Normal Cell Surface Research Articles

Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

BackgroundSialylation of glycoproteins and glycolipids is important for biological processes such as cellular communication, cell migration and protein function. Biosynthesis of CMP-sialic acid, the essential substrate, comprises five enzymatic steps, involving ManNAc and sialic acid and their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood. Methods and resultsWe aimed to study the mechanisms of sialic acid metabolism in knockouts (KO) of the sialic acid pathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), NANS (sialic acid synthase) and CMAS (N-acylneuraminate cytidylyltransferase) genes, but was largely unaffected in NANP (N-acylneuraminate-9-phosphatase) KO, as studied by MAA and PNA lectin binding. NANP is the third enzyme in sialic acid biosynthesis and dephosphorylates sialic acid 9-phosphate to free sialic acid. LC-MS analysis of sialic acid metabolites showed that CMP-sialic acid was dramatically reduced in GNE and NANS KO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP-sialic acid levels in NANP KO were comparable to WT cells, even though sialic acid 9-phosphate, the substrate of NANP accumulated. Metabolic flux analysis with 13C6-labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid. ConclusionsOur data provide evidence that NANP activity is not essential for de novo sialic acid production and point towards an alternative phosphatase activity, bypassing NANP. General significanceThis report contributes to a better understanding of sialic acid biosynthesis in humans.

Immunoreceptor NKG2D and its ligand-mediated cellular immunity and its relationship with diseases

Natural killer (NK) cells play an important role in host defense.NKG2D is expressed as a homodimer on the surface of NK cells and is an important lymphocyte activating receptor.NKG2D ligands are negatively or poorly expressed on the surface of normal cells, but when the cells are in a state of oxidative stress, malignant transformation, autoimmune or inflammatory stimulation, these ligands will be induced on the cell surface.The NKG2D receptor and its ligand-mediated cellular immune response play an important role in immune surveillance of tumor cells, and at the same time it is also closely related to certain autoimmune diseases. Key words: NKG2D; NKG2D ligand; Cellular immunity; Autoimmune diseases

Inhibition of ezrin causes PKCα-mediated internalization of erbb2/HER2 tyrosine kinase in breast cancer cells

Unlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKCα-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKCα-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface.

Study of erythrocytes as a novel drug carrier for the delivery of artemether

Resealed erythrocytes have been explored in various dimensions of drug delivery, owing to their high biocompatibility and inability to initiate immune response. The present research was designed to evaluate the drug delivery potential of erythrocytes by loading a hydrophobic anti-malarial drug, Artemether. Three different loading techniques were applied to achieve maximum optimized drug loading. A HPLC method was validated for drug quantification in erythrocytes. The relatively high loading was achieved using hypotonic treatment was 31.39% as compared to other two methods. These, drug loaded erythrocytes were characterized for membrane integrity via ESR showing higher ESR values for drug loaded cells as compared to normal cells. Moreover, microscopic evaluation was done to observe morphological changes in erythrocytes after successful loading which showed swollen cells with slight rough surface as compared to smooth surface of normal cells. Drug release was studied for 8 h which showed more than 80% release within 3-7 h from erythrocytes treated with different hypotonic methods. Overall, the study revealed a potential application of erythrocytes in delivery of hydrophobic drugs using hypotonic treatment as compared to other methods.

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells.

BackgroundTo improve the targeting ability of antitumor drugs, we identified the antigens with high expression on the surface of tumor cells associated with tumor escape, such as the complement regulatory protein CD55 molecule, which is also known as the decay accelerating factor. In this study, phage display technology was used to screen and identify CD55-specific ligand peptide (CD55sp) bound to CD55 molecule on the surface of cervical cancer HeLa cells. We then explored the role of this peptide in inhibiting the growth of cervical cancer cells in vitro. Our characterization of CD55sp will provide implication for tumor target therapy.MethodsThe phage bound to the surface of HeLa cells were isolated by phage display technology. Positive phage clones were identified by ELISA. Phage was then amplified and determined by agarose gel electrophoresis after monoclonal DNA extraction. DNA sequencing and bioinformatical analysis were conducted to obtain specific ligand peptides. Flow cytometry and immunofluorescence were used to measure the expression of CD55 molecule on the surface of tumor and normal cells. Subsequently, the effects of CD55sp on the proliferation and apoptosis of HeLa and SiHa cells were determined by Cell Counting Kit-8 (CCK-8), flow cytometry, and TUNEL assay, respectively. The morphology of apoptotic cells was examined by electron microscope. The distribution of Cleaved caspase-3 was detected by immunofluorescence. The expression of bcl-2 and Cleaved caspase-3 were determined by Western blot.ResultsThe results showed that the peptide (QVNGLGERSQQM) can bind to the CD55 molecule on the surface of cervical cancer HeLa and SiHa cells as a ligand peptide. It can also effectively inhibit the proliferation of cervical cancer cells and induce cell apoptosis.ConclusionThis study demonstrates that CD55sp screened by phage display technology plays a strong antitumor role.

Abstract B28: Targeting the tumor-associated carbohydrate antigen STn with humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibits ovarian cancer tumor growth in vitro and in vivo

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies in the U.S. The current standard of care is tumor debulking followed by chemotherapy. This treatment results in approximately 70% of patients achieving an initial complete clinical response. However, many of these patients will unfortunately relapse with chemoresistant disease developing in part to the presence of cancer stem cells (CSCs) within the tumor. Indeed, ovarian CSCs have been identified and shown to be resistant to chemo- and radiotherapy. Cancer cell surface glycans, called tumor-associated carbohydrate antigens (TACAs), are a class of attractive cancer-specific targets found on the cell surface of many solid tumors. Siamab is targeting a glycan structure (Sialyl-Tn, STn) that is cancer-specific and a major reported constituent of two well-known CSC biomarkers, CD44 and MUC1, residing on both CSCs and mature malignant cells in some cancer types. Although CD44 and MUC1 are also present on normal tissue and normal stem cell surfaces, tumor and CSC specificity is conferred the presence of STn-glycosylated variants of these CSC markers and other cell surface proteins that are essentially absent from normal human tissues. Importantly, STn can be a component of CA-125 (MUC16), conferring additional specificity to this diagnostic marker for ovarian carcinoma. The elevated presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. In addition, elevations in STn expression are linked to chemotherapy resistance and enable tumors to evade the host immune system. We have observed that STn levels are elevated in cancer cells following chemotherapy treatment in both in vitro and in vivo model systems and STn is present on specific subtype of tumor-infiltrating myeloid-derived suppressor cells. Both observations support the role of STn in tumor chemoresistance and immunomodulation. While attempts have been made to target STn clinically with a cancer vaccine, efficacy has been limited. Siamab has developed a unique immunotherapeutic solution aimed at eradicating human neoplasias by targeting both CSCs and bulk tumor to enable whole tumor killing. We have identified and humanized a panel of mouse monoclonal novel antibodies (Abs) that specifically target with high affinity the STn glycan independent of its carrier protein. These Abs are highly selective in binding assays demonstrating robust nanomolar EC50s, possess specificity on tissues as determined with neuraminidase treatment, and exhibit STn-specific glycan binding on Siamab’s proprietary glycan array. Antitumor efficacy was evaluated in vitro and in vivo utilizing humanized anti-STn antibody drug conjugated (ADC) material. Cell cytotoxicity was demonstrated in a panel of STn-expressing cell lines with low nanomolar IC50s. We confirmed that the decrease in proliferation in vitro can be translated to a reduction in tumor size in vivo, through a series of cell line and patient-derived ovarian cancer xenograft models. Inhibition of tumor progression were observed in all models, with complete regressions observed in some treatment arms. No significant weight loss was observed for any treatment groups, indicating the therapy was well tolerated by all the groups. A decrease in STn expression was noted in the tumors following treatment, indicating that we were hitting the target against which these Abs were generated. In addition, we have begun the development of both tissue- and serum-based biomarker assays utilizing these selective anti-STn Abs. Our data demonstrate that high-affinity, STn-selective humanized mAbs show promise as therapies for ovarian tumors, as well as tools for patient stratification and pharmacodynamic biomarker assessments. Citation Format: Daniel T. Dransfield, Jillian M. Prendergast, David A. Eavarone, Rawan Nazer, Linah Al-Alem, Jenna Stein, Jeff Behrens, Bo Rueda. Targeting the tumor-associated carbohydrate antigen STn with humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibits ovarian cancer tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B28.

Visualization of Ovarian Cancer Cells with Peptide VEGEGEEGEEY

The binding of peptide VEGEGEEGEEY to the surface of ovarian cancer cells was studied by confocal microscopy. It has been demonstrated that the peptide competes with hyaluronan for the binding to RHAMM (receptor for hyaluronan-mediated motility). It was found that peptide VEGEGEEGEEY specifically bound to RHAMM on ovarian cancer cell surface, and this interaction was blocked by anti-RHAMM antibodies. The peptide did not bind to RHAMM-knockout fibroblasts RHAMM(–/–) but interacted with transfected fibroblasts RHAMM(+/+), which further confirms the binding of the peptide to RHAMM. Evaluation of the peptide binding selectivity showed that the peptide reacts with cancer cells and does not bind to the surface of fibroblasts and normal cells. Thus, the specificity of binding of peptide FITC-VEGEGEEGEEY to RHAMM suggests a possibility of its application as a molecular probe for imaging and diagnosis of ovarian cancer at early stages.

The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology.

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20-25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.

Glucose-bridged silver nanoparticle assemblies for highly sensitive molecular recognition of sialic acid on cancer cells via surface-enhanced raman scattering spectroscopy

The expression levels of glycans on the surfaces of cancer and normal cells show different, however, this difference is not noticeable enough to distinguish them directly. So, herein, based on the targeted molecular recognition of the glycans on cell surfaces by 4-mercaptophenyl boronic acid (MPBA), a novel surface-enhanced Raman scattering (SERS) nanoprobe (glucose-MPBA@AgNPs) was prepared by inducing controllable assembly of MPBA decorated Ag nanoparticles (MPBA@AgNPs) in a certain level via the bridge of glucose to amplify such a limited difference in SERS measurements. On the basis of the aggregation-induced 3D SERS hot spot effect, this multi-particle nanoprobe possesses over 10 times stronger SERS enhancement ability than the individual MPBA@AgNPs. As the different sialic acid (SA) expression on the surfaces of cancer and normal cells led to the different accumulation of glucose-MPBA@AgNPs, the results we obtained (mean intensities recorded from five cells) indicate the SA amounts on two kinds of cells can provide 5–7 times signal contrast grade in SERS band intensities (P < 0.001). Compared with the monodispersed nanoprobe, our developed nanoprobe amplifies the SA expression difference on cell surfaces and supports high sensitivity for cancer cell recognition, which might be useful in providing highly effective recognition of the edges of tumor tissues in clinic field.

Anti-MUC1 aptamer: A potential opportunity for cancer treatment.

Mucin 1 (MUC1) is a protein usually found on the apical surface of most normal secretory epithelial cells. However, in most adenocarcinomas, MUC1 is overexpressed, so that it not only appears over the entire cell surface, but is also shed as MUC1 fragments into the blood stream. These phenomena pinpoint MUC1 as a potential target for the diagnosis and treatment of cancer; consequently, interest has increased in MUC1 as a molecular target for overcoming cancer therapy challenges. MUC1 currently ranks second among 75 antigen candidates for cancer vaccines, and different antibodies or aptamers against MUC1 protein are proving useful for tracing cancer cells in the emerging field of targeted delivery. The unique properties of MUC1 aptamers as novel targeting agents, and the revolutionary role that MUC1 now plays in cancer therapy, are the focus of this review. Recent advancements in MUC1-targeted cancer therapy are also assessed.

FOLATE PREVENTS THE DETRIMENTAL EFFECTS OF OLIGOMERIC Aβ ON INSULIN RECEPTOR LOCALIZATION AND FUNCTION AND LONG-TERM POTENTIATION

Oligomeric Aβ peptide toxicity may contribute to neuronal dysfunction and neurodegeneration in Alzheimer's disease (AD). For example, we showed that Aβ toxicity may underlie AD-associated “Type 3 Diabetes” by inhibiting Kinesin-5, thereby preventing the normal localization and function of the insulin receptor and the glucose transporter (AAIC, 2016). Aβ treatment acutely inhibits long-term potentiation (LTP) and results in synaptic loss, which is mediated in part by Aβ-mediated inhibition of Kinesin-5, likely via mis-localization of key neurotransmitter receptors. In view of evidence that folic acid/folate supplementation may delay/reduce the incidence of AD, we sought to determine whether folate can prevent the toxic effects of Aβ on insulin receptor localization and function and on LTP. N2A cells in culture were co-treated with folate (2 μM) and Aβ42 peptide (2 μM), and insulin receptor localization, function, and phosphorylation were assessed by immunocytochemistry following insulin challenge. LTP in the stratum radiatum was also assessed in mouse brain slices by response to high frequency (2 x 100 Hz) stimulation (HFS) of the Schaffer collateral axons. LTP was strongly inhibited by 100 nM oligomeric Aβ peptide. In the presence of Aβ peptide, folate (2 μM) was added either for 40 min before the HFS (short) or for at least 60 min prior to the HFS and for the remaining 60 min of the recording period (long). N2A cells treated with oligomeric Aβ (48 h) alone exhibited decreased insulin receptor activation (phosphorylation), and the majority of the insulin receptor localized to small aggregates associated with the cis-Golgi, rather to the cell surface. N2A cells co-treated with folate and Aβ maintained normal cell surface localization and function/phosphorylation of the insulin receptor in response to insulin stimulation. ‘Long’ treatment of the mouse brain slices, but not ‘short’ treatment, with folate restored LTP in the presence of oligomeric Aβ. Folate pre-treatment blocks oligomeric Aβ-mediated inhibition of neuronal metabolism and memory function. These results provide a mechanism by which folate can prevent the Aβ toxicity and reinforce the concept that folate supplementation may slow the development of AD.

Altered expression of lysosomal associated membrane protein 1 in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most common cancers. LAMP1, major protein components of lysosome, is primarily located on the lysosomal membrane and rarely expressed on the surface of normal cells, playing an important role in the lysosome-mediated physiological processes. Previous studies confirmed that LAMP1 showed high expression in astrocytoma. The purpose of this study was to investigate the expression levels of LAMP1 and to discuss its roles in ESCC. MethodsWe collected 610 tissue samples of ESCC patients to construct tissue microarrays, which were subsequently stained by immunohistochemistry with LAMP1 antibody. ResultsAfter immunohistochemical staining, a total of 584 patients, including 453 men and 131 women, were analysed. The positive immunostaining was mainly located at the cytoplasm. The LAMP1 expression levels were significantly different between different T status (P<0.001), TNM stages (P<0.01) and degrees of tumor histological differentiation (P<0.001). Besides, LAMP1 expression levels were positively correlated with TNM stages (P<0.05). The higher the TNM stages, the higher the LAMP1 expression levels. Similar results also appeared in degrees of tumor histological differentiation (P<0.01), but not in ages, genders, tumor size, T status, lymphatic metastasis and tumor locations (P>0.05). ConclusionLAMP1 is involved in the TNM stages and histological differentiation of the ESCC. Targeted therapy for LAMP1 may be a promising novel therapeutic strategy against poorly differentiated ESCC.

Self-Targeting, Immune Transparent Plasma Protein Coated Nanocomplex for Noninvasive Photothermal Anticancer Therapy.

Cancer cells exhibit specific physiological differences compared to normal cells. Most surface membranes of cancer cells are characterized by high expression of given protein receptors, such as albumin, transferrin, and growth factors that are also present in the plasma of patients themselves, but are lacking on the surface of normal cells. These distinct features between cancer and normal cells can serve as a niche for developing specific treatment strategies. Near-infrared (NIR)-light-triggered therapy platforms are an interesting novel avenue for use in clinical nanomedicine. As a photothermal agent, conducting polymer nanoparticles, such as polypyrrole (PPy), of great NIR light photothermal effects and good biocompatibility, show promising applications in cancer treatments through the hyperthermia mechanism. Autologous plasma proteins coated PPy nanoparticles for hyperthermia therapy as a novel core technology platform to treat cancers through secreted protein acid and rich in cysteine targeting are developed here. This approach can provide unique features of specific targeting toward cancer cell surface markers and immune transparency to avoid recognition and attack by defense cells and achieve prolonged circulation half-life. This technology platform unveils new clinical options for treatment of cancer patients, supporting the emergence of innovative clinical products.

Lysosomal Quality Control in Prion Diseases.

Prion diseases are transmissible, familial or sporadic. The prion protein (PrP), a normal cell surface glycoprotein, is ubiquitously expressed throughout the body. While loss of function of PrP does not elicit apparent phenotypes, generation of misfolded forms of the protein or its aberrant metabolic isoforms has been implicated in a number of neurodegenerative disorders such as scrapie, kuru, Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Sträussler-Scheinker and bovine spongiform encephalopathy. These diseases are all phenotypically characterised by spongiform vacuolation of the adult brain, hence collectively termed as late-onset spongiform neurodegeneration. Misfolded form of PrP (PrPSc) and one of its abnormal metabolic isoforms (the transmembrane CtmPrP) are known to be disease-causing agents that lead to progressive loss of structure or function of neurons culminating in neuronal death. The aberrant forms of PrP utilise and manipulate the various intracellular quality control mechanisms during pathogenesis of these diseases. Amongst these, the lysosomal quality control machinery emerges as one of the primary targets exploited by the disease-causing isoforms of PrP. The autophagosomal-lysosomal degradation pathway is adversely affected in multiple ways in prion diseases and may hence be regarded as an important modulator of neurodegeneration. Some of the ESCRT pathway proteins have also been shown to be involved in the manifestation of disease phenotype. This review discusses the significance of the lysosomal quality control pathway in affecting transmissible and familial types of prion diseases.

A comprehensive lipid binding and activity validation of a cancer-specific peptide-peptoid hybrid PPS1

We recently identified a peptide-peptoid hybrid, PPS1, which recognizes lipids that have an overall negative charge, such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylinositol (PI), but that does not bind to neutral lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). The simple dimeric version of PPS1, PPS1D1, displayed strong cytotoxicity to cancer cells over normal cells in vitro and tumor burden in vivo. In this study, we comprehensively characterized the direct binding and activity of PPS1 on PS, PG, and PA using liposome-based assays and lung cancer cell lines that express these negatively charged lipids. First, the fluorescence polarization (FP) binding studies of fluoresceinated-PPS1 (PPS1-FITC) to PS-, PG-, and PA-containing PC-liposomes showed that the binding of PPS1 to PC-liposomes increased as concentrations of these lipids increased. In terms of activity, PPS1D1 induced the release of calcein from large, unilamellar PC-liposomes containing 15–30% PS, PG, and PA. PPS1D1 had no activity when the liposomes were composed of 100% PC. This effect was higher at 30% lipids than 15%, and the EC50 for PG and PA were higher than that of PS, indicating that PPS1D1 is more specific towards PS. PPS1D1 binds to and induces significant cytotoxicity in lung cancer cell lines H1693, HCC95, and H1395, which express negatively charged lipids, but had no effect on normal HBEC30KT cells, which has mostly PC in the outer layer. In addition, a series of previously developed PPS1D1 derivatives, which retain or lose activity, were tested with these liposome-based assays, and the data were equivalent to previous observations. This study provides comprehensive binding and activity validations of a unique peptide-peptoid hybrid, PPS1, on negatively charged lipids PS, PA, and PG that are elevated on cancer cell surfaces relative to normal human cell surfaces.

SC05.04 Lung Cancer Vaccines: An Update

SC05.04 Lung Cancer Vaccines: An Update

IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression.Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice.In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through Filipin based cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of lipid rafts formation with decreased levels of ganglioside GM1. Thereby, Inadequate CD19/BCR co-receptor signaling caused by disruption of lipid rafts homeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo.Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining lipid rafts and CD19 surface expression. IFITM3-dependent lipid raft stability and CD19 surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Comparison of the diagnostic utility of ADA and CA125 in tuberculous effusion

BackgroundPleural effusion can be due to various pleural infections like TB as well as neoplasia. CA125 is a tumor marker found on the surface of ovarian and other normal cells as pleural cells. CA125 has been found to increase in serum and hence pleural fluid of patients with pleural effusion due to malignancy as well as due to TB.This study was conducted to evaluate the utility of CA125 in the diagnosis of pleural effusion resulting from TB, malignancy and pneumonia as well as to evaluate and compare the diagnostic utility of CA125 and ADA in the diagnosis of TB effusion. Patients and methods20 patients with tuberculous effusion (group I), 20 patients with malignant effusion (group II) and 20 patients with parapneumonic effusions (group III) were evaluated for the levels of CA125 and ADA in their pleural fluid. In malignant cases, diagnosis was made through microscopic inspection of pleural biopsy samples and cytology of pleural fluid. For diagnosis of tuberculosis, Ziehl Neelsen sputum smear, pleural fluid smear and/ or culture. Parapneumonic effusions were confirmed by pleural fluid cell count and culture & sensitivity. ResultsThe mean±SD level of CA125 in pleural fluid was 41.732±20.744U/ml, 309.27±79.564U/ml and 7.040±5.601U/ml in tuberculous, malignant and parapneumonic effusions respectively; which showed a statistically significant difference between the three groups (p<0.01). Pleural fluid CA125 was significantly higher in group II than group I (P1=0.000), and group III (P3=0.000). Pleural fluid CA125 was significantly higher in group I than group III (P2=0.000). Pleural fluid ADA was significantly higher in group I than group II (P1=0.000) and group III (P2=0.000). For diagnosing TB, CA125 showed a sensitivity and specificity of 74.1%, 76.9%, respectively while ADA demonstrated a sensitivity and specificity of 75% and 75% respectively. ConclusionCA-125 levels in pleural fluid may be used for differentiation between TB, pneumonic, and malignancy-induced effusions.Also CA125 may be added to the diagnostic workup of pleural fluid for accurate diagnosis of TB effusion.

Abstract 3917: Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target

Abstract This project investigates the role of Thymidine Kinase 1 (TK1) as a possible target for cancer therapeutics in breast cancer. Currently, therapeutic cancer treatments frequently damage bystander cells and have a devastating impact on the immune system. Many attempts have been made to engineer therapeutic techniques that are specific to cancer cells, unfortunately, resulting in few breakthroughs. TK1 is a phosphotransferase enzyme that is consistently present at abnormally high levels in cancer patient serum. TK1 assists in DNA repair and is characteristically expressed in the cytosol. We have discovered that TK1 is overexpressed on the surface of breast cancer cells lines MCF-7, MDA-MB-231, and Sk-Br-3. Interestingly, TK1 is not found on the surface of normal cells. We developed a unique monoclonal anti-TK1 antibody specific to human TK1 (A72). A72 was used in conjunction with flow cytometry, confocal microscopy, electron microscopy, and immunohistochemistry to test for TK1 on the cell surface. We stained each breast cancer cell line with A72-FITC conjugate and prepared appropriate controls. The samples were then analyzed using an Attune Flow Cytometer showing a 6% binding of A72 on the normal lymphocytes, while A72 binding was elevated to 20% of the breast cancer cells. Further testing using confocal microscopy was performed. The cells were treated with A72-FITC conjugate and Cell Mask Deep Red (a red membrane dye), and consecutively imaged. Results showed that A72-FITC associate with the cells in the same region of the red membrane stain, demonstrating that A72 binds to the cell membrane. This was further confirmed using electron microscopy by probing the cells with anti-TK1 conjugated to biotin that complexed with gold-streptavidin. The normal cells expectedly showed no significant binding, while gold staining was clearly seen on the surface of the each breast cancer cell line. Immunohistochemistry was completed to confirm the presence of TK1 in actual breast tumors from patients. Normal breast tissues along with breast tumor tissues were obtained from the Utah Valley Regional Medical Center. After treating the slides with anti-TK1 antibodies in conjunction with MACH 4 HRP secondary, DAP peroxidase was used to develop the slides. The tissues were examined using a light microscope. Cancer tissues appeared brown, indicating an overexpression of TK1. As expected, healthy breast tissues showed no significant staining, suggesting little to no presence of TK1. These data strongly suggest that TK1 is located on the surface of breast cancer cells, but is not found on the surface of normal cells. Further testing may open the possibilities of using TK1 as a new breast cancer biomarker. Ultimately, this also indicates the possible use of TK1 monoclonal antibodies in a therapeutic approach. Citation Format: Evita G. Weagel, Rachel A. Brog, Michelle H. Townsend, Edwin J. Velazquez, Toshiko A. Becker, Camilo A. Mejia, Michael R. Downey, Juan A. Arroyo, Richard A. Robison, Kim L. O’Neill. Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3917.