In the field of cell-matrix adhesion, an emerging concept is that cells respond differently to matrices that are configured in three-dimensions, compared to the """"""""classic"""""""" 2D configuration that has been studied for several years. As our understanding of cell-matrix interactions increases, it is becoming apparent that cells recognize not only the matrix composition and the 3D configuration, but also the compliance vs. stiffness of the surrounding extracellular matrix environment. We were among the first to demonstrate that breast epithelial cells contract compliant matrices through Rho and its effector, ROCK, and that this contraction is necessary for mammary ductal morphogenesis in vitro (Wozniak et al, 2003). Importantly, the stiffness of the matrix feeds back to regulate Rho activity, but the mechanism by which this mechano-signaling occurs is unknown. Moreover, although much is known about the role of Rho in a host of responses to the ECM, surprisingly little is known about which Rho GEFs and GAPs might be linked to mechanical signals. We hypothesize that Rho is the central conduit by which cells sense the stiffness of their environment, and respond to that stiffness. More specifically, we hypothesize that regulation of Rho occurs by activation due to specific GEFs under conditions of matrix stiffness, and the inactivation by specific GAPs under conditions of matrix compliance. Therefore, the goal of this proposal is to investigate how these molecules function to regulate Rho in response to matrix stiffness:
Aim 1 : Determine mechanisms of Rho regulation by p190RhoGAP-B in response to compliant matrices. We find that p190RhoGAP-B, but not-A, mediates Rho down-regulation and ductal morphogenesis in a compliant matrix. We will test the hypothesis that p190RhoGAP-B is a suppressor of invasion, and that it regulates Rho via binding to p120 Catenin and Rac 1.
Aim 2 : Identify and investigate the role of specific Rho GEFs in activating Rho in response to stiff matrices. The contribution of GEF-H1 and p115RhoGEF to regulation of Rho will be determined. In addition to this candidate approach, an siRNA screen of all RhoGEFs will be performed to identify additional GEFs that regulate Rho in response to matrix stiffness. RhoGEFs identified by these means will be assessed for their role in linking Rho to downstream effects: cellular contractility, matrix remodeling, and tumor invasion.
Aim 3 : Determine mechanisms by which filamin regulates contractility through Rho and invasion into 3D matrices in vitro and in vivo. Filamin increases cellular contractility and matrix remodeling, and tunes cellular response to a stiff matrix. We will determine whether filamin enhances invasion in vitro and in vivo, and investigate whether binding of filamin to its partners Trio, ROCK, or PAK mediates its regulation of Rho and contractility.
Understanding the role the physical properties of the extracellular matrix plays in cancer progression is of great health relevance as breast density accounts for a 4-6 fold increase in carcinoma risk. These experiments are designed to understand the underlying molecular mechanisms by which the dense extracellular matrix regulates breast cell behavior and could suggest future targets for therapy.
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