Mechanisms Driving Cortical Cytoskeleton Dynamics in Cancer Cell Invasion

描述(申请人提供)：许多注意力集中在Rho家族的GTP酶上，它们在肌动蛋白重塑中发挥基础作用，并且经常在人类癌症中表现出增强的表达和/或激活。然而，在理解关键的下游效应蛋白如何在癌细胞中传播Rho信号方面存在着根本的差距。这些研究的长期目标是了解推动侵袭性癌细胞前沿皮质肌动蛋白细胞骨架重排的分子和细胞机制。本应用的目的是在基础分子和细胞水平上确定Rho效应哺乳动物透明相关形成蛋白(MDia2)及其调节因子透明相互作用蛋白(DIP)在三维(3D)基质中乳腺癌细胞迁移中的作用。通过DIP对mDia2活性的干扰会导致细胞形态变圆和细胞膜起泡。膜起泡是促进阿米巴细胞运动(ACM)的生理过程。ACM不同于涉及灶性粘连和基质金属蛋白酶(MMPs)的间叶型细胞运动；ACM是癌细胞迁移的一种特殊方式，被认为在转移中发挥重要作用。中心假设是DIP和mDia2控制3D癌细胞迁移过程中与变形虫转变相关的皮质肌动蛋白组装的变化。这项研究的基本原理是，针对间叶型细胞迁移的治疗方法在乳腺癌和其他癌症的临床治疗中基本上失败了，这表明细胞也可以利用不依赖于蛋白酶的机制进行体内迁移；因此，必须针对多种细胞迁移模式来有效地阻止转移。这项拟议的研究与NIH的使命有关，该使命涉及发展基础知识，以潜在地帮助减轻人类残疾的负担。我们将追求三个特定的目标：1.在2D基质中确定mDia2相关蛋白在起泡癌细胞中的时空调节；2.评估DIP/mDia2节点在体外驱动肿瘤细胞黏附、迁移和侵袭的需求；3.确定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，Tet诱导的MDA-MB-231细胞，一种高侵袭性的腺癌细胞系，将被培养成表达野生型(Wt)或突变型DIP和/或mDia2荧光融合蛋白，或DIP或mDia2导向的miRNA的细胞。在目标1和目标2中，定量活细胞共聚焦、FRET和TIRF成像将测试二维和三维基质中乳腺癌细胞迁移中对mDia2和DIP的需求及其空间/时间调节。在目标3中，乳房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，该分析利用组织学检测和全动物成像对肿瘤的3D光学成像。这项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迫切需要的替代治疗靶点。 公共卫生相关性：彻底了解细胞迁移和侵袭对于诊断和治疗转移和其他与细胞迁移或侵袭密切相关的疾病状态至关重要。此外，了解细胞前沿皮质细胞骨架的动态调节，无论是在迁移的胚胎成纤维细胞、再生轴突中的生长锥体或由转移的恶性肿瘤产生的癌细胞的背景下，都是细胞生物学的主要目标，在疾病和发育的研究中具有广泛的意义。我们预计，我们的实验将阐明在不同细胞类型的不同行为过程中细胞骨架重构的基本和保守的机制。

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. PUBLIC HEALTH RELEVANCE: A thorough understanding of cell migration and invasion is essential for progress in diagnosis and therapy of metastasis and other disease states in which cell migration or invasion is centrally involved. Moreover, understanding the dynamic regulation of the cortical cytoskeleton at the cell's leading edge, whether in the context of a migrating embryonic fibroblast, an advancing growth cone in a regenerating axon or a cancer cell spawned by metastatic malignancy, is a major goal in cell biology with widespread implications in the study of disease and development. We anticipate that our experiments will shed light on basic and conserved mechanisms of cytoskeletal remodeling during various behaviors in diverse cell types.