhibition of STAT3 reversed the impacts of GRP78 on cell proliferation, apoptosis, and migration. These findings indicate that STAT3 mediates sGRP78-promoted breast cancer cell growth and migration.

The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M.

BackgroundEmerging data have suggested that cell surface GRP78 is a multifunctional receptor and has been linked to proliferative and antiapoptotic signaling cascades. Activated α2−macroglobin (α2M*) is a natural circulating ligand of cell surface GRP78. Association of cell surface GRP78 with α2M* is involved in the regulation of cell proliferation, survival and apoptosis in human cancers.MethodsThe invasion and metastasis of HCC cells were examined using transwell and wound healing assay; Cell surface expression of GRP78 was detected by in cell western assay. Translocation of GRP78 from cytosol to cell surface was observed by transfection of GRP78-EGFP plus TRIRC-WGA staining. The levels of Src, phosphor-Src, FAK, phospho-FAK, EGFR, phospho-EGFR, phospho-Cortactin, phospho-Paxillin were determined by western blot. Cell surface expression of GRP78 in HCC tissue samples was observed by immunofluorescence. The distribution of Paxillin and Cortactin in HCC cells was also observed by immunofluorescence. The interaction between GRP78 and Src were detected by far-western blot, co-immunoprecipitation and GST pulldown. GRP78 mRNA was detected by RT-PCR.ResultsIn the current study, we showed that association of cell surface GRP78 with α2M* stimulated the invasion and metastasis of HCC. Cell surface GRP78 could interact directly with c-Src, promoted the phosphorylation of c-Src at Y416. Inhibition of the tyrosine kinase activity of c-Src with PP2 reverted the stimulatory effect caused by association of cell surface GRP78 with α2M*. Moreover, association of cell surface GRP78 with α2M* facilitates the interaction between EGFR and c-Src and consequently phosphorylated EGFR at Y1101 and Y845, promoting the invasion and metastasis of HCCs. However, inhibition of the tyrosine kinase of c-Src do not affect the interaction between EGFR and Src.Conclusionc-Src plays a critical role in the invasion and metastasis of HCC induced by association of cell surface GRP78 with α2M*. Cell surface GRP78 directly binds and phosphorylates c-Src. As a consequence, c-Src phosphorylated EGFR, promoting the invasion and metastasis of HCCs.

Isthmin is a novel vascular permeability inducer that functions through cell-surface GRP78-mediated Src activation

Isthmin (ISM) is a recently identified 60 kDa secreted angiogenesis inhibitor. Two cell-surface receptors for ISM have been defined, the high-affinity glucose-regulated protein 78 kDa (GRP78) and the low-affinity αvβ5 integrin. As αvβ5 integrin plays an important role in pulmonary vascular permeability (VP) and ISM is highly expressed in mouse lung, we sought to clarify the role of ISM in VP. Recombinant ISM (rISM) dose-dependently enhances endothelial monolayer permeability in vitro and local dermal VP when administered intradermally in mice. Systemic rISM administration through intravenous injection leads to profound lung vascular hyperpermeability but not in other organs. Mechanistic investigations using molecular, biochemical approaches and specific chemical inhibitors revealed that ISM-GRP78 interaction triggers a direct interaction between GRP78 and Src, leading to Src activation and subsequent phosphorylation of adherens junction proteins and loss of junctional proteins from inter-endothelial junctions, resulting in enhanced VP. Dynamic studies of Src activation, VP and apoptosis revealed that ISM induces VP directly via Src activation while apoptosis contributes indirectly only after prolonged treatment. Furthermore, ISM is significantly up-regulated in lipopolysaccharide (LPS)-treated mouse lung. Blocking cell-surface GRP78 by systemic infusion of anti-GRP78 antibody significantly attenuates pulmonary vascular hyperpermeability in LPS-induced acute lung injury (ALI) in mice. ISM is a novel VP inducer that functions through cell-surface GRP78-mediated Src activation as well as induction of apoptosis. It induces a direct GRP78-Src interaction, leading to cytoplasmic Src activation. ISM contributes to pulmonary vascular hyperpermeability of LPS-induced ALI in mice.

Activated α2-Macroglobulin Binding to Human Prostate Cancer Cells Triggers Insulin-like Responses

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy.

Characterization and Mechanism of Stress-induced Translocation of 78-Kilodalton Glucose-regulated Protein (GRP78) to the Cell Surface

Glucose-regulated protein (GRP78)/BiP, a major chaperone in the endoplasmic reticulum, is recently discovered to be preferably expressed on the surface of stressed cancer cells, where it regulates critical oncogenic signaling pathways and is emerging as a target for anti-cancer therapy while sparing normal organs. However, because GRP78 does not contain classical transmembrane domains, its mechanism of transport and its anchoring at the cell surface are poorly understood. Using a combination of biochemical, mutational, FACS, and single molecule super-resolution imaging approaches, we discovered that GRP78 majorly exists as a peripheral protein on plasma membrane via interaction with other cell surface proteins including glycosylphosphatidylinositol-anchored proteins. Moreover, cell surface GRP78 expression requires its substrate binding activity but is independent of ATP binding or a membrane insertion motif conserved with HSP70. Unexpectedly, different cancer cell lines rely on different mechanisms for GRP78 cell surface translocation, implying that the process is cell context-dependent.

Targeting the glucose-regulated protein-78 abrogates Pten-null driven AKT activation and endometrioid tumorigenesis.

Rates of the most common gynecologic cancer, endometrioid adenocarcinoma (EAC), continue to rise, mirroring the global epidemic of obesity, a well-known EAC risk factor. Thus, identifying novel molecular targets to prevent and/or mitigate EAC is imperative. The prevalent Type 1 EAC commonly harbors loss of the tumor suppressor, Pten, leading to AKT activation. The major endoplasmic reticulum (ER) chaperone, GRP78, is a potent pro-survival protein to maintain ER homeostasis, and as a cell surface protein, is known to regulate the PI3K/AKT pathway. To determine whether targeting GRP78 could suppress EAC development, we created a conditional knockout mouse model utilizing progesterone receptor (PR)-Cre-recombinase to achieve Pten and Grp78 (cPtenf/fGrp78f/f) deletion in the endometrial epithelium. Mice with a single Pten (cPtenf/f) deletion developed well-differentiated EAC by 4 weeks. In contrast, no cPtenf/fGrp78f/f mice developed EAC, even after more than 8 months of observation. Histologic examination of uteri from cPtenf/fGrp78f/f mice also revealed no complex atypical hyperplasia (CAH), a well-established EAC precursor. These histologic observations among the cPtenf/fGrp78f/f murine uteri also corresponded to abrogation of AKT activation within the endometrium. We further observed that GRP78 co-localized with activated AKT on the surface of EAC thus providing an opportunity for therapeutic targeting. Consistent with previous findings that cell surface GRP78 is an upstream regulator of PI3K/AKT signaling, we show here that in vivo short-term systemic treatment with a highly specific monoclonal antibody against GRP78 suppressed AKT activation and increased apoptosis in the cPtenf/f tumors. Collectively, these findings present GRP78-targeting therapy as an efficacious therapeutic option for EAC.

Pharmacological induction of cell surface GRP78 contributes to apoptosis in triple negative breast cancer cells.

Breast cancer tumor with triple-negative receptors (estrogen, progesterone and Her 2, receptors) is the most aggressive and deadly subtype, with high rates of disease recurrence and poor survival. Here, we show that induction in cell surface GRP78 by doxorubicin and tunicamycin was associated with CHOP/GADD153 upregulation and increase in apoptosis in triple negative breast cancer tumor cells. GRP78 is a major regulator of the stress induced unfolded protein response pathway and CHOP/GADD153 is a pro-apoptotic transcription factor associated exclusively with stress induced apoptosis. The blocking of cell surface GRP78 by anti-GRP78 antibody prevented apoptosis, suggesting that induction of cell surface GRP78 by doxorubicin and tunicamycin is required for apoptosis. A better understanding of stress induction of apoptotic signaling in triple negative breast cancer cells may help to define new therapeutic strategies.