Delineating and rationally perturbing signaling mechanisms involved in metastasis.

摘要

Metastasis is the dissemination of tumor cells from the primary locus of formation to other organs. During migration from the primary tumor, tumor cells need to traverse the vasculature, a process termed extravasation. Extravasation is a critical step in the metastatic cascade and nevertheless a poorly understood phenomenon. Endothelial cells form a barrier, which prevents cells and plasma constituents from moving into interstitial tissues. The disruption of the endothelial barrier leads to increased barrier permeability resulting in enhanced cancer cell extravasation.;Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor uniquely activated by proteases. PAR1 increases endothelial permeability when activated by the protease thrombin. Strikingly, PAR1 signaling can also mediate decreases in endothelial permeability when activated by activated protein C (APC), an anti-coagulant protease. In the first two chapters of this dissertation I examined the mechanism responsible for protease-selective signaling by PAR1. I specifically examined the effect of APC and thrombin on the activation of RhoA and Rac1 that differentially regulate endothelial permeability. In chapter 2 of this dissertation I also investigated whether compartmentalization of PAR1 in caveolae was critical for APC selective signaling and I demonstrated that caveolae are required for APC-selective signaling to Rac1 activation and endothelial barrier protection. Furthermore, in chapter 3 of this dissertation I asked whether APC protection from thrombin-induced increased permeability involved desensitization of PAR1. And I reported that APC induces PAR1 phosphorylation and desensitizes endothelial cells to thrombin.;The metastatic process also requires degradation of extracellular matrices by proteases present in the tumor microenvironment, especially serine proteases. Inhibition of these proteases has remarkable therapeutic effects against tumor progression.;Maspin is an atypical member of the family of serine proteases inhibitors. Maspin inhibits the serine protease urokinase activated plasminogen and suppresses tumor growth and metastasis. Interestingly, maspin is silenced by epigenetic mechanisms in cancer cells. In chapter 4 of my dissertation I used artificial transcription factors (ATFs) as a novel strategy to re-activate maspin in breast cancer cells. I showed that re-expression of maspin by ATFs leads to reduction of tumor growth and metastasis in an in vivo xenograft animal model.