Inhibition of the mammalian target of rapamycin (mTOR) through the use of drug eluting stents releasing rapamycin or its analogs has proven effective in preventing in-stent restenosis. However, recent reports demonstrate delayed stent endothelialization with these devices, increasing late stent thrombosis and decreasing their overall efficacy. In contrast, these same anti-angiogenic properties are being exploited to decrease tumor growth in the treatment of various cancers. Our understanding of the anti-angiogenic potential of mTOR inhibition is limited, with no clear pathway between mTOR signal transduction and the cytoskeletal events that form the basis of cell motility. The mTOR pathway is intricate, beginning with the ability of mTOR to form two functionally distinct complexes, mTORC1 and mTORC2. Furthermore, a link between mTOR and a key effector of mTOR signal transduction, the cyclin-dependent kinase inhibitor p27Kip1, is still unclear. This proposal will describe a novel connection between mTOR and angiogenesis, and also elucidate the mechanism through which mTOR regulates p27Kip1 protein levels. We hypothesize that inhibition of mTORC2 increases levels of p27Kip1, which in turn inhibits activation of the small GTPase, Rho. Rho is a key mediator of the cytoskeletal reorganization required for cell motility and angiogenesis. This proposal will demonstrate this through three aims, each examining a more focused level of regulation. Aim 1 will measure the effects of mTOR inhibition on endothelial cell migration and tube formation, comparing the various mTOR inhibitors and using siRNA techniques to identify which mTOR complex is responsible its effects. Aim 2 focuses on the protein level, measuring increases in p27Kip1 following mTOR inhibition and using siRNA methods to demonstrate the dependence of these effects on p27Kip1. As p27Kip1 subcellular localization and degradation is regulated through phosphorylation of specific residues, Aim 3 will measure changes in the phosphorylation of p27Kip1 in response to inhibition of mTOR. Together, these studies will elucidate the molecular changes that occur in response to mTOR inhibition that inhibit endothelial cell migration and tube formation, providing a basis for future drug eluting stent design and development of mTOR inhibitors as cancer therapeutics.
|Effective start/end date||07/01/2008 → 06/30/2010|