Mechanisms Driving Cortical Cytoskeleton Dynamics in Cancer Cell Invasion

癌细胞侵袭中皮质细胞骨架动力学的驱动机制

• 批准号: 8294993
• 负责人: KATHRYN M EISENMANN
• 金额: $ 30.15万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294993
• 关键词: Actins; Adenocarcinoma Cell; Animals; Attention; Automobile Driving; Axon; Behavior; Biological Assay; Breast; Breast Cancer Cell; Bulla; Cancer cell line; Cell Adhesion; Cell Line; Cells; Cellular Membrane; Cellular Morphology; Cellular biology; Chimeric Proteins; Clinic; Complex; Coupled; Cytoskeleton; DNA Sequence Rearrangement; Detection; Development; Diagnosis; Disease; Embryo; Energy Transfer; Exhibits; Family; Fatty acid glycerol esters; Fibroblasts; Focal Adhesions; Goals; Growth Cones; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Human; Image; In Vitro; Knowledge; Life; Light; Link; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediating; Membrane; Mesenchymal; MicroRNAs; Microfilaments; Microscopy; Mission; Molecular; Natural regeneration; Neoplasm Metastasis; Normal Cell; Peptide Hydrolases; Physiological Processes; Play; Proteins; Regulation; Research; Role; Signal Transduction; Testing; Tetanus Helper Peptide; Time; Total Internal Reflection Fluorescent; Tumor Cell Invasion; ameboid movement; base; cancer cell; cell motility; cell type; disability; formin-2; in vivo; insight; migration; mouse model; mutant; neoplastic cell; novel; optical imaging; public health relevance; research study; rho; therapeutic target; tumor; tumor growth; tumorigenesis

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.

描述（由申请人提供）：很多注意力集中在 GTP 酶的 Rho 家族上，其在肌动蛋白重塑中发挥重要作用，并且通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 信号传导方面存在根本性差距。这些研究的长期目标是了解驱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。本申请的目的是在基本分子和细胞水平上定义 Rho 效应哺乳动物透明相关形式 (mDia2) 及其调节器透明相互作用蛋白 (DIP) 在乳腺癌细胞三维 (3D) 迁移中的作用。 ) 体外和体内基质。通过 DIP 扰动 mDia2 活性可诱导圆形细胞形态和膜起泡。膜起泡是一种促进变形虫细胞运动（ACM）的生理过程。 ACM 不同于涉及粘着斑和基质金属蛋白酶 (MMP) 的间充质型细胞运动； ACM 是癌细胞迁移的一种特殊模式，被认为在转移中发挥重要作用。中心假设是 DIP 和 mDia2 控制与 3D 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。拟议研究的基本原理是，针对间充质型细胞迁移的疗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阻止转移。拟议的研究与 NIH 的使命相关，即发展基础知识以潜在地帮助减轻人类残疾的负担。我们将追求三个具体目标： 1. 确定二维矩阵中起泡癌细胞中 mDia2 相关蛋白的空间和时间调节； 2；评价DIP/mDia2节点在体外驱动肿瘤细胞粘附、迁移和侵袭的需要； 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，将开发表达野生型 (wt) 或突变型 DIP 和/或 mDia2 荧光融合蛋白，或 DIP 或 mDia2 定向 miRNA 的 Tet 诱导型 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 mDia2 和 DIP 在 2D 和 3D 矩阵中迁移乳腺癌细胞的需求和空间/时间调节。在目标 3 中，乳腺脂肪垫小鼠模型将利用组织学检测和通过整体动物成像对肿瘤进行 3D 光学成像的多平台分析来评估肿瘤发生/转移。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。 公共卫生相关性：对细胞迁移和侵袭的透彻了解对于转移和细胞迁移或侵袭主要参与的其他疾病状态的诊断和治疗取得进展至关重要。此外，了解细胞前缘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、再生轴突中前进的生长锥还是转移性恶性肿瘤产生的癌细胞的背景下，都是细胞生物学的一个主要目标在疾病和发育的研究中具有广泛的影响。我们预计我们的实验将揭示不同细胞类型的各种行为期间细胞骨架重塑的基本和保守机制。