epidermal-growth-factor and Sarcoma--Ewing

Ewing sarcoma (ES) is a type of childhood cancer probably arising from stem mesenchymal or neural crest cells. The epidermal growth factor receptor (EGFR) acts as a driver oncogene in many types of solid tumors. However, its involvement in ES remains poorly understood.. Human SK-ES-1 and RD-ES ES cells were treated with EGF, the EGFR inhibitor tyrphostin (AG1478), or phosphoinositide 3-kinase (PI3K) or extracellular-regulated kinase (ERK)/mitogen-activated kinase (MAPK) inhibitors. Cell proliferation survival, cycle, and senescence were analyzed. The protein content of possible targets of EGFR manipulation was measured by Western blot.. Cell proliferation and survival were increased by EGF and inhibited by AG1478. The EGFR inhibitor also altered the cell cycle, inducing arrest in G1 and increasing the sub-G1 population, reduced polyploidy and increased the population of senescent cells. In addition, AG1478 reduced the levels of phosphorylated AKT (p-AKT), ERK, p-ERK, cyclin D1, and brain-derived neurotrophic factor (BDNF), while enhancing p53 levels. Cell proliferation was also impaired by inhibitors of PI3K or ERK, alone or combined with AG1478.. Our findings reveal novel aspects of EGFR regulation of ES cells and provide early evidence for antitumor activities of EGFR inhibitors in ES.

Topics: Bone Neoplasms; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinazolines; Sarcoma, Ewing; Signal Transduction; Tumor Suppressor Protein p53; Tyrphostins

Ewing's sarcoma family of tumours (ESFT) is a malignant small round-cell tumour of the bone and soft tissues. It is characterised by a strong tendency to invade and form metastases. The microenvironment of the bone marrow is a large repository for many growth factors, including the basic fibroblast growth factor (bFGF). However, the role of bFGF in the invasive and metastatic phenotype of ESFT has not been investigated.. The motility and invasion of ESFT cells were assessed by a wound-healing assay, chemotaxis assay, and invasion assay. The expression and activation of FGF receptors (FGFRs) in ESFT cell lines and clinical samples were detected by RT-PCR, western blotting, and immunohistochemistry. The morphology of ESFT cells was investigated by phase-contrast microscopy and fluorescence staining for actin. Activation of Rac1 was analysed by a pull-down assay.. bFGF strongly induced the motility and invasion of ESFT cells. Furthermore, FGFR1 was found to be expressed and activated in clinical samples of ESFT. Basic FGF-induced cell motility was mediated through the FGFR1-phosphatidylinositol 3-kinase (PI3K)-Rac1 pathway. Conditioned medium from bone marrow stromal cells induced the motility of ESFT cells by activating bFGF/FGFR1 signalling.. The bFGF-FGFR1-PI3K-Rac1 pathway in the bone microenvironment may have a significant role in the invasion and metastasis of ESFT.

Topics: Bone Marrow; Cell Line, Tumor; Cell Movement; Cytoskeleton; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; rac1 GTP-Binding Protein; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 3; Recombinant Proteins; Sarcoma, Ewing; Wound Healing

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Culture Media, Serum-Free; Demecolcine; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Neoplasm Proteins; Oncogene Proteins, Fusion; Platelet-Derived Growth Factor; Prostatic Neoplasms; Proto-Oncogene Protein c-fli-1; Receptors, Fibroblast Growth Factor; RNA-Binding Protein EWS; Sarcoma, Ewing; Thymidine; Transcription Factors; Translocation, Genetic; Tumor Cells, Cultured

The ability of high-density lipoprotein (HDL) to support the growth of an established tumor cell line exposed to defined medium supplemented with transferrin has been examined. Low-density A-431 carcinoma cells maintained on extracellular matrix- or fibronectin-coated dishes proliferated actively when exposed to a synthetic medium supplemented with HDL, 500 micrograms protein per ml. Epidermal growth factor added at concentrations above 0.5 ng/ml inhibited cell growth, while at concentrations above 5 ng/ml it was cytotoxic. Among the various substrata tested for their ability to support the active proliferation of low-density A-431 cells when exposed to transferrin and HDL, plastic was the least efficient. On fibronectin-coated dishes, cells ceased to proliferate after 8 population doublings, while on extracellular matrix-coated dishes cells could be passaged for 50 population doublings. In the case of colon carcinoma, rhabdomyosarcoma, and Ewing's sarcoma cells exposed to medium supplemented with transferrin, the addition to the cultures of HDL alone resulted in a growth rate and final cell density which were similar to those observed when cells were exposed to serum-supplemented medium. In the case of the mammary carcinoma cell lines MCF-7 and ZR-75-1, HDL also supported cell growth, although to a lesser extent than did serum. The present study therefore indicates that HDL is capable of supporting, either totally or partially, the in vitro proliferation of tumor cells.

Topics: Blood; Breast Neoplasms; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; Epidermal Growth Factor; Fibronectins; Humans; Insulin; Lipoproteins, HDL; Neoplasms; Rhabdomyosarcoma; Sarcoma, Ewing; Transferrin

Growth of human tumor cells (hepatocarcinoma, Ewing's sarcoma) on an extracellular matrix (ECM) produced by bovine corneal endothelial cells is associated with the adoption of a morphological appearance and growth properties that are not expressed when the cells are maintained on plastic. Within minutes after seeding cell aggregates onto an ECM, the aggregates attached firmly. Active cell migration leading to the formation of flattened and nonoverlapping cell clusters was subsequently observed. In contrast, no firm attachment, migratory activity or disorganization of cell aggregates was observed when the same cells were maintained on plastic. Cells seeded on ECM, instead of growing as floating or loosely attached aggregates, formed a cell monolayer composed of firmly attached, highly flattened and closely apposed epithelioid-like cells. Cell overlapping and subsequent detachment were observed only late at confluence. Cells maintained on ECM had a higher growth rate as well as a lower serum requirement than those maintained on plastic. These results demonstrate that the phenotypic expression as well as the proliferation of tumor cells can be modulated by their adhesive interaction with the extracellular matrix. Both tumor cells and normal cells of epithelial origin are more likely to resemble their in vivo counterparts when maintained on extracellular matrix than on plastic, and when so maintained can therefore provide a better model for oncogenic studies.

Topics: Cell Adhesion; Cell Aggregation; Cell Division; Cell Line; Cell Movement; Cornea; Endothelium; Epidermal Growth Factor; Humans; Liver Neoplasms; Plastics; Sarcoma, Ewing