An antibody that targets cell-surface glucose-regulated protein-78 inhibits expression of inflammatory cytokines and plasminogen activator inhibitors by macrophages.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

Physiological Roles of the Autoantibodies to the 78-Kilodalton Glucose-Regulated Protein (GRP78) in Cancer and Autoimmune Diseases.

The 78 kDa glucose-regulated protein (GRP78), a member of the 70 kDa heat-shock family of molecular chaperones (HSP70), is essential for the regulation of the unfolded protein response (UPR) resulting from cellular endoplasmic reticulum (ER) stress. During ER stress, GRP78 evades retention mechanisms and is translocated to the cell surface (csGRP78) where it functions as an autoantigen. Autoantibodies to GRP78 appear in prostate, ovarian, gastric, malignant melanoma, and colorectal cancers. They are also found in autoimmune pathologies such as rheumatoid arthritis (RA), neuromyelitis optica (NMO), anti-myelin oligodendrocyte glycoprotein antibody-associated disorder (AMOGAD), Lambert-Eaton myasthenic syndrome (LEMS), multiple sclerosis (MS), neuropsychiatric systemic lupus erythematosus (NPSLE) and type 1 diabetes (T1D). In NMO, MS, and NPSLE these autoantibodies disrupt and move across the blood-brain barrier (BBB), facilitating their entry and that of other pathogenic antibodies to the brain. Although csGRP78 is common in both cancer and autoimmune diseases, there are major differences in the specificity of its autoantibodies. Here, we discuss how ER mechanisms modulate csGRP78 antigenicity and the production of autoantibodies, permitting this chaperone to function as a dual compartmentalized receptor with independent signaling pathways that promote either pro-proliferative or apoptotic signaling, depending on whether the autoantibodies bind csGRP78 N- or C-terminal regions.

Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders.

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.

Cell surface GRP78 signaling: An emerging role as a transcriptional modulator in cancer.

Cancer cells acquire dysregulated gene expression to establish specific transcriptional dependencies and their underlying mechanisms that are ultimately responsible for this addictions have not been fully elucidated. Glucose-regulated protein 78 (GRP78) is a stress-inducible, multifunctional, prosurvival, endoplasmic reticulum chaperone in the heat shock protein 70 family. Expression of cell surface GRP78 (CS-GRP78) is associated with increased malignant behavior and resistance to chemotherapy and radiotherapy by endowing various cancer cells with increased proliferative ability, altered metabolism, improved survival, and augmented invasive and metastatic potential. Emerging evidence has highlighted an unusual role of CS-GRP78 in regulating transcription factors (TFs) by mediating various signaling pathways involved in malignant transformation, metabolic reprogramming, and tumor progression. During the last decade, we targeted CS-GRP78 with C38 monoclonal antibody (C38 Mab) in numerous studies, which have highlighted the epigenetic interplay between CS-GRP78 and various TFs including c-MYC, Yes-associated protein/transcriptional coactivator with PDZ-binding motif, c-Fos, and histone acetylation to potentiate subsequent modulation of tumorigenesis, invasion, and metastasis. Here, we summarize the current state of knowledge about the role of CS-GRP78 in cancer development and progression, including epigenetic regulation and sheds light on CS-GRP78 as vulnerable target for cancer therapy. Overall, this review focuses on the mechanisms of TFs that are behind the transcriptional dysregulation in cancer and lays the groundwork for rational therapeutic use of C38 Mab based on CS-GRP78 biology.

Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion.

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

Targeting cell surface GRP78 enhances pancreatic cancer radiosensitivity through YAP/TAZ protein signaling.

Ionizing radiation (IR) can promote migration and invasion of cancer cells, but the basis for this phenomenon has not been fully elucidated. IR increases expression of glucose-regulated protein 78kDa (GRP78) on the surface of cancer cells (CS-GRP78), and this up-regulation is associated with more aggressive behavior, radioresistance, and recurrence of cancer. Here, using various biochemical and immunological methods, including flow cytometry, cell proliferation and migration assays, Rho activation and quantitative RT-PCR assays, we investigated the mechanism by which CS-GRP78 contributes to radioresistance in pancreatic ductal adenocarcinoma (PDAC) cells. We found that activated α2-Macroglobulin (α2M*) a ligand of the CS-GRP78 receptor, induces formation of the AKT kinase (AKT)/DLC1 Rho-GTPase-activating protein (DLC1) complex and thereby increases Rho activation. Further, CS-GRP78 activated the transcriptional coactivators Yes-associated protein (YAP) and tafazzin (TAZ) in a Rho-dependent manner, promoting motility and invasiveness of PDAC cells. We observed that radiation-induced CS-GRP78 stimulates the nuclear accumulation of YAP/TAZ and increases YAP/TAZ target gene expressions. Remarkably, targeting CS-GRP78 with C38 monoclonal antibody (Mab) enhanced radiosensitivity and increased the efficacy of radiation therapy by curtailing PDAC cell motility and invasion. These findings reveal that CS-GRP78 acts upstream of YAP/TAZ signaling and promote migration and radiation-resistance in PDAC cells. We therefore conclude that, C38 Mab is a promising candidate for use in combination with radiation therapy to manage PDAC.

Adipose stem cell crosstalk with chemo-residual breast cancer cells: implications for tumor recurrence.

PURPOSE: Most triple-negative breast cancer (TNBC) patients exhibit an incomplete response to neoadjuvant chemotherapy, resulting in chemo-residual tumor cells that drive tumor recurrence and patient mortality. Accordingly, strategies for eliminating chemo-residual tumor cells are urgently needed. Although stromal cells contribute to tumor cell invasion, to date, their ability to influence chemo-residual tumor cell behavior has not been examined. Our study is the first to investigate cross-talk between adipose-derived stem cells (ASCs) and chemo-residual TNBC cells. We examine if ASCs promote chemo-residual tumor cell proliferation, having implications for tumor recurrence. METHODS: ASC migration toward chemo-residual TNBC cells was tested in a transwell migration assay. Importance of the SDF-1α/CXCR4 axis was determined using neutralizing antibodies and a small molecule inhibitor. The ability of ASCs to drive tumor cell proliferation was analyzed by culturing tumor cells ± ASC conditioned media (CM) and determining cell counts. Downstream signaling pathways activated in chemo-residual tumor cells following their exposure to ASC CM were studied by immunoblotting. Importance of FGF2 in promoting proliferation was assessed using an FGF2-neutralizing antibody. RESULTS: ASCs migrated toward chemo-residual TNBC cells in a CXCR4/SDF-1α-dependent manner. Moreover, ASC CM increased chemo-residual tumor cell proliferation and activity of extracellular signal-regulated kinase (ERK). An FGF2-neutralizing antibody inhibited ASC-induced chemo-residual tumor cell proliferation. CONCLUSIONS: ASCs migrate toward chemo-residual TNBC cells via SDF-1α/CXCR4 signaling, and drive chemo-residual tumor cell proliferation in a paracrine manner by secreting FGF2 and activating ERK. This paracrine signaling can potentially be targeted to prevent tumor recurrence.

Serum cholesterol levels and tumor growth in a PTEN-null transgenic mouse model of prostate cancer.

BACKGROUND: Some, but not all, epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone synthesis, in prostate cancer. Using a PTEN-null transgenic mouse model of prostate cancer, we tested the effect of modifying serum cholesterol levels on prostate tumor development and growth. We hypothesized that serum cholesterol reduction would lower tumor androgens and slow prostate cancer growth. METHODS: PTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (30 mg/kg/day), or no intervention, and sacrificed at 2, 3, or 4 months of age. Serum cholesterol and testosterone were measured by ELISA and intraprostatic androgens by mass spectrometry. Prostate histology was graded, and proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL, respectively. RESULTS: Ezetimibe-treated mice had lower serum cholesterol at 4 months (p = 0.031). Serum cholesterol was positively correlated with prostate weight (p = 0.033) and tumor epithelial proliferation (p = 0.069), and negatively correlated with tumor epithelial apoptosis (p = 0.004). Serum cholesterol was unrelated to body weight (p = 0.195). Tumor stromal cell proliferation was reduced in the ezetimibe group (p = 0.010). Increased serum cholesterol at 4 months was associated with elevated intraprostatic DHEA, testosterone, and androstenedione (p = 0.043, p = 0.074, p = 0.031, respectively). However, cholesterol reduction did not significantly affect adenocarcinoma development at 2, 3, or 4 months of age (0, 78, and 100% in ezetimibe-treated vs. 0, 80, and 100% in mice not receiving ezetimibe). CONCLUSIONS: Though serum cholesterol reduction did not significantly affect the rate of adenocarcinoma development in the PTEN-null transgenic mouse model of prostate cancer, it lowered intraprostatic androgens and slowed tumor growth. These findings support a role for serum cholesterol in promoting prostate cancer growth, potentially via enhanced tumor androgen signaling, and may provide new insight into cholesterol-lowering interventions for prostate cancer treatment.

ROS-independent Nrf2 activation in prostate cancer.

In prostate cancer, oxidative stress and the subsequent Nrf2 activation promote the survival of cancer cells and acquired chemoresistance. Nrf2 links prostate cancer to endoplasmic reticulum stress, an event that triggers the unfolded protein response, aiming to restore cellular homeostasis as well as an adaptive survival mechanism. Glucose-regulated protein of 78 kD /immunoglobulin heavy chain binding protein (GRP78/BiP) is a key molecular chaperone in the endoplasmic reticulum that, when expressed at the cell surface, acts as a receptor for several signaling pathways enhancing antiapoptotic and proliferative signals. We showed GRP78/BiP translocation to PC3 cell surface in the presence of tunicamycin, an ER stress inductor, and demonstrated the existence of a GRP78/BiP-dependent non-canonical Nrf2 activation, responsible for increased resistance to ER-stress induced apoptosis. We found that, even in the absence of ROS production, tunicamycin causes Nrf2 activation, and activates Akt signaling, events bulnted by anti-GRP78/BiP antibody treatment. The presence of GRP78/BiP at the cell surface might be exploited for the immunotherapeutic strategy of prostate cancer since its blockage by anti-GRP78/BiP antibodies might promote cancer death by suppressing some of the several molecular protective mechanisms found in aggressive cancer cells.

Autoantibodies against the cell surface-associated chaperone GRP78 stimulate tumor growth via tissue factor.

Tumor cells display on their surface several molecular chaperones that normally reside in the endoplasmic reticulum. Because this display is unique to cancer cells, these chaperones are attractive targets for drug development. Previous epitope-mapping of autoantibodies (AutoAbs) from prostate cancer patients identified the 78-kDa glucose-regulated protein (GRP78) as one such target. Although we previously showed that anti-GRP78 AutoAbs increase tissue factor (TF) procoagulant activity on the surface of tumor cells, the direct effect of TF activation on tumor growth was not examined. In this study, we explore the interplay between the AutoAbs against cell surface-associated GRP78, TF expression/activity, and prostate cancer progression. First, we show that tumor GRP78 expression correlates with disease stage and that anti-GRP78 AutoAb levels parallel prostate-specific antigen concentrations in patient-derived serum samples. Second, we demonstrate that these anti-GRP78 AutoAbs target cell-surface GRP78, activating the unfolded protein response and inducing tumor cell proliferation through a TF-dependent mechanism, a specific effect reversed by neutralization or immunodepletion of the AutoAb pool. Finally, these AutoAbs enhance tumor growth in mice bearing human prostate cancer xenografts, and heparin derivatives specifically abrogate this effect by blocking AutoAb binding to cell-surface GRP78 and decreasing TF expression/activity. Together, these results establish a molecular mechanism in which AutoAbs against cell-surface GRP78 drive TF-mediated tumor progression in an experimental model of prostate cancer. Heparin derivatives counteract this mechanism and, as such, represent potentially appealing compounds to be evaluated in well-designed translational clinical trials.

Cell surface GRP78 promotes tumor cell histone acetylation through metabolic reprogramming: a mechanism which modulates the Warburg effect.

Acetyl coenzyme A (acetyl-CoA) is essential for histone acetylation, to promote cell proliferation by regulating gene expression. However, the underlying mechanism(s) governing acetylation remains poorly understood. Activated α2-Macroglobulin (α2M*) signals through tumor Cell Surface GRP78 (CS-GRP78) to regulate tumor cell proliferation through multiple signaling pathway. Here, we demonstrate that the α2M*/CS-GRP78 axis regulates acetyl-CoA synthesis and thus functions as an epigenetic modulator by enhancing histone acetylation in cancer cells. α2M*/CS-GRP78 signaling induces and activates glucose-dependent ATP-citrate lyase (ACLY) and promotes acetate-dependent Acetyl-CoA Synthetase (ACSS1) expression by regulating AKT pathways to acetylate histones and other proteins. Further, we show that acetate itself regulates ACLY and ACSS1 expression through a feedback loop in an AKT-dependent manner. These studies demonstrate that α2M*/CS-GRP78 signaling is a central mechanism for integrating glucose and acetate-dependent signaling to induce histone acetylation. More importantly, targeting the α2M*/CS-GRP78 axis with C38 Monoclonal antibody (Mab) abrogates acetate-induced acetylation of histones and proteins essential for proliferation and survival under hypoxic stress. Furthermore, C38 Mab significantly reduced glucose uptake and lactate consumption which definitively suggests the role of aerobic glycolysis. Collectively, besides its ability to induce fatty acid synthesis, our study reveals a new mechanism of epigenetic regulation by the α2M*/CS-GRP78 axis to increase histone acetylation and promote cell survival under unfavorable condition. Therefore CS-GRP78 might be effectively employed to target the metabolic vulnerability of a wide spectrum of tumors and C38 Mab represents such a potential therapeutic agent.

Evidence for Feedback Regulation Following Cholesterol Lowering Therapy in a Prostate Cancer Xenograft Model.

BACKGROUND: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS: Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS: Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS: Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

Chemotherapy enriches for an invasive triple-negative breast tumor cell subpopulation expressing a precursor form of N-cadherin on the cell surface.

BACKGROUND: Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others' indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC. METHODS: The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. RESULTS: TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment. CONCLUSIONS: TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease.

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.

Catalytic autoantibodies against myelin basic protein (MBP) isolated from serum of autistic children impair in vitro models of synaptic plasticity in rat hippocampus.

Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD.

Ascites Increases Expression/Function of Multidrug Resistance Proteins in Ovarian Cancer Cells.

Chemotherapy resistance is the major reason for the failure of ovarian cancer treatment. One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells. As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression. However, whether ascites drives multidrug resistance in ovarian cancer cells awaits elucidation. Here, we demonstrate that when cultured with ascites derived from ovarian cancer-bearing mice, a murine ovarian cancer cell line became less sensitive to paclitaxel, a first line chemotherapeutic agent for ovarian cancer patients. Moreover, incubation of murine ovarian cancer cells in vitro with ascites drives efflux function in these cells. Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)]. To demonstrate relevance of our findings to ovarian cancer patients, we studied relative efflux in human ovarian cancer cells obtained from either patient ascites or from primary tumor. Immortalized cell lines developed from human ascites show increased susceptibility to efflux inhibitors (MRP1, BCRP) compared to a cell line derived from a primary ovarian cancer, suggesting an association between ascites and efflux function in human ovarian cancer. Efflux in ascites-derived human ovarian cancer cells is associated with increased expression of ABC transporters compared to that in primary tumor-derived human ovarian cancer cells. Collectively, our findings identify a novel activity for ascites in promoting ovarian cancer multidrug resistance.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

INTRODUCTION: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)]. METHODS: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy. RESULTS: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells. CONCLUSION: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

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.

Syngeneic Murine Ovarian Cancer Model Reveals That Ascites Enriches for Ovarian Cancer Stem-Like Cells Expressing Membrane GRP78.

Patients with ovarian cancer are generally diagnosed at FIGO (International Federation of Gynecology and Obstetrics) stage III/IV, when ascites is common. The volume of ascites correlates positively with the extent of metastasis and negatively with prognosis. Membrane GRP78, a stress-inducible endoplasmic reticulum chaperone that is also expressed on the plasma membrane ((mem)GRP78) of aggressive cancer cells, plays a crucial role in the embryonic stem cell maintenance. We studied the effects of ascites on ovarian cancer stem-like cells using a syngeneic mouse model. Our study demonstrates that ascites-derived tumor cells from mice injected intraperitoneally with murine ovarian cancer cells (ID8) express increased (mem)GRP78 levels compared with ID8 cells from normal culture. We hypothesized that these ascites-associated (mem)GRP78(+) cells are cancer stem-like cells (CSC). Supporting this hypothesis, we show that (mem)GRP78(+) cells isolated from murine ascites exhibit increased sphere forming and tumor initiating abilities compared with (mem)GRP78(-) cells. When the tumor microenvironment is recapitulated by adding ascites fluid to cell culture, ID8 cells express more (mem)GRP78 and increased self-renewing ability compared with those cultured in medium alone. Moreover, compared with their counterparts cultured in normal medium, ID8 cells cultured in ascites, or isolated from ascites, show increased stem cell marker expression. Antibodies directed against the carboxy-terminal domain of GRP78: (i) reduce self-renewing ability of murine and human ovarian cancer cells preincubated with ascites and (ii) suppress a GSK3α-AKT/SNAI1 signaling axis in these cells. Based on these data, we suggest that (mem)GRP78 is a logical therapeutic target for late-stage ovarian cancer.

Binding of tissue-type plasminogen activator to the glucose-regulated protein 78 (GRP78) modulates plasminogen activation and promotes human neuroblastoma cell proliferation in vitro.

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.