Differentiation resistance is a phenotype commonly associated with enhanced malignancy. For the past 10 years we have been studying mechanisms of ras-mediated inhibition of cellular differentiation. We have defined a novel pathway by which a ras oncogene controls cell differentiation and tumorigenicity. The transcription factor AP-2 is overexpressed in ras transformed human teratocarcinoma, PA-1 cells. We have demonstrated that in ras transformable PA-1 target cells overexpression of AP-2, a downstream target of ras, is sufficient for transformation. These PA-1 target cells can also be transformed by overexpression of the AP-2 activation domain fused to a GAL4 DNA binding domain (AP-2AD-GAL4) alone. Whether transformation is mediated by ras, AP-2 or AP-2AD, the endogenous activity of AP-2 is dramatically reduced. Therefore the activation domain is sufficient for self-inhibition of endogenous AP-2 activity. The mechanism by which oncogenic transcription factors and ultimately ras oncogenes transform cells is not understood. Does this mechanism involve transcription factor-specific gene targets normally regulated by the factor which are now aberrantly expressed in transformed cells and then this specific dysregulation of gene expression gives rise to transformation? Or alternatively is transformation due to a more general effect exerted by the overexpressed transcription factor interacting with protein components of the transcriptional machinery? Because the AP-2AD without the AP-2 DNA binding domain can transform these cells the latter hypothesis is more likely. To prove this latter hypothesis, during the previous funding period, we identified AP-2 interacting proteins and proved that one of these Cofactors, PC4, modulates ras mediated transformation via relief of squelched AP-2 activity. In ras-transformed cells PC4 overexpression restores AP-2 activity which results in slow growth, a restoration of retinoic acid induced differentiation and suppression of tumorigenicity in nude mice. This unique observation indicates that the penultimate target of ras transformation is transcriptional in nature as transformation can be reversed by a purely transcriptional mechanism. This novel finding impacts current dogma regarding the mechanism by which transcriptional oncogenes and ras inhibit differentiation and induce neoplasia. The goals for this application are to study this novel mechanism of how an imbalance of cofactors results in transformation and develop strategies to manipulate the cofactor PC4 to inhibit transformation. We will analyze PC4/AP-2 protein/protein interactions. We will determine the motifs in AP-2 and PC4 that are essential for this interaction and test their effect on transforming activity. We will ascertain whether this interaction of PC4 with AP-2 is necessary or sufficient for suppression of ras transformation. Likewise we will derive mutations in PC4 that block and/or enhance its coactivator activity, its transformation reversion activity or both. Our molecular characterization of PC4-mediated transformation suppression will provide a novel approach for future therapies to abrogate differentiation resistance and transformation mediated by ras-associated signal transduction.
