Mechanisms Driving Cortical Cytoskeleton Dynamics in Cancer Cell Invasion

描述（由申请人提供）：GTPases的rho家族备受关注，它在肌动蛋白重塑中起着重要作用，并且经常在人类癌症中表现出增强的表达和/或激活。然而，在了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前沿驱动皮层肌动蛋白细胞骨架重排的分子和细胞机制。本应用程序的目的是在基本的分子和细胞水平上定义Rho效应物哺乳动物透明相关双胍（mDia2）及其调节物透明相互作用蛋白（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扰动mDia2活性可诱导圆形细胞形态和膜起泡。膜起泡是促进变形虫细胞运动（ACM）的生理过程。ACM不同于涉及局灶黏附和基质金属蛋白酶（MMPs）的间充质型细胞运动；ACM是癌细胞迁移的一种特殊模式，被认为在转移中起重要作用。中心假设是DIP和mDia2控制与三维癌细胞迁移过程中变形虫转变相关的皮质肌动蛋白组装的变化。提出这项研究的基本原理是，针对间充质型细胞迁移的治疗方法在临床治疗乳腺癌和其他癌症方面基本上失败了，这表明细胞也可以利用蛋白酶独立的体内迁移机制；因此，必须针对多种细胞迁移模式来有效阻断转移。拟议的研究与NIH的使命有关，即发展基础知识，以潜在地帮助减轻人类残疾的负担。我们将努力实现三个具体目标：目的：探讨二维基质中mdia2相关蛋白在泡状癌细胞中的时空调控；2;探讨DIP/mDia2淋巴结在体外驱动肿瘤细胞粘附、迁移和侵袭的必要性；和3。探讨mDia2在体内乳腺肿瘤生长、侵袭和转移中的功能需求。为了实现这一目标，将开发一种高侵袭性腺癌细胞系——tet诱导的MDA-MB-231细胞，表达野生型（wt）或突变型DIP和/或mDia2荧光融合蛋白，或DIP或mDia2定向miRNA。在目标1和目标2中，定量活细胞共聚焦、FRET和TIRF成像将测试在二维和三维基质中迁移乳腺癌细胞对mDia2和DIP的需求和时空调节。在Aim 3中，一个乳腺脂肪垫小鼠模型将通过多平台分析来评估肿瘤的发生/转移，利用全动物成像的组织学检测和肿瘤的3D光学成像。这项研究具有重要意义，因为了解阿米巴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.