Mechanisms Driving Cortical Cytoskeleton Dynamics in Cancer Cell Invasion

描述（由申请人提供）：许多注意力集中在GTP酶的Rho家族上，其在肌动蛋白重塑中起重要作用，并且通常在人类癌症中表现出增强的表达和/或活化。然而，在理解关键下游效应蛋白如何在癌细胞中传播Rho信号方面存在根本性的差距。这些研究的长期目标是了解在侵袭性癌细胞的前沿驱动皮质肌动蛋白细胞骨架重排的分子和细胞机制。本申请的目的是在基本的分子和细胞水平上确定Rho效应物哺乳动物透明相关蛋白（mDia 2）及其调节剂透明相互作用蛋白（DIP）在体外和体内三维（3D）基质中乳腺癌细胞迁移中的作用。通过DIP干扰mDia 2活性诱导圆形细胞形态和膜起泡。膜起泡是一种促进阿米巴样细胞运动（ACM）的生理过程。ACM与涉及局灶性粘附和基质金属蛋白酶（MMP）的间充质型细胞运动不同; ACM是癌细胞迁移的专门模式，并且被认为在转移中起重要作用。中心假设是DIP和mDia 2控制与3D癌细胞迁移期间的变形虫转变相关的皮质肌动蛋白组装的变化。拟议研究的基本原理是，靶向间充质型细胞迁移的治疗方法在治疗乳腺癌和其他癌症的临床中基本上失败了，这表明细胞也可以利用蛋白酶非依赖性机制进行体内迁移;因此，必须靶向多种细胞迁移模式以有效阻断转移。拟议的研究与NIH的使命有关，即开发基础知识，以帮助减轻人类残疾的负担。我们将追求三个具体目标：1。确定2D基质中起泡癌细胞中mDia 2相关蛋白的空间和时间调节; 2;评估DIP/mDia 2结在体外驱动肿瘤细胞粘附、迁移和侵袭的需要;以及3。确定mDia 2在体内乳腺肿瘤生长、侵袭和转移中的功能需求。为了实现这一点，将开发表达野生型（wt）或突变DIP和/或mDia 2荧光融合蛋白或DIP或mDia 2定向miRNA的Tet-inducible MDA-MB-231细胞（一种高度侵袭性腺癌细胞系）。在目标1和2中，定量活细胞共聚焦、FRET和TIRF成像将测试mDia 2和DIP在2-和3D基质中迁移的乳腺癌细胞中的需求和空间/时间调节。在目标3中，乳腺脂肪垫小鼠模型将通过多平台分析评估肿瘤发生/转移，该分析利用组织学检测和通过整体动物成像进行肿瘤的3D光学成像。拟议的研究具有重要意义，因为了解变形虫运动的分子基础将为控制癌细胞迁移的机制提供新的见解，并可能突出转移性疾病急需的替代治疗靶点。

DESCRIPTION (provided by applicant): Much attention has focused upon the Rho-family of GTPases, which play fundamental roles in actin remodeling and often exhibit enhanced expression and/or activation in human cancers. However, there is a fundamental gap in understanding how key downstream effecter proteins propagate Rho signaling in cancer cells. The long-term goal of these studies is understand the molecular and cellular mechanisms driving rearrangements of the cortical actin cytoskeleton at the leading edge of invasive cancer cells. The objective of this application is to define at the basic molecular and cellular level the role of the Rho effecter mammalian Diaphanous-related formin (mDia2) and its regulator Diaphanous-interacting protein (DIP) in breast cancer cell migration in three-dimensional (3D) matrices both in vitro and in vivo. Perturbation of mDia2 activity via DIP induces a rounded cellular morphology and membrane blebbing. Membrane blebbing is a physiological process that promotes amoeboid cell motility (ACM). ACM is distinct from the mesenchymal-type of cell motility involving focal adhesions and matrix metalloproteinases (MMPs); ACM is a specialized mode of cancer cell migration and is proposed to play an essential role in metastasis. The central hypothesis is that DIP and mDia2 control changes in cortical actin assembly associated with an amoeboid transition during 3D cancer cell migration. The rationale for the proposed research is that therapeutics targeting mesenchymal-type cell migration have largely failed in the clinic for treating breast and other cancers, suggesting that cells can also utilize protease-independent mechanisms for in vivo migration; therefore, multiple modes of cell migration must be targeted to effectively block metastasis. The proposed research is relevant to NIH's mission pertaining to developing fundamental knowledge to potentially help reduce the burdens of human disability. We will pursue three specific aims: 1. To determine the spatial and temporal regulation of mDia2-associated proteins in blebbing cancer cells in 2D matrices; 2; To evaluate the requirement for the DIP/mDia2 node for driving tumor cell adhesion, migration and invasion in vitro; and 3. To determine the functional requirement for mDia2 in breast tumor growth, invasion and metastasis in vivo. To achieve this, Tet-inducible MDA-MB-231 cells, a highly invasive adenocarcinoma cell line, will be developed expressing wild-type (wt) or mutant DIP and/or mDia2 fluorescent fusion proteins, or DIP- or mDia2-directed miRNA. In Aims 1 and 2, quantitative live cell confocal, FRET and TIRF imaging will test the requirement for and spatial/temporal regulation of mDia2 and DIP in migrating breast cancer cells in 2- and 3D matrices. In Aim 3, a mammary fat pad mouse model will assess tumorigenesis/metastasis by multiple platform analyses utilizing histological detection and 3D optical imaging of tumors by whole animal imaging. The proposed research is significant as understanding the molecular basis of amoeboid motility will lend novel insight into mechanisms controlling cancer cell migration and may highlight critically needed alternative therapeutic targets for metastatic disease.