Search

Coenzyme A (CoA) is an essential co-factor at the intersection of diverse metabolic pathways. Cellular CoA biosynthesis is regulated at the first committed step— phosphorylation of pantothenic acid—catalyzed by pantothenate kinases (PANK1,2,3 in humans, PANK3 being the most highly expressed). Despite the critical importance of CoA in metabolism, the differential roles of PANK isoforms remain poorly understood. Our investigations of PANK proteins as precision oncology collateral lethality targets (PANK1 is co-deleted as part of the PTEN locus in some highly aggressive cancers) were severely hindered by a dearth of commercial antibodies that can reliably detect endogenous PANK3. While we successfully validated commercial antibodies for PANK1 and PANK2 using CRISPR knockout cell lines, we found no commercial antibody that could detect endogenous PANK3. We therefore set out to generate a mouse monoclonal antibody against human PANK3 protein. We demonstrate that clone (Clone MDA-299-62A) can reliably detect endogenous PANK3 protein in cancer cell lines, with band-specificity confirmed by CRISPR PANK3 knockout cell lines. Sub-cellular fractionation indicates that PANK3 is overwhelmingly cytosolic and expressed broadly across cancer cell lines. PANK3 monoclonal antibody MDA-299-62A should prove a valuable tool for researchers investigating this understudied family of metabolic enzymes in health and disease.

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte-associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a β-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain-containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy.

Anterior gradient 2 (AGR2) promotes cancer growth, metastasis, and resistance to therapy via unknown mechanisms. We investigated the effects of extracellular AGR2 signaling through the orphan glycosylphosphatidylinositol-linked receptor C4.4A in pancreatic ductal adenocarcinoma (PDAC). Proliferation, migration, invasion, and apoptosis were measured using colorimetric, Boyden chamber, and FACS analyses. We developed blocking mAbs against AGR2 and C4.4A and tested their effects, along with siRNAs, on cancer cell functions and on orthotopic tumors in nude mice. Extracellular AGR2 stimulated proliferation, migration, invasion, and chemoresistance of PDAC cell lines. AGR2 interacted with C4.4A in cell lysates and mixtures of recombinant proteins. Knockdown of C4.4A reduced migration and resistance to gemcitabine. PDAC tissues, but not adjacent healthy pancreatic tissues, expressed high levels of AGR2 and C4.4A. AGR2 signaling through C4.4A required laminins 1 or 5 and integrin β1. Administration of antibodies against AGR2 and C4.4A reduced growth and metastasis and caused regression of aggressive xenograft tumors, leading to increased survival of mice. These data support a model in which AGR2 binds and signals via C4.4A in an autocrine loop and promotes the growth of pancreas tumors in mice. Blocking mAbs against AGR2 and C4.4A may have therapeutic potential against PDAC. Mol Cancer Ther; 14(4); 941–51. ©2015 AACR.

Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-α production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-α and IL-6 production and NF-κB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.

The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 x 10(9) M(-1) and 1.11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.

High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas-IIB-MEL-J, IIB-MEL-LES, and lIB-MEL-IAN-express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA, Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The over- all data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA–induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

IIB-MEL-J is a highly heterogeneous newly established human melanoma cell line. The addition of 3 mM L-tyrosine to the culture medium produced (1) a great decrease in the cell growth rate, (2) a loss of the anchor-age-independent growth capacity, and (3) a change in the morphology of the cells to a fibroblastoid aspect. Coincident with these changes, an increase in subpopulations I and II and a decrease in subpopulations III and IV took place. In view of this evidence we consider that the cells have differentiated. The melanin production was not increased by the L-tyrosine treatment, suggesting that differentiation and melanin expression are not strictly correlated.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive–metastatic phenotype development.

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter Fc epsilon RI gamma. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with Fc epsilon RI gamma, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.

The MCF-7 breast carcinoma cell line can be separated by Percoll density gradient centrifugation into several subpopulations, A to F, one of which (E) has been previously suggested to be highly enriched in stem cells. The anchorage-independent growth of the different fractions and its sensitivity to estradiol (E2) and tamoxifen (TAM) was assayed. The anchorage-independent growth capacity of the different fractions was E greater than A greater than B greater than D greater than C,F. The E fraction had the highest clonogenic index (6.62 +/- 1.18) and was unaffected by E2 or TAM. The karyotypic analysis of the E fraction revealed features similar to those of the unfractionated cell line. It is suggested that the high growth rate of fraction E is due to an enrichment in stem cells and not to the existence of a different clone.

The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminishes the efflux of the drug in previously loaded cells. It is suggested that the combination of inhibitors of the de novo pathway for pyrimidine biosynthesis, such as MTX, with inhibitors of the salvage pathway, such as dipyridamole, may increase the cytotoxic activity of MTX alone.

Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.

Background Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. Results We used Rae-1–overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti–Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Conclusions Our cell line–based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.