Rac1 regulates protrusion and invasion of breast cancers in 3D

描述（由申请人提供）：在浸润性乳腺腺癌中，入侵细胞通过基质爬行的机制涉及两个不同的过程，这两个过程必须很好地协调才能影响有效的入侵。基质降解突起（侵入性足）必须能够有效地降解基质，收缩，并允许运动假足/板足突出到基质中的降解孔中，并且这个过程必须循环以实现持续的侵入性迁移。侵殖虫组装过程的分子基础已被充分记录，涉及小gtpase的p21 Rho家族，包括Cdc42， RhoA和RhoC；然而，在基质降解完成后，侵过体是如何分解的，以及表型是如何从基质降解转变为体积机车突出的，目前尚不清楚。在这里，我们提出p21 Rho家族GTPase成员Rac1 GTPase在浸润性乳腺腺癌中参与侵足的解体，同时在2维和3维上驱动假足/板足的突出。此外，我们假设Rac1通过与两个不同的下游效应靶点相互作用激活两个不同的下游通路，分别负责影响每个过程。我们进一步假设，在二维培养条件下，我们所做的关于Rac1在侵入性板足和前缘板足中的作用的观察可以扩展到三维侵入，其中基质降解和大量运动突出室会聚到同一空间。我们将使用我们的新的Rac1 GTPase基因编码生物传感器来解决这个问题，该传感器能够在亚细胞分辨率下实时报告Rac1的激活动态，我们将利用最先进的Rac1 GTPase光检测技术来进一步解决Rac1在invadopodia中的激活作用。这项研究将提供第一个证据，证明单个GTPase Rac1的激活循环如何在2D和3D侵袭环境中有效地将侵袭性/运动性机械的表型从基质降解转变为大块运动性突起。

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.