Rac1 regulates protrusion and invasion of breast cancers in 3D

描述(申请人提供)：在浸润性乳腺腺癌中，侵袭细胞在基质中爬行的机制涉及两个不同的过程，必须很好地协调才能影响有效的侵袭。基质降解突起(侵袭足)必须能够有效地降解基质，收缩，并允许运动性伪足/片脂突起伸入基质中降解的孔中，并且这一过程必须循环以实现持续的侵入性迁移。Invadodidia组装过程的分子基础已经被很好地记录下来，涉及p21Rho家族的小GTP酶，包括CDC42，RhoA和RhoC；然而，invadodia在完成基质降解时如何分解，以及表型如何从基质降解转换为大量机车突起尚不清楚。在这里，我们认为p21Rho家族的GTPase成员rac1 GTPase在侵袭性乳腺癌中参与了侵袭性足部的分解，同时也驱动了伪足/片状脂体在二维和三维的突起。此外，我们假设rac1通过与两个不同的下游效应器靶点相互作用，激活了两个不同的单独的下游通路，负责分别影响每个过程。我们进一步假设，在2维培养条件下，我们关于rac1在不定足动物体内相对于前沿片层的作用的观察可以扩展到3维入侵，其中基质降解和大部分运动突起间隔会聚到同一空间。我们将使用我们新的针对rac1 GTPase的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的激活动力学，并将利用最先进的rac1 GTPase光老化技术来进一步解决rac1激活在invadodia中的作用。这项研究将提供第一个证据，说明单个GTPase rac1的激活循环如何有效地将侵袭性/运动性机械的表型从基质降解转换为2D和3D侵袭环境中的大量运动性突起。

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.