MOLECULAR MECHANISMS OF MALIGNANT TRANSFORMATION

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We have devised and utilized an expression cloning strategy to isolate novel oncogenes. By the analysis of signaling pathways involving the oncogenes isolated by this strategy, we are attempting to clarify the molecular mechanisms of malignant transformation. In this fiscal year, two oncogenes we previously isolated were characterized in detail. (1) A dual receptor system has been proposed where interaction of basic fibroblast growth factor (bFGF) with cell surface proteoglycans is required for high affinity binding of basic FGF to FGF receptor-1 (FGFR1). During a course of expression cloning to identify transforming genes from an osteosarcoma cDNA library, we identified a unique isoform of FGFR2 containing a dual receptor system in a single molecule. This receptor is modified by heparan sulfate glycosaminoglycan at a site encoded by an alternative exon. Moreover, presence of a sequence encoded by another alternative exon abrogated this modification. This high affinity receptor is required for increased and sustained mitogen- activated protein kinase activity, ternary complex factor-independent gene expression, and especially for DNA synthesis. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification. (2) A search of transforming cDNAs from a rat brain cDNA expression library led to the isolation of an isoform of the ost oncogene, which we previously identified in osteosarcomas. In addition to the motifs found in the original Ost isoform, this isoform from brain contained an extended N-terminal domain, an SH3 domain and an additional unique domain which was exclusively expressed in brain. In contrast, an isoform containing the SH3 domain, but not the brain-specific domain, was ubiquitously expressed in various tissues. Genomic analysis suggested that these isoforms were generated by tissue- specific alternative RNA splicing events. Whereas deletion of the N- terminal domain activated the transforming activity of Ost, presence of the SH3 or brain-specific domain did not affect this activity. The transforming activity of Ost was inhibited by the coexpression of dominant negative forms of Rho family proteins. Transcription regulated by the serum responsive element was potently induced by expression of the truncated form of Ost. Expression of truncated Ost also modestly induced an invasive phenotype in NIH/3T3 fibroblasts, suggesting that it may play a role in cytoskeletal organization. Titled changed from Isolation of Novel Oncogenes by an Efficient Expression Cloning System.