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IGF-IR plays an essential role in the establishment and maintenance of the transformed phenotype of ES cells and interference with the IGF-IR pathways causes reversal of the malignant potential of this neoplasm. In this report, we stably transfected a dominant negative IGF-IR expression plasmid in an ES cell line to determine the effectiveness of this strategy against the in vitro and in vivo growth of ES cells. DXR sensitivity of TC-71 cells expressing dominant negative mutants of IGF-IR was also examined. The mutated IGF-IR that we used carries a mutation in the ATP-binding domain of the intracellular β subunit, while the extracellular, ligand-binding α subunit remains unchanged. Cells carrying the dominant mutant IGF-IR had a marked decrease in proliferation, a significant increase in anoikis-induced apoptosis and a severely reduced ability to form colonies in soft agar. In vivo, when cells carrying dominant negative IGF-IR were injected into nude mice, the tumor formation and metastatic abilities of ES cells were reduced and survival increased. Furthermore, transfected clones showed significantly higher sensitivity to DXR, a major drug in the treatment of ES. These results indicate that the IGF/IGF-IR stimulation of ES cells may be inhibited by expression of mutated IGF-IR on their surfaces and that this strategy may be considered a possible alternative to impair this important target of ES cells, whose therapeutic potential was further confirmed. © 2002 Wiley-Liss, Inc.

CD99 is a Mr 32,000 transmembrane molecule that shows a high level of expression on cells of the hemopoietic system as well as on Ewing tumor cells. Within the hematopoietic system, CD99 has been implicated in cell adhesion and cell death, participating in this way in the differentiation of T-cell precursors. In this study, we demonstrate that engagement of CD99 significantly inhibits the in vitro and in vivo growth ability of Ewing tumor cells by delivering an apoptotic stimulus and reducing the malignant potential of these cells. Moreover, we show that anti-CD99 monoclonal antibodies may be advantageously used in association with conventional anticancer agents. These results provide a novel entry site for therapeutic intervention, which may have application in the care of patients with Ewing tumor, and warrant additional studies to clarify the molecular mechanisms activated by CD99 engagement.

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.