CD44/VARIANT CYTOSKELETON IN BREAST CANCER PROGRESSION

在这个继续的研究计划中，我们计划验证CD44变体（CD44V）和细胞骨架蛋白锚蛋白之间的跨膜相互作用在促进癌原性（例如tiam1催化的RhoA激活和rho激酶（ROK）介导的细胞骨架功能）导致肿瘤细胞粘附生长中起重要作用的假设。乳腺癌进展过程中的侵袭和迁移。这项研究的结果将为更好地理解人类乳腺癌转移和进展的细胞和分子机制提供帮助。具体来说，我们计划使用各种生化、细胞生物学和分子生物学技术来阐明CD44变体（CD44V）结合所需的细胞骨架蛋白锚蛋白的结合位点多样性。重点将放在CD44V亚型和转移性乳腺肿瘤细胞中锚蛋白重复结构域（ARD）之间的结构和功能关系上。我们还将探索新的CD44V和锚蛋白连接的致癌信号通路，特别关注鸟嘌呤核苷酸交换因子，Tiam1 （T淋巴瘤侵袭和转移）催化的RhoA信号和RhoA激酶（ROK）在转移性乳腺肿瘤细胞中的激活。此外，我们将采用一种新的信号扰动策略，通过首先构建各种缺乏特定功能域的Tiam1和/或ROK缺失和位点定向突变体（例如显性阴性突变体），来破坏Tiam1- rhoa信号传导和/或ROK特异性细胞骨架功能。这些显性阴性Tiam1和/或ROK突变多肽随后将在转移性乳腺上皮细胞中表达，以测试它们下调CD44V亚型介导的转移性肿瘤细胞行为（如肿瘤细胞粘附生长、迁移和侵袭）的能力。在提案的第二部分，我们将使用免疫组织化学分析CD44变体（如CD44V3和CD44V10-containing亚型）与锚蛋白和某些致癌信号分子（如Tiam1或ROK）在人乳腺癌中的共表达，以建立有用的结构/功能相关标记物，用于乳腺癌检测和预后。最后，我们计划建立一种治疗性反义策略，特异性抑制某些CD44亚型（如CD44V3/V10）的mRNA编码，以有效阻断CD44V亚型的表达及其下游致癌信号事件，从而导致乳腺癌转移性肿瘤的进展。我们相信这些实验的结果将更好地理解CD44V异构体与锚蛋白的相互作用，锚蛋白调节细胞骨架功能和各种转移性肿瘤细胞特性（如肿瘤细胞粘附、生长、侵袭和迁移）所需的Tiam1-RhoA信号和ROK激活。本提案获得的信息可能将CD44V（如CD44V3/ cd44v10 -containing isoforms）与锚蛋白和某些致癌信号分子（如Tiam1或ROK）共表达作为早期发现和评估致癌潜力的重要肿瘤标志物。最重要的是，我们提出的新策略使用了一种新的Tiam1/ rok相关信号扰动，以及CD44V3/V10亚型的特异性反义构建物和某些致癌分子，可能会发现抑制乳腺癌转移和癌症进展的新药物靶点。

In this continuation research proposal, we plan to test the hypothesis that transmembrane interaction between CD44 variants (CD44V) and the cytoskeletal protein, ankyrin, plays an important role in promoting onocongenic [e.g. Tiam1-catalyzed RhoA activation and Rho-Kinase (ROK)-mediated cytoskeleton function] leading to tumor cell adhesion growth. invasion and migration during breast cancer progression. The results of this research will definitely provide a better understanding of the cellular and molecular mechanisms involved in human breast cancer metastasis and progression. Specifically, we plan to use a variety of biochemical, cell biological and molecular biological techniques to elucidate the binding site diversity on the cytoskeletal protein, ankyrin, required for CD44 variant (CD44V) association. An emphasis will be placed on the structural and functional relationships between the CD44V isoforms and the ankyrin repeat domain (ARD) in metastatic breast tumor cells. We will also explore a new CD44V and ankyrin-linked oncogenic signaling pathway with a special focus on the guanine nucleotide exchange factor, Tiam1 (T lymphoma invasion and metastasis)-catalyzed RhoA signaling and Rho-Kinase (ROK) activation in metastatic breast tumor cells. In addition, we will employ a novel signaling perturbation strategy to impair Tiam1-RhoA signaling and/or ROK-specific cytoskeleton function by first constructing various Tiam1 and/or ROK deletion and site-directed mutants lacking specific functional domains (e.g. dominant negative mutants). These dominant-negative Tiam1 and/or ROK mutant polypeptides will then be expressed in metastatic breast epithelial cells to test their ability to downregulate CD44V isoform-mediated metastatic tumor cell behaviors (e.g. tumor cell adhesion growth, migration and invasion). In the second part of the proposal, we will use immunohistochemistry to analyze the coexpression of CD44 variants (e.g. CD44V3 and CD44V10-containing isoforms) with ankyrin and certain oncogenic signaling molecules (e.g. Tiam1 or ROK) in human breast carcinomas in order to establish useful structure/function-related markers for breast cancer detection and prognosis. Finally, we plan to establish a therapeutic antisense strategy to specifically inhibit the mRNA encoding for certain CD44 isoforms (e.g. CD44V3/V10) in order to effectively block the expression of CD44V isoforms and their downstream oncogenic signaling events which lead to metastatic breast tumor progression. We believe the results of these proposed experiments will provide a much better understanding of the CD44V isoform interaction with ankyrin which regulates Tiam1-RhoA signaling and ROK activation required for cytoskeleton function and various metastatic tumor cell properties (e.g. tumor cell adhesion, growth, invasion and migration). The information obtained from this proposal may establish the coexpression of CD44V (e.g. CD44V3/CD44V 10-containing isoforms) with ankyrin and certain oncogenic signaling molecules (e.g. Tiam1 or ROK) as an important tumor marker for early detection and evaluation of oncogenic potential. Most importantly, our proposed new strategy using a novel Tiam1/ROK-related signaling perturbation, and specific antisense constructs of CD44V3/V10 isoforms together with certain oncogenic molecules, may identify new drug targets for the inhibition of breast tumor metastasis and cancer progression.