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MemGRP78 positive tumor cells have a lower proliferation rate

Characterization of ID8 cell line

Ascites from ovarian cancer-bearing mice contributes to a faster progression of the cancer.

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Selected genes

Stemness genes

Supplementary Data from The Effects of Varying Dietary Carbohydrate and Fat Content on Survival in a Murine LNCaP Prostate Cancer Xenograft Model

Optimization of ascites treatment conditions

MemGRP78 positive tumor cells have a lower proliferation rate

Purpose: Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model.Experimental Design: Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 × 106 LNCaP cells (day 0) and sacrificed when tumors were ≥1,000 mm3.Results: Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29-0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37-0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors.Conclusions: These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.

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.

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.

Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrixproteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose(HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation anddownstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiatethis signaling cascade. Importantly, increased α2M was observed in diabetic patients’ serum, saliva,and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessedusing siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita andCD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein weresignificantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M*was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seenin normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibroticsignaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, andTGFβ1). In patients with established DKD, urinary α2M* and TGFβ1 levels were correlated. Thesedata reveal an important role for α2M* in the pathogenesis of DKD and support further investigationas a potential novel therapeutic targe Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients’ serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFβ1). In patients with established DKD, urinary α2M* and TGFβ1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.

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 α(2)-Macroglobulin (α(2)M*) 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.

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.

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.

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.

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.

Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys91 residue of the MBP NH2-terminal region Asp82 -Pro99, and the binding of Pg via a lysine-dependent mechanism to the Lys122 residue of the MBP COOH-terminal region Leu109-Gly126. In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology.

Heat shock proteins (HSP) play an important regulatory role in the physiology and pathology of both the urokinase-type plasminogen (Pg) activator (uPA) and tissue-type Pg activator (tPA) systems in the central nervous system (CNS). All the components of the Pg activator system are expressed in the CNS and their co-localization with Hsp27, Hsp70 or the 78-kDa glucose-regulated protein (GRP78) makes them essential players in processes such as synaptic transmission, synapse plasticity, neuroinflammation and nervous system repair. The Hsp participate in the neuroprotection of the CNS and their connections with both Pg activator systems play a regulatory role in these processes. The conversion of Pg to the active protease plasmin by both Pg activators are part of mechanisms used by neurons, microglia and astrocyte cells to stimulate cell growth and synaptic plasticity. In the CNS, the activity of plasmin is tightly regulated via mechanisms operating under the coordinated expression of HSP and components of the Pg activator system during normal conditions or in response to tissue injury. This chapter reviews the roles of Hsp27, Hsp70 and GRP78 in these mechanisms.

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 © 2016 Wiley Periodicals, Inc.

The Glucose Regulated Protein 78,000 (GRP78) is a member of the heat shock protein family in the endoplasmic reticulum to regulate protein quality control and degradation. We and others demonstrated that GRP78 is preferentially translocated onto the cell surface (CS-GRP78) and plays a novel role in cellular signaling, proliferation, migration, invasion, apoptosis, inflammation, and immunity which have major implications in cancer progression. CS-GRP78 acts as a multifunctional receptor by interacting with various ligands and other cell surface proteins to control key oncogenic signaling pathways. Activated α2-macroglobulin (α2M*) signals predominately through CS-GRP78 to regulate transcriptional activation of target genes, and, modulates metabolic regulation which might be an adaptive mechanism in rapidly proliferating cancer cells. Therapeutic agents against CS-GRP78 have the potential to target tumor cells with minimal harmful effects on normal cells. This review will focus on our understanding of CS-GRP78 and their influence on fundamental biological processes as well as its targeting agents which hold great therapeutic promise.

This chapter will present a historical perspective with respect to the presence of Glucose Regulated Protein 78,000 (GRP78) on the cell surface. Its presence there was completely unexpected since this protein normally functions as a molecular chaperone within the endoplasmic reticulum. When it appears on the cell surface, most particularly of cancer cells, it functions as a cell surface receptor coupled to signaling cascades which are pro-proliferative, antiapoptotic, and promigratory. Studies have identified antibodies in the plasma of cancer patients which bind to the amino-terminal domain of GRP78 where natural ligands also bind, functioning as agonistics. These antibodies are markers suggesting a poor prognosis. By contrast, antibodies which bind to the carboxyl-terminal domain block cancer cell proliferation and migration while promoting apoptosis. Thus, cell surface-associated GRP78 offers a new target for cancer therapy, particularly employing monoclonal antibodies.

Background: Ligation of cancer cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes proliferation and blocks apoptosis. Results: α2M* treatment of prostate cancer cells enhances the Warburg effect and up-regulates lipid metabolism in an insulin-like manner. Conclusion: α2M* exerts insulin-like effects on prostate cancer cells. Significance: Targeting cell surface GRP78-dependent cancer cell regulation, such as by antibodies, offers a unique potential therapeutic approach. 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-[14C]glucose or 1-[14C]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 [14CH3]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.

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.

// Ilaria Bellezza 1 , Paolo Scarpelli 1 , Salvatore V. Pizzo 2 , Silvia Grottelli 1 , Egidia Costanzi 1 and Alba Minelli 1 1 Department of Experimental Medicine, University of Perugia, Perugia, Italy 2 Duke University School of Medicine, Durham, NC, USA Correspondence to: Ilaria Bellezza, email: ilaria.bellezza@unipg.it Paolo Scarpelli, email: paolo.scarpelli@unipg.it Keywords: GRP78/BiP, Nrf2, Akt, Anti-GRP78/BiP antibody, dexamethasone Received: April 12, 2017 Accepted: May 23, 2017 Published: June 28, 2017 ABSTRACT 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.

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.

α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule.

In this study, we reported the effect of the ATP binding site competitive inhibitor Torin1 on activated α2-macroglobulin (α2M*)-induced cell proliferation and activation of mTORC1 and mTORC2 signaling in prostate cancer cells. Torin1 significantly inhibited α2M*-induced cellproliferation as measured by protein and DNA synthesis. Translational activity, a major cellular response in malignant cells,is coordinately regulated by the mTORC1-S6-kinaseand mTORC1-4EBP1 axes. Torin1 significantly inhibited α2M*- and insulin-induced activation of mTORC1 as determined by phosphorylation of S6-kinaseat Thr389 and 4EBP1 at Thr37/46 compared to untreated cells employing Raptor immunoprecipitates. Torin1 also significantly inhibited α2M*- and insulin-induced upregulation of p-AktT308 and p-AktS473 in prostate cancer cells. The effect was comparable to that of insulin employed as a positive control. Finally, Torin1 inhibited α2M*- and insulin-induced activation of mTORC2 kinase assayas measured by phosphorylation of Akt at Ser473 inRictor immunoprecipitates of prostate cancer cells.

Background Resveratrol (RESV) is a naturally occurring compound that may possess anticancer capabilities in both prostate carcinoma and melanoma. Methods The in vitro and in vivo cytotoxic activity of RESV and 3,5-dihydroxy-4'-acetoxy- trans -stilbene (4-ACE) was tested using cellular assays and a xenograft model. Five prostate carcinoma cell lines were used for in vitro evaluation. A melanoma cell line (Duke melanoma 738 [DM738]) and the prostate carcinoma line CWR22 were used for in vivo experiments. Mice were randomized to osmotic mini pumps with 200 μL of RESV (250 mg/mL), 4-ACE (335 mg/mL), or vehicle (50% dimethyl sulfoxide, 50% polyethylene glycol). Serum drug and metabolite levels were calculated by high-performance liquid chromatography with diode-array detection. Western blots were performed on treated tumors. Results were analyzed using a student’s t-test, analysis of variance, and the Mann–Whitney rank sum test. Results RESV and 4-ACE were cytotoxic in a time- and dose-dependent manner in all prostate carcinoma cell lines tested. Enhanced growth compared with controls was seen at the 24 h time point in four lines treated with RESV and two lines treated with 4-ACE ( P s In vivo , no difference in either tumor growth or postmortem tumor weight was detected in either DM738 ( P = 0.555, P = 0.562) or CWR22 ( P = 0.166, P = 0.811) xenografts treated with either drug. Serum drug levels did not correlate with tumor growth rates for any treatment group (all P s > 0.11). Treated tumors demonstrated protein changes by western blot. Conclusion Although in vitro data were promising, RESV and 4-ACE have limited potential as single agents in the treatment of prostate carcinoma and melanoma.

// Erik R. Nelson 1,5 , Shenduo Li 4 , Margaret Kennedy 4 , Sturgis Payne 4 , Kelly Kilibarda 4 , Jeffrey Groth 4 , Michelle Bowie 4 , Edgardo Parilla-Castellar 4 , Gustaaf de Ridder 4 , Paul Kelly Marcom 4 , Matthew Lyes 4 , Bercedis L. Peterson 2 , Michael Cook 3 , Salvatore V. Pizzo 4 , Donald P. McDonnell 5 and Robin E. Bachelder 4 1 Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign, Urbana and University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL, USA 2 Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, NC, USA 3 Department of Immunology, Duke University School of Medicine, Durham, NC, USA 4 Department of Pathology, Duke University Medical Center, Durham, NC, USA 5 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Correspondence to: Robin E. Bachelder, email: // Keywords : Precursor-N-cadherin, triple-negative breast cancer, invasion, metastasis, chemotherapy resistance Received : August 16, 2016 Accepted : October 07, 2016 Published : October 28, 2016 Abstract 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.

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.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 × 10Simvastatin 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.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.

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.

There has been an increasing recognition that mitochondrial perturbations play a central role in human heart failure. Mitochondrial networks, whose function is to maintain the regulation of mitochondrial biogenesis, autophagy ('mitophagy') and mitochondrial fusion/fission, are new potential therapeutic targets. Yet our understanding of the molecular underpinning of these processes is just emerging. We recently identified a role of the SWI/SNF ATP-dependent chromatin remodeling complexes in the metabolic homeostasis of the adult cardiomyocyte using cardiomyocyte-specific and inducible deletion of the SWI/SNF ATPases BRG1 and BRM in adult mice (Brg1/Brm double mutant mice). To build upon these observations in early altered metabolism, the present study looks at the subsequent alterations in mitochondrial quality control mechanisms in the impaired adult cardiomyocyte. We identified that Brg1/Brm double-mutant mice exhibited increased mitochondrial biogenesis, increases in 'mitophagy', and alterations in mitochondrial fission and fusion that led to small, fragmented mitochondria. Mechanistically, increases in the autophagy and mitophagy-regulated proteins Beclin1 and Bnip3 were identified, paralleling changes seen in human heart failure. Evidence for perturbed cardiac mitochondrial dynamics included decreased mitochondria size, reduced numbers of mitochondria, and an altered expression of genes regulating fusion (Mfn1, Opa1) and fission (Drp1). We also identified cardiac protein amyloid accumulation (aggregated fibrils) during disease progression along with an increase in pre-amyloid oligomers and an upregulated unfolded protein response including increased GRP78, CHOP, and IRE-1 signaling. Together, these findings described a role for BRG1 and BRM in mitochondrial quality control, by regulating mitochondrial number, mitophagy, and mitochondrial dynamics not previously recognized in the adult cardiomyocyte. As critical to the pathogenesis of heart failure, epigenetic mechanisms like SWI/SNF chromatin remodeling seem more intimately linked to cardiac function and mitochondrial quality control mechanisms than previously realized.

BACKGROUND Resveratrol increases lifespan and decreases the risk of many cancers. We hypothesized resveratrol will slow the growth of human prostate cancer xenografts. METHODS SCID mice were fed Western diet (40% fat, 44% carbohydrate, 16% protein by kcal). One week later, human prostate cancer cells, either LAPC-4 (151 mice) or LNCaP (94 mice) were injected subcutaneously. Three weeks after injection, LAPC-4 mice were randomized to Western diet (control group), Western diet plus resveratrol 50 mg/kg/day, or Western diet plus resveratrol 100 mg/kg/day. The LNCaP mice were randomized to Western diet or Western diet plus resveratrol 50 mg/kg/day. Mice were sacrificed when tumors reached 1,000 mm3. Survival differences among groups were assessed using Cox proportional hazards. Serum insulin and IGF axis were assessed using ELISAs. Gene expression was analyzed using Affymetrix gene arrays. RESULTS Compared to control in the LAPC-4 study, resveratrol was associated with decreased survival (50 mg/kg/day—HR 1.53, P = 0.04; 100 mg/kg/day—HR 1.22, P = 0.32). In the LNCaP study, resveratrol did not change survival (HR 0.77, P = 0.22). In combined analysis of both resveratrol 50 mg/kg/day groups, IGF-1 was decreased (P = 0.05) and IGFBP-2 was increased (P = 0.01). Resveratrol induced different patterns of gene expression changes in each xenograft model, with upregulation of oncogenic pathways E2F3 and beta-catenin in LAPC-4 tumors. CONCLUSION Resveratrol was associated with significantly worse survival with LAPC-4 tumors, but unchanged survival with LNCaP. Based on these preliminary data that resveratrol may be harmful, caution should be advised in using resveratrol for patients until further studies can be conducted. Prostate 73: 754–762, 2013. © 2012 Wiley Periodicals, Inc.

Worldwide regional disparities in the occurrence of prostate cancer (PCa) have been attributed in part to the effect of diet, with Asian men having up to a six fold decreased incidence over their western counterparts [1]. Epidemiologic observations, however, have been inconclusive about which specific dietary components contribute to PCa [2–5]. A logical extension is that if diet can affect the development of cancer, it can also influence the growth of an established tumor, and indeed this has been demonstrated in animal studies [6–9]. While traditionally a low fat diet was thought to be optimal, we previously hypothesized that a reduced carbohydrate diet was likewise beneficial [10]. In a prior study we found that mice consuming a no-carbohydrate ketogenic diet (NCKD) had increased overall survival and slower tumor growth, compared to mice on a western diet, a benefit not observed in mice consuming a low fat diet [11]. These findings were then replicated in a similar xenograft model with the finding that a low-fat diet was also beneficial [12]. The favorable effect in the NCKD mice was thought to be from decreased signaling of the insulin/ insulin-like growth factor (IGF) axis, a pathway integral in the progression of PCa [8, 10, 13–18]. These provocative results were further explored by another group with diets not completely devoid of carbohydrates, but rather with lower proportions (such as might be reasonably tolerated in human subjects) which have also been shown to delay tumor growth in a prostate xenograft model [19]. However, in that prior study a low-carbohydrate diet was not directly compared to a NCKD. To address this, we recently tested low-carbohydrate diets (10 and 20% carbohydrate kcals, respectively) vs. a NCKD in a mouse xenograft model and found all carbohydrate restricted diets had similar effects on tumor growth, overall survival, and IGF axis signaling, suggesting that low carbohydrate diets may be as effective as a NCKD [20]. While these dietary changes appear to be promising for slowing tumor growth, the best established therapy for stunting PCa involves chemical or surgical castration. The deleterious effects of androgen deprivation on weight, diabetes and cardiovascular health make dietary decisions under this circumstance of particular interest [21–23]. Thus we sought to explore the effect of low- and no-carbohydrate diets on tumor growth and survival as an adjunct to androgen deprivation therapy in a PCa xenograft model.

What's known on the subject? and What does the study add? It is known that both lactate inhibition and carbohydrate restriction inhibit tumour growth. What is unknown is whether the two work synergistically together. This study adds that though the combination of lactate inhibition and carbohydrate restriction did not synergistically slow tumour growth in our model, we confirmed that carbohydrate restriction started after tumour inoculation slowed tumour growth. Moreover, lactate inhibition resulted in changes in the tumour microenvironment that may have implications for future metabolic targeting of prostate cancer growth. OBJECTIVE • To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer. MATERIALS AND METHODS • 120 nude athymic male mice (aged 6–8 weeks) were injected s.c. in the flank with 1.0 × 105 LAPC-4 prostate cancer cells. • Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via α-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh; or NCKD and MCT1 inhibition. • Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights. • Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal–Wallis analysis of all four groups. Independent effects of treatment (NCKD vs WD and CHC vs Veh) on tumour volume were tested using linear regression analysis. • All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal–Wallis and linear regression analysis, respectively. RESULTS • There were no significant differences in tumour volumes among the four groups (P= 0.09). • When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P= 0.026), while CHC administration was not (P= 0.981). However, CHC was associated with increased necrotic fraction (P < 0.001). CONCLUSIONS • Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD. • MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction.

Administered in vivo, covalent receptor-recognized α2-macroglobulin (α2M*)-antigen complexes enhance humoral and cell-mediated immunity. We hypothesized that in vivo α2M*-encapsulation could be promoted in the setting of vaccines that co-deliver α2M* with unbound antigen, thereby eliminating the need to prepare complexes in vitro. Mice immunized intradermally with co-delivered α2M* and OVA demonstrated antigen-specific immune responses, including anti-tumor responses, similar to those elicited by conjugated α2M*-OVA complexes. Enhanced immunity appears to result from in vivo α2M*-encapsulation of antigen. This finding represents a significant advancement in the development of α2M* as an antigen delivery vehicle capable of enhancing the presentation of subunit vaccines.

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.