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.

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

Background: Medullary thyroid carcinoma management consists mainly of surgical resection and is largely chemoresistant There is ongoing effort to discover novel therapies for medullary thyroid carcinoma. Increased levels of heat shock proteins have been associated with multiple cancers and are being studied as potential therapeutic targets. The purpose of this study was to determine the expression levels of heat shock proteins 90 and 70 and of glucose related protein 78 in medullary thyroid carcinoma tissues compared with normal thyroid tissues.Methods: 20 tissue specimens of medullary thyroid carcinoma and 10 specimens of thyroids without malignancy were analyzed by immunohistochemistry.Results: Medullary thyroid carcinoma specimens showed 27% higher expression level of heat shock protein 90 immunostaining, and a 43% higher expression level of heat shock protein 70 immunostaining versus normal controls. These differences, however, were not statistically significant. A significantly higher expression level was noted for glucose related protein 78 in the medullary thyroid carcinoma specimens than in the controls.Conclusion: This study indicates increased expression levels of heat shock proteins 90 and 70 and glucose related protein 78 levels in medullary thyroid carcinoma. These findings, though preliminary imply that these proteins may have a role in medullary thyroid carcinoma's tumor biology and may have and future therapeutic options. Larger cohorts are needed to corroborate these results. (C) 2016 Elsevier Inc. All rights reserved.

BACKGROUND AND OBJECTIVES: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.METHODS: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.RESULTS: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.CONCLUSIONS:We demonstrate that administration of the peptide ADoPep to diabetic mice improved type 1 diabetes by preventing pancreatic β cell apoptosis.

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.

Physiological stress takes place in the endoplasmic reticulum（ER） of cells where activation and up-regulation of genes and proteins are primarily induced to enhance pro-survival mechanisms such as the unfolded protein response（UPR）.A dominant protein in the UPR response is the heat shock GRP78 protein.Although GRP78 is primarily located in the ER,under certain conditions it is transported to the cell surface,where it acts as a receptor inducing pathways of cell signaling such as proliferation or apoptosis.In the prolonged chronic stress transportation of the GRP78 from the ER to the cell membrane is a major event where in addition to the presentation of the GRP78 as a receptor to various ligands,it also marks the cells that will proceed to apoptotic pathways.In the normal cell that under stress acquires cell surface GRP78 and in the tumor cell that already presents cell surface GRP78,cell surface GRP78 is an apoptotic flag.The internalization of GRP78 from the cell surface in normal cells by ligands such as peptides will enhance cell survival and alleviate cardiovascular ischemic diseases.The absence of cell surface GRP78 in the tumor cells portends proliferative and metastatic tumors.Pharmacological induction of cell surface GRP78 will induce the process of apoptosis and might be used as a therapeutic modality for cancer treatment.

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.

Introduction Glucose-regulated protein 78 (GRP78) has emerged as an important regulator of tumour cell signalling and viability. We have analysed cell surface GRP78 in benign and malignant parotid salivary tumours in correlation to apoptosis. Materials and methods Primary cell-cultures were produced from tissue biopsies. Cell surface GRP78 stained by anti GRP78 antibody was analysed by fluorocytometry. Apoptosis was determined by Annexin V/PI. Results A significant decrease in the percentage of cell surface GRP78 was determined in the benign parotid tumour in comparison to normal tissue. Cell surface GRP78 expression in malignant tumours was significantly higher and correlated with decrease in apoptosis. Conclusion This study is the first report describing cell surface GRP78 expression in parotid salivary tumour. The evidence of high surface GRP78 expression in malignant parotid tissue in contrast to the benign tumour might add a diagnostic tool and serve as a future target for treatment of malignant parotid tumours.

e22179 Background: Glucose-regulated protein (GRP78) is a key regulator of the unfolding protein response. Cancer cells overexpress GRP78, inducing its translocation to the cell surface. Little is known about cell surface GRP78 expression in breast cancer. The aim of this study was to assess the potential prognostic impact and predictive value of GRP78 expression in patients with early and locally advanced breast cancers. Methods: Cytoplasmic and cell-surface expression of GRP78 was examined in two patient groups: Group 1 - patients with operable T1,2, estrogen receptor-positive, node-negative cancer who were assessed with the Oncotype-DX gene profile in addition to the standard pathology assessment; Group 2 - patients mostly with locally advanced disease who received neoadjuvant systemic treatment. Association analyses were performed between GRP78 expression and prognostic variables of grade, estrogen receptor and progesterone receptor expression, and HER2 and Ki67 status. GRP78 values were compared between Group 1 , Group 2 before systemic treatment and Group 2 after systemic treatment. Results: The cohort included 48 patients, 21 in Group 2 (20 with locally advanced breast cancer). Rates of cell-surface GRP78 expression were 44% in Group 1, 4.8% in Group 2 before systemic treatment, and 23.8% in Group 2 after systemic treatment. The difference among the groups was statistically significant (p<0.003). This was not true for cytoplasmic GRP78 expression. Positive cell-surface GRP78 expression was associated with known good prognostic markers of high progesterone receptor expression (p=0.04) and low Ki67 percentage (p=0.018). There was a trend toward an inverse correlation between positive cell-surface GRP78 and the Oncotype-DX gene profile score, (p=0.11). Conclusions: Cell surface GRP78 positivity is a good prognostic marker in breast cancer and may serve as a potential marker of benefit from chemotherapy. Prospective studies of neoadjuvant treatment are needed using serial biopsies with a correlation to response in order to confirm these findings.

Background The identification of new biomarkers is required for early diagnosis of colorectal carcinoma patients (CRC), since about 20% of these patients are initially diagnosed with a distant metastatic disease. GRP78, a heat shock protein, functions also as a cell surface signaling receptor of cells under physiological stress. GRP78 was found to be expressed on the cell surface of various tumor cells. The presence of autoantibodies to GRP78 in cancer patient's serum was found to be correlated with a poor prognosis. In this study we aimed to identify anti-GRP78 antibodies in the serum of 85 patients diagnosed by colonoscopy, as an early detection biomarker. Methods We developed an ELISA assay with recombinant GRP78 immobilized on 96-well culture plates and used an anti-IgG antibody to measure the sole anti-GRP78 IgGs. Results Testing for anti-GRP78 showed a significant increase in antibody titer in patients with a polyp and in CRC patients (p<0.001) compared to healthy subjects. Conclusions This is the first study showing the presence of anti-GRP78 at the very early stages of CRC.

The better understanding of the mechanism in which the immune system responds to the developing cancer provided the outcome in a new era in cancer immunotherapy. The tumor suppressive effect on the immune system is caused by negative T cell receptor signaling that abrogate immunity against the cancer cells. Novel monoclonal antibodies that target co-inhibitory receptors on T cells block the tumor induced inhibition of the immune system and enable the immune system to eradicate the tumors. The development of such antibodies started twenty years ago by the preparation of a monoclonal antibody termed BAT. A single administration of the antibody to tumor bearing mice resulted in striking anti tumor activity that was mediated by the lymphocytes. These studies provided a basis for the new era of cancer immunotherapy. The present review summarizes twenty years to the discovery of monoclonal antibodies harnessing the immune system to eradicate tumors.

Background The glucose regulated heat shock protein 78 (GRP78) is a central regulator of ER (endoplasmic reticulum) stress due to its pro-survival property. Up regulated GRP78 expression in tumor cells has been correlated with aggressive malignancies whereas some reports have predicted an improved prognosis. Over-expression of GRP78 in the ER promotes its localization to the cell surface on several cell types including tumor cells. Methods In order to elucidate whether GRP78 receptor positive and negative tumor cells manifest different properties in colorectal cancer, we first artificially separated GRP78 positive and negative sub-populations from HM7 and HCT116 cell lines using anti GRP78 antibody coated magnetic beads. Results Only GRP78 negative cells were highly proliferative, induced significant growth in tumor size in nude mice and metastasized to the liver in a human metastatic colorectal carcinoma model in mice. In contrast, GRP78 positive cells manifested reduced proliferation, colony formation, tumor growth and liver metastases. The reduced tumorigenicity of GRP78 positive subpopulation was abrogated by silencing GRP78 expression using siRNA oligomers. In our efforts to induce cell surface GRP78, we subjected the cells to doxorubicin and taxol that increased significantly the percent of GRP78 positive population. Cells pre-incubated with doxorubicin exhibited reduced proliferation and tumor growth in mice. Conclusion This study demonstrates the significance of cell surface GRP78 in colon cancer, which may be used as a marker for reduced tumorigenicity.

Myocardial ischemia is a severe stress condition that causes extensive biochemical changes triggering cardiac cell death. The 78-kDa glucose-regulated protein (GRP78), a heat shock protein present in all cells and a widely used marker of endoplasmic reticulum stress, functions in controlling the structural maturation of nascent glycoproteins. However, GRP78 was also found to be expressed on the cell surface of several cells such as endothelial cells, macrophages, and tumor cells where it functions as a receptor for a variety of ligands in signaling pathways. Recently, we have identified peptides from two different sources that specifically bind GRP78 protein. We have shown that binding of these peptides to endothelial cell surface GRP78 resulted in angiogenesis. In this study, we first established the presence of cell surface GRP78 on cardiac myocytes. Analysis of cardiomyocytes under hypoxia determined the significant increase in cell surface GRP78 in addition to gene expression and total protein. Apoptosis that was significantly increased in cardiomyocytes under hypoxic conditions was inhibited by the presence of the peptide-binding GRP78 during hypoxia. Inhibition of apoptosis was mediated by the binding of the peptide to cardiomyocytes cell surface GRP78 resulting in blocking caspase-3/7 activation. Silencing GRP78 RNA that reduced GRP78 receptor abrogated the peptide activity. Apoptosis of cardiac cells induced by myocardial infarction in a mouse model was also significantly inhibited by the administration of the peptide to mouse hearts. Our findings may make ADoPep1 a useful therapeutic tool for relieving of ischemia.

We have previously identified several angiogenic peptides that bind cell surface proteins by screening a phage display peptide library on human umbilical endothelial cells exposed to hypoxic conditions. In this study we describe one of the selected peptides, SP. We found by protein precipitation of endothelial cell lysates that the 12 amino acid SP peptide binds cell surface vimentin. Surprisingly, vimentin was detected on the cell surface of about 30% of intact endothelial cells under both normoxic and hypoxic conditions, as was demonstrated by fluorocytometric analysis on viable cells. The assessment of SP in the induction of angiogenesis was established by a significant increase in endothelial cell proliferation and tube formation under hypoxic conditions and not under normoxic conditions. Cell proliferation and tube length increased two-fold in endothelial cells in the presence of 10 ng/ml SP peptide when compared to controls. The specificity of SP binding to vimentin was demonstrated by SP inhibition of anti-vimentin binding and by the inhibition of tube formation in cells transfected with siRNA against vimentin. Local intramuscular administrations of the peptide SP to ischemic hind limbs using the mouse hind limb ischemia model, demonstrated that SP inoculated at 1 and 10 μg, improved blood perfusion compared to inoculations with an irrelevant peptide or PBS. The recovery of blood perfusion correlated with the increase in the number of detectable capillaries in the ischemic limb. The development of novel peptides for the induction of pro-angiogenic activity may pave the way for new therapeutic strategies in the treatment of cardiovascular ischemic diseases.

OBJECTIVES:It is a common clinical observation that collateral vessel development is impaired in diabetic patients with ischaemic vascular diseases. Consequently, alternative revascularisation strategies in diabetic patients are needed. This study presents the effect and mechanism of new peptide therapeutic angiogenesis in an ischaemic and diabetic mouse model.DESIGN:Streptozocin-injected mice that had undergone hind-limb ischaemia were treated with angiogenic peptides. Blood flow restoration was calculated by laser Doppler imager and corroborated by histological section. For the mechanism study, endothelial cells were exposed to hypoxia and high glucose concentrations to study the effect of the peptides on proliferation and anti-apoptosis.RESULTS:The peptides significantly restored blood perfusion 21 days after surgery in the diabetic mice (p < 0.01) by neo-vascularisation, corroborated by an increase in capillary density. In addition, the peptides induced the proliferation of hypoxic endothelial cells (p < 0.01) and protected the cells from apoptosis in high glucose cultures.CONCLUSIONS:This is the first approach for treatment of ischaemic vascular disease with peptides in a diabetic mouse model.