Title: Experimental models of transformation as platforms for oncogene discovery
Description: The successful development of rational anti-neoplastic therapeutics relies both upon an improved understanding of the genetic pathways that conspire to program cellular transformation and the identification of specific transforming molecules withín these pathways. My dissertation focuses on the construction of increasingly relevant in vitro model systems of human cell transformation and the characterization of their utility for oncogene discovery. Murine embryo fibroblasts are readily transformed by the introduction of specific combinations of oncogenes; however, the expression of those same oncogenes in human cells fails to convert such cells to tumorigenicity. Using normal human and murine embryonic fibroblasts, I show that the transformation of human cells requires several additional alterations beyond those required to transform comparable murine cells. These genetic differences involve the pathways regulated by telomerase, as well as the RB and PTEN tumor suppressor proteins. These experiments permitted the development of transformed human fibroblasts with genetic alterations similar to those found associated wíth human cancers and defined specific differences in the susceptibility of human and murine fibroblasts to experimental transformation.

These in vitro model systems of human cell transformation were further leveraged in both candidate- and screening-based discovery efforts. I demonstrate that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to replace H-RAS V12 ( RAS ) in the transformation of human cells. Using a library of activated kinases, I identified several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses revealed that one of these kinases, IKBKE (IKK[varepsilon]), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKK[varepsilon] expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKK[varepsilon] overexpression activates the nuclear factor kappa B (NF-[kappa]B) transcription factor, consistent with the observed activation of this pathway in many breast cancers. These observations implicate the NF-[kappa]B pathway as an essential downstream mediator of PI3K. Furthermore, they provide a framework for integrated genomic approaches in oncogene discovery and demonstrate the utility and relevance for cell-based models of human malignancy.