We have continued to focus on the role of insulin-like growth factors (IGF's) and tumor specific translocations in the pathogenesis of pediatric sarcomas, specifically rhabdomyosarcomas (RMS), Ewing's sarcomas/PNET (Ewing's family of tumors EFT), and osteogenic sarcomas (OS). We have demonstrated that loss of imprinting (LOI) of IGF2 occurs uniformly in RMS and occasionally in EFT and are now studying methylation patterns in the IGF2/H19 locus to determine whether this contributes to LOI. We have demonstrated that wild-type p53 inhibits transcription from the P3 and P4 promoters of IGF2 by distinct mechanisms and mutant forms of p53 (commonly found in RMS) appear to have lost this activity potentially contributing to overexpression of IGF2 in these tumors. We have transfected a dominant negative IGF1 receptor (IGFIR) construct into RMS cells and shown that this receptor appears to inhbit the growth and tumorigenicity of these cells, further confirming the importance of this signaling pathway in these tumors. We have recently used IGFIR negative mouse fibroblasts obtained by knock-out to demonstrate that the EWS-FLI-1 fusion found in most EFT's requires the presence of the IGFIR to transform cells and these same experiments appear to show that mouse fibroblasts that contain normal IGFIR and the fusion protein hyperphosphorylate IRS-1. Further studies are ongoing to determine the importance of this phenomenon. We have characterized 4 spontaneously metastatic murine OS cell lines with regard to their growth and metastasis characteristics and in response to a non-calcitropic Vit D analog and found that this treatment leads to growth inhibition with alterations in components of the TGF-beta signaling pathway. We have also demonstrated a novel germline p53 mutation at codon 220 in a family with Li-Fraumeni and osteosarcomas in 3 siblings. We have continued ours studies aimed at utilizing the t(11;22) translocations of EFT and the t(2;13) translocation in alveolar RMS to generate tumor specific CTL. In addition to peptide vaccination strategies, we have generated vaccinia virus vectors containing these translocation cDNA's and plasmids contianing the translocation peptides preceeded by an Adenovirus leader sequence in order to maximize CTL generation in murine models. Finally, we have continued our development of metastatic sarcoma animal models and have recently completed characterization of human RMS subclones with varying metastatic potential with the ultimate aim of using these reagents to identify critical genetic differences in metastatic versus non-metastatic cells.
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