The long term goals of this project are to characterize the signaling pathways involved in transformationby activated Src, and to extend these studies to an analysis of the role of Src and its effectors in human cancer. The first specific aim concerns the mechanisms by which Src induces cellular invasion of theextracellular matrix. We have observed that the small GTPase Rho is required for the formation of invasive adhesions, termed podosomes or invadopodia, and propose to identify the Rho isoforms and Rho effectors required for the induction of podosomes by activated Src. We have also observed that atypical PKC activity is required for matrix invasion by Src-transformed cells, and will identify aPKC substrates required for Src-induced matrix invasion. In addition we will examine the effects of Src-dependent tyrosine phosphorylation of actin binding proteins such as Eps8 and vinculin on actin dynamics within podosomes. Src induces entry into S phase in part by inducing the expression of the transcription factor Myc. We have recently observed that Src abrogates the Myc requirement for GO/G1but not the Myc requirement for G1/S. The second specific aim concerns the mechanism by which Src abrogates the Myc requirement for GO/G1. We hypothesize that chromatin changes at Myc target loci underlie both this phenomenon and the phenomenon of myc oncogene addiction. We will test this hypothesis by characterization of chromatin modifications at the promoters of Myc target loci. Src is overexpressed and/or activated in many types of human cancer. We have shown that inhibition of Src induces reversion of some aspects of the malignant phenotype of human breast cancer cell lines; in certain lines acinar morphogenesis in three-dimensional basement membrane cultures is restored, while in others invadopodium formation and matrix degradation are blocked. We will examine the roles of different Src effectors in mediating these effects. Inhibitors of Src are in clinical trials as anti-cancer agents, and inhibitors of Myc are also under development. A deeper understanding of how these proteins act to induce cancer and how their functions are inter-related will provide insights into how these agents should be deployed.
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