The taxanes are among the most effective agents for the treatment of advanced cancers, and their direct cytotoxic effects on tumor cells have been well documented and are presumed to be the primary mechanism for their clinical activity. In our recently published Preliminary Studies, we found that these microtubule-disrupting agents blocked endothelial cell migration in vitro and angiogenesis in vivo. Taxotere in particular, produced some of these actions in vitro at extremely low concentrations (10 pM) that had no effect on endothelial cell proliferation or on gross microtubule structure, suggesting that taxotere has a mechanism of action that has not been previously described and that differs from its well-described effects on mitosis, apoptosis, and cell proliferation. The overall objectives of this proposal are to determine the molecular mechanism(s) by which taxotere inhibits endothelial cell migration, and obtain information which will provide for its optimal clinical use as an anti-angiogenic agent. This application proposes the hypothesis that taxotere inhibits angiogenesis by modulating a critical early step in the signaling pathways which mediate directed cell migration. To test this hypothesis, we will document signal transduction events in endothelial cells stimulated to undergo migration, and the effect of taxotere on these events, including: a) upregulation and surface clustering of integrins; b) phosphorylation of specific tyrosine residues of focal adhesion kinase (FAK), paxillin, and p130cas; c) spatial and temporal association of these molecules to form focal adhesions; d) the extent to which these molecules localize at the centrosome (also known as the microtubule-organizing center, MOC); e) the role of the MOC in directed cell migration (using time-lapse measurements of MOC repositioning in live cells); and f) changes in the extent and intracellular localization of (i.e. in the migratory front) of tyrosinated tubulin. The rationale for these experiments are previous studies which demonstrated that key components of these signaling pathways, in particular FAK and paxillin, were associated intracellularly with microtubules, 7-tubulin, and the MOC. Therefore we will test, both in cell-free systems and in intact cells, the hypothesis that taxotere specifically disrupts the binding of components of the cell signaling/cell skeleton pathways to microtubules and/or tubulin, ultimately leading to the observed inhibition of cell migration. An additional hypothesis, that taxotere affects the microtubule-dependent activation of the Rho family of GTPases, will also be examined. In addition to understanding its mechanism of anti-angiogenic action, there are therapeutic implications to our demonstration of a potent effect of taxotere on endothelial cell migration. There is a need for further exploration of the possibility that taxotere's anti-angiogenic actions contribute to its clinical anti-tumor activity, and for the determination of the conditions under which taxotere optimally inhibits angiogenesis. In these studies, we will test the hypothesis that a """"""""metronomic"""""""" dosing schedule of taxotere will increase its efficacy in vivo. We will also use in vivo xenografts of breast and ovarian tumors which are either sensitive or resistant to taxotere's direct cytotoxic actions, so as to distinguish between taxotere's effect on endothelial cells and angiogenesis from its direct effects on the tumor cells.
