epidermal-growth-factor and Multiple-Myeloma

Topics: Aminoacridines; Amsacrine; Animals; Anthracenes; Antineoplastic Agents; Blood Physiological Phenomena; Bone Marrow; Breast Neoplasms; Colony-Forming Units Assay; Drug Evaluation; Drug Evaluation, Preclinical; Drug Stability; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Interferons; Karyotyping; Leukemia; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Ovarian Neoplasms; Platelet-Derived Growth Factor; Suspensions; Transplantation, Heterologous; Tumor Stem Cell Assay

Drug-resistance is a major problem preventing a cure in patients with multiple myeloma (MM). Previously, we demonstrated that activated-leukocyte-cell-adhesion-molecule (ALCAM) is a prognostic factor in MM and inhibits EGF/EGFR-initiated MM clonogenicity. In this study, we further showed that the ALCAM-EGF/EGFR axis regulated the MM side population (SP)-mediated drug-resistance. ALCAM-knockdown MM cells displayed an enhanced ratio of SP cells in the presence of bone marrow stromal cells (BMSCs) or with the supplement of recombinant EGF. SP MM cells were resistant to chemotherapeutics melphalan or bortezomib. Drug treatment stimulated SP-genesis. Mechanistically, EGFR, primed with EGF, activated the hedgehog pathway and promoted the SP ratio; meanwhile, ALCAM inhibited EGFR downstream pro-MM cell signaling. Further, SP MM cells exhibited an increased number of mitochondria compared to the main population. Interference of the mitochondria function strongly inhibited SP-genesis. Animal studies showed that combination therapy with both an anti-MM agent and EGFR inhibitor gefitinib achieved prolonged MM-bearing mice survival. Hence, our work identifies ALCAM as a novel negative regulator of MM drug-resistance, and EGFR inhibitors may be used to improve MM therapeutic efficacy.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Animals; Antigens, CD; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Fetal Proteins; Hedgehog Proteins; Humans; Mice; Multiple Myeloma

Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis.. Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14. We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14. In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs.

Topics: Amphiregulin; Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Exosomes; Humans; Interleukin-8; Ligands; Mesenchymal Stem Cells; Mice; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteogenesis; RAW 264.7 Cells; Tumor Microenvironment

In human multiple myeloma (MM), the tumor cells exhibit strict dependence on bone marrow (BM) stromal elements. It has been suggested that, in turn, MM cells modify multipotent stromal cells (MSCs), diverting them to support the myeloma. We investigated MM-derived MSCs by comparing their toll-like receptor (TLR) responses to those of MSCs derived from healthy controls. We now report that MM-derived MSCs manifested intact proliferation responses and IL-6 secretion and their adipose and osteogenic differentiation responses to TLR ligands were also similar to those of healthy controls, ranging from augmentation to inhibition. However, MM-derived MSCs were found to be defective in IL-8 secretion and ERK1/2 phosphorylation following TLR-2 activation. Moreover, MM-derived MSCs failed to respond to EGF by elevation of ERK1/2 phosphorylation. The persistence of these changes in extensively cultured MM-derived MSCs, suggests that these cells are stably, if not irreversibly modified.

Topics: Adult; Biomarkers; Cell Differentiation; Cell Membrane; Cell Proliferation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Interleukin-8; Kinetics; Ligands; Lipoproteins; Male; Mesenchymal Stem Cells; Middle Aged; Multiple Myeloma; Phosphorylation; Toll-Like Receptors

The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-alpha were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) - neuregulin-1, amphiregulin, HB-EGF - promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 10(5) molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy.

Topics: B-Lymphocytes; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gene Expression; Gene Expression Profiling; Hematopoietic Stem Cells; Heparan Sulfate Proteoglycans; Humans; Ligands; Middle Aged; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Plasma Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Syndecan-1

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Blotting, Western; Bone Marrow Cells; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Separation; Cells, Cultured; Dexamethasone; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Hematopoietic Stem Cells; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-6; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Monocytes; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Receptor, ErbB-4; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

To determine the difference in gene expression between human multiple myeloma (MM) and normal bone marrow.. cDNA chip was used to detect the mRNA of mononuclear cell from 10 untreated MM patients and 10 cases of normal bone marrow.. Of the 2,048 genes, we found 566 different expression genes, of which the expression of 237 genes was higher and 329 lower in MM than in the normal bone marrow.. Many genes may be involved in the pathogenesis of MM. cDNA chip technology is an effective tool in the study of tumor related genes.

Topics: Adult; Aged; Boronic Acids; Bortezomib; Epidermal Growth Factor; Female; Gene Expression; Gene Expression Profiling; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Multiple Myeloma; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Protein Tyrosine Phosphatases; Pyrazines; RNA, Messenger

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.

Topics: Antigens, CD; Apoptosis; Autocrine Communication; Cell Division; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-6; Kinetics; Membrane Glycoproteins; Models, Biological; Multiple Myeloma; RNA, Neoplasm; Tetraspanin 29; Tumor Cells, Cultured

Interleukin 6 (IL-6) has been shown to be a key growth factor for myeloma cells. To study IL-6 signal transduction in multiple myeloma (MM), we employed chimeric receptors composed of the epidermal growth factor receptor (EGFR) extracellular domain, gp130 transmembrane domain, and full-length or truncated gp130 cytoplasmic domains lacking regions previously shown to be necessary for MAPK, STAT1, and STAT3 activation. The IL-6-dependent KAS-6/1 MM cell line was transfected with various chimeric receptor constructs and assayed for EGF responsiveness. EGF stimulation surprisingly stimulated DNA synthesis in all transfectants, regardless of receptor length. When cell proliferation was assayed instead, only transfectants capable of inducing high levels of STAT3 activation proliferated in response to EGF. Additional studies revealed that EGF stimulation resulted in tyrosine phosphorylation of endogenous gp130 in cells expressing the chimeric receptor. Replacing the gp130 transmembrane region with the EGFR transmembrane domain diminished but did not disrupt this interaction. This receptor interaction was also observed in the IL-6-dependent MM cell line ANBL-6. In summary, although our results suggest that STAT activation is crucial in gp130-mediated proliferation of myeloma cells, these results must be interpreted with caution given our demonstration of the interaction between chimeric and endogenous receptors in myeloma cells. Importantly, this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-MM cells.

Topics: Amino Acid Sequence; Antigens, CD; Cell Division; Cytokine Receptor gp130; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Interleukin-6; MAP Kinase Signaling System; Membrane Glycoproteins; Multiple Myeloma; Phosphorylation; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Deletion; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transfection; Tumor Cells, Cultured

In multiple myeloma (MM), the growth of primary plasma cells depends not only on interleukin-6 (IL-6), but also on additional unidentified signals delivered by the bone marrow environment. Using Atlas complementary DNA (cDNA) arrays comprising 268 genes coding for intercellular signaling molecules, this study identified genes that are overexpressed in myeloma cells compared to autologous B-lymphoblastoid cell lines. These genes encode the oncogenic Tyro3 tyrosine kinase receptor, the heparin-binding epidermal growth factor-like growth factor (HB-EGF) that is an epithelial autocrine tumor growth factor, the thrombin receptor (TR) that is linked to HB-EGF and syndecan-1 processing and to cell invasion, chemokine receptors CCR1 and CCR2, the Wnt pathway actor Frizzled-related protein (FRZB), and the Notch receptor ligand Jagged 2. These data, obtained with the Atlas cDNA array, were confirmed by reverse transcriptase-polymerase chain reaction or protein analysis or both. Furthermore, Tyro3, HB-EGF, TR, and FRZB gene expression was documented in purified primary malignant plasma cells from patients with plasma cell leukemia or MM. HB-EGF and FRZB were poorly expressed in purified polyclonal plasma cells. Finally, HB-EGF was proved to be an essential autocrine growth factor for the XG-1 myeloma cells. This study shows the potency and the biologic relevance of cDNA arrays used to analyze simultaneously a large panel of intercellular signaling genes and, by identifying several genes overexpressed in malignant plasma cells, opens new fields of investigation in MM biology. (Blood. 2001;98:771-780)

Topics: B-Lymphocytes; Cell Division; Epidermal Growth Factor; Flow Cytometry; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Plasma Cells; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Cells, Cultured

The overexpression of the human epidermal growth factor receptor (EGFR) has been demonstrated in many malignancies like squamous cell carcinoma of the head and neck, cervix, breast etc. which are most prevalent in India. This is often associated with poor prognosis and high mortality in these patients. Monoclonal antibodies generated against EGFR which inhibit binding of ligands like EGF to their receptor have anti-tumor activity and hence therapeutic application. One such monoclonal antibody designated as CIBCNSH3 generated in our laboratory has been found to recognize an epitope in the extracellular domain of EGFR by immunoprecipitation. By immunoperoxidase test this antibody exhibited strong reactivity to EGFR in head and neck cancers and breast cancers studied. It also inhibited the binding of Epidermal Growth Factor (EGF) to its receptor on MDA MB468 breast cancer cells rich in EGFR as revealed by competitive binding assay using 125I EGF, indicating its anti-tumor activity. The in vivo therapeutic efficacy has been demonstrated by injecting i.p. into tumor bearing mice 200 micrograms of the antibody for 4 consecutive days and then 100 micrograms twice a week resulting in complete regression of tumors of initial tumor size of 0.5-1.0 cm diameter. These results were compared with a control antibody against EGFR and also a nonspecific antibody which were administered to different groups of animals. In vivo studies performed using cell lines in culture like MDA MB468, MDA MB157 and HN5 with overexpression of EGFR revealed 98% cell death when incubated with different concentrations of the antibody. This monoclonal antibody seems to have a promising future application as therapeutic agent for tumors which overexpress EGFR.

Topics: Animals; Antibodies, Monoclonal; Binding, Competitive; Breast Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Cell Division; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Epitopes; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunization, Passive; Mammary Neoplasms, Experimental; Mice; Multiple Myeloma; Neoplasm Proteins; Radioimmunodetection; Transplantation, Heterologous; Tumor Cells, Cultured

The influence of human hepatocyte growth factor (HGF) on the transforming growth factor beta (TGF-beta) bioassay CCL-64 was examined. HGF induced proliferation of the CCL-64 cells and potently counteracted TGF-beta-induced growth inhibition. HGF was not inactivated by transient acidification to pH 2, a commonly used procedure to activate latent TGF-beta. HGF was a stronger mitogen for the mink lung cells than epidermal growth factor (EGF), a known stimulator of CCL-64 cell growth. Costimulation of the cells by these two cytokines resulted in an additive effect on proliferation. In complex biological fluids containing large amounts of HGF, the TGF-beta concentration can be underestimated when determined by the CCL-64 assay. When a fixed amount of TGF-beta is added, the CCL-64 cells can be used as a reliable bioassay for HGF with a sensitivity of about 1 ng/ml.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Division; Cell Line; Epidermal Growth Factor; Growth Inhibitors; Hepatocyte Growth Factor; Humans; Lung; Mice; Mink; Multiple Myeloma; Transforming Growth Factor beta

A human myeloma cell line, PCM6, was newly established from peripheral blood of a patient with advanced IgG myeloma by addition of recombinant interleukin-6 (IL-6) in culture. PCM6 cells had a morphology typical of mature plasma cells. Cytogenetic and surface marker studies confirmed that PCM6 cells were identical to fresh myeloma cells. Coculture of PCM6 cells with normal bone marrow mononuclear cells resulted in increased colony size of bone marrow-derived fibroblastoid colony-forming cells (CFU-F). Conditioned medium of PCM6 (PCM6-CM) cells increased the CFU-F colony size in a dose-dependent manner. The activity was labile to trypsin treatment but was heat stable (60 degrees C, 30 minutes). Molecular weight of the activity was approximately 165 kd by Sephacryl S-300 gel filtration. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), and IL-1 beta were not detectable in the conditioned medium. These findings suggest that in some myeloma cases, bone marrow stroma may be affected by CFU-F growth-promoting activity.

Topics: Antigens, Surface; Base Sequence; Bone Marrow; Cells, Cultured; Chromatography, Gel; Culture Media, Conditioned; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblasts; Genome, Viral; Hematopoietic Stem Cells; Herpesvirus 4, Human; Humans; Immunoglobulin G; Interleukin-1; Interleukin-6; Middle Aged; Molecular Sequence Data; Multiple Myeloma; Platelet-Derived Growth Factor; Recombinant Proteins; Trypsin; Tumor Cells, Cultured

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Calcium; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Hybridomas; Hydrogen-Ion Concentration; Inositol Phosphates; Kinetics; Mice; Multiple Myeloma; Phorbol Esters; Phosphorylation; Temperature; Tumor Cells, Cultured

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth.

Topics: Animals; Antibodies, Monoclonal; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Mice; Mice, Inbred BALB C; Multiple Myeloma; Phosphorylation; Receptors, Cell Surface; Thymidine