DESCRIPTION (provided by applicant): In invasive breast adenocarcinomas, the mechanism by which the invading cells crawl through the matrix involves two distinct processes that must be well coordinated to affect an efficient invasion. The matrix degrading protrusions (invadopodia) must be able to efficiently degrade the matrix, retract, and allow for the protrusion of the locomotory pseudopodia/lamellipodia into the degraded hole in the matrix, and this process must cycle to achieve a continuous invasive migration. The molecular basis underlying the assembly process of invadopodia is well documented, involving the p21 Rho family of small GTPases including Cdc42, RhoA and RhoC; however how the invadopodia disassembles upon completion of the matrix degradation and how the phenotype switches from that of matrix degradation to bulk locomotive protrusion is not yet clear. Here, we propose that the p21 Rho family GTPase member Rac1 GTPase is critically involved in the disassembly of invadopodia in invasive breast adenocarcinomas, and at the same time drives the protrusion of the pseudopodia/lamellipodia in 2- and 3-dimensions. Furthermore, we hypothesize that Rac1 activates two distinct and separate downstream pathways through interacting with two different downstream effector targets, responsible for affecting each of the processes separately. We further hypothesize that the observations we make regarding the role of Rac1 in invadopodia versus the leading edge lamellipodia in 2 dimensional culture conditions can be extended to 3 dimensional invasion where the matrix degrading and the bulk locomotive protrusion compartments converge into the same space. We will approach this problem using our new genetically encoded biosensor for Rac1 GTPase, capable of reporting the activation dynamics of Rac1 in real time at subcellular resolutions, and we will utilize the state-of-the-art photouncaging technologies for Rac1 GTPase to further address the role of Rac1 activation at invadopodia. This study will provide the first evidence into how the activation cycling of a singl GTPase Rac1, can efficiently switch the phenotype of invasive/motility machinery from matrix degradation to bulk locomotory protrusion in 2D and in 3D invasion settings.

PUBLIC HEALTH RELEVANCE: In invasive breast adenocarcinomas, the process of invasion through the extracellular matrix during metastasis is tightly regulated and coordinated to balance the activities of the matrix degrading invadopodia versus the locomotory pseudopodia protrusions. These processes are driven and regulated by the Rho-family of p21 small GTPases, including Cdc42, RhoA and RhoC. Here, we will show for the first time, the direct involvement of Rac1 GTPase during invadopodia function. We hypothesize that Rac1, though activation of p21-activated kinase 1 downstream of Rac1, initiates the cascade of signaling pathway that ultimately culminates in disassembly of invadopodia. We will show this and perturb this signaling mechanism through the use of our novel, genetically encoded single-chain biosensor for Rac1 based on FRET, as well as using the state-of-the-art photo-uncaging approaches to modulate the local activation states of Rac1 at invadopodia. We further hypothesize that Rac1, through two distinct and separate downstream pathways, regulates the disassembly of invadopodia and protrusion of the leading edge protrusions at the same time. These two compartments are separated in space in the 2-dimensional culture setting, but we will address this in 3-dimensional setting to show for the first time that Rac1 is the single molecular "switch" that turns off the invasive protrusions and turns on the locomotory protrusions, thus contributing to cycling of these two types of processes critical for an efficient migration through the extracellular matrix.