SIGNALING PATHWAYS OF TRANSFORMING ONCOGENES

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Many transforming oncoproteins are localized outside the nucleus. Tumorigenesis requires that these proteins transmit their advertent signals to the nucleus where the stable genetic changes associated with transformation are executed. We are interested in identifying the nuclear factors which mediate these changes and propose that, at least in some instances, they consist of the products encoded by nuclear proto-oncogenes and immediate early response genes (collectively referred to as the "nuclear effector" genes). This family consists of members which may themselves be oncogenes (c-myc, c-fos, c-jun) or which are induced by growth stimuli but have no reported oncogenic potential (egr-1/zif268, NGF- 1B/nur77). We will test our proposal by the conditional expression of the nuclear effector gene "antisense" transcripts in NIH 3T3 cell transformed by four different types of oncogenes (raf, src, ras, and cis). Glucocorticoid inducible antisense constructs will be transfected into these cells and the resultant colonies examined for morphological reversion. Individual clones will be examined for biological properties associated with transformation such as growth in soft agar and tumorigenic potential in nude mice. Preliminary results with v-K-ras-transformed NIH 3T3 cells indicate that the depletion of either c-myc or NGF-1B/nur77 induces morphological reversion although only c-myc antisense clones lose the ability to grow in soft agar or low serum. In addition to affirming the feasibility of the proposed work, these results suggest that there exists a hierarchy of nuclear effector genes, each controlling different aspects of the transformed phenotype. We expect to use the cell lines generated by these studies to examine the intactness of other pathways that are disrupted by transformation. This may allow us to determine whether these pathways are casually or causally linked to the ras transformation pathway. Additional proposed studies are aimed at further characterization of the means by which one extranuclear oncoprotein (src) is able to induce the expression of the nuclear effector gene junB. Thus, the work proposed in this application is relevant for the following reasons: 1) it will identify a role for nuclear effector genes in mediating transformation by extranuclear oncoproteins; 2) it promises to demonstrate a hierarchy of nuclear effector genes; 3) by demonstrating the requirement for common nuclear effector genes, it promises to reveal heretofore unappreciated relationships among extranuclear oncogenes; 4) it will allow us to determine the relevance to transformation of other signalling pathways; and 5) it will identify potential targets for therapeutic intervention.