Cancer cells adopt many strategies to maintain their growth and survival in their host. Some of these strategies allow the sustained growth of a primary tumor; others promote the process of metastasis. To metastasize tumor cells need to be motile, and to be able to cross the basement membrane which encloses blood vessels and organs. It is generally thought that basement membrane is degraded by extracellular proteases, provided by both tumor and host cells, that are activated by the tumor. In recent years, growing attention has focused on the role of specialized membrane protrusions called podosomes or invadopodia in control of basement membrane proteolysis. The outer surface of the podosome is rich in proteases, including those of the metallo-, serine- and cysteine protease families, which act to degrade the extracellular matrix (ECM). We recently identified a Src substrate and adaptor protein called Tks5 which is localized to podosomes/ invadopodia in both normal and cancer cells. Reducing Tks5 expression with siRNA inhibits the formation of podosomes/invadopodia and invasion, even though proteases are still secreted, in keeping with the importance of this structure in the invasive phenotype. The mechanisms which control podosome/invadopodia formation are not yet well understood. We have found that incubation of cancer cells in 1% oxygen increased the formation of podosomes/invadopodia. Furthermore, podosome formation was prevented by treating the cells with anti-oxidants or the flavoprotein inhibitor DPI, suggesting that reactive oxygen species (ROS) were involved. The level of Tks5 mRNA was upregulated by Src transformation, by hypoxia, and in cancer cells that had undergone an epithelial mesenchymal transition. Our hypothesis is that pro-invasive signal transduction pathways upregulate the level of Tks5, and that this in turn promotes podosome formation the production of ROS. Together, Tks5 and ROS increase the formation of podosomes/invadopodia and thus invasion. To test this hypothesis, we propose the following specific aims: 1. to evaluate the transcriptional control of Tks5. 2. To elucidate the composition and role of NADPH oxidases in the formation of ROS. 3. To characterize the mechanisms by which ROS promotes invasion. The significance of this research is that it will increase our understanding of the mechanisms by which cancer cell invasion is regulated. The impact of the research lies in the opportunity to elucidate new molecular targets for metastasis control. ? ? ? ?
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