CD44/VARIANT CYTOSKELETON IN BREAST CANCER PROGRESSION

在这一继续研究方案中，我们计划测试CD44变异体(CD44V)和细胞骨架蛋白ankyrin之间的跨膜相互作用在促进肿瘤细胞黏附生长的原癌[例如Tiam1催化的RhoA激活和Rho-Kinase(韩国)介导的细胞骨架功能]中发挥重要作用的假设。乳腺癌进展过程中的侵袭和迁移。本研究的结果将有助于更好地了解乳腺癌转移和进展的细胞和分子机制。具体地说，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变异体(CD44V)结合所需的细胞骨架蛋白ankyrin上的结合位点多样性。重点将放在转移性乳腺肿瘤细胞中CD44V亚型和Ankyrin重复结构域(ARD)之间的结构和功能关系。我们还将探索一种新的CD44V和锚蛋白连接的致癌信号通路，特别是在转移的乳腺肿瘤细胞中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Rho-Kinase(ROK)激活。此外，我们将采用一种新的信号扰动策略来削弱Tiam1-RhoA信号和/或韩国特有的细胞骨架功能，方法是首先构建各种Tiam1和/或韩国缺失和缺乏特定功能结构域的定点突变(例如显性负突变)。然后，这些显性阴性的Tiam1和/或韩国突变多肽将在转移的乳腺上皮细胞中表达，以测试它们下调CD44V异构体介导的转移肿瘤细胞行为的能力(例如，肿瘤细胞的黏附生长、迁移和侵袭)。在建议的第二部分，我们将利用免疫组织化学方法分析CD44变异体(如CD44V3和CD44V10亚型)与Ankyrin和某些致癌信号分子(如Tiam1或Rok)在人类乳腺癌中的共表达，以建立有用的结构/功能相关标志物，用于乳腺癌的检测和预后。最后，我们计划建立一种治疗反义策略，特异性地抑制某些CD44亚型(如CD44V3/V10)的mRNA编码，从而有效地阻断CD44V亚型及其下游致癌信号事件的表达，从而导致转移性乳腺癌的进展。我们相信，这些拟议的实验结果将使我们更好地理解CD44V与ankyrin的相互作用，后者调控Tiam1-RhoA信号和细胞骨架功能所需的韩国激活，以及各种转移的肿瘤细胞属性(例如，肿瘤细胞的黏附、生长、侵袭和迁移)。从这一建议获得的信息可能建立CD44V(例如，CD44V3/CD44V10的亚型)与Ankyrin和某些致癌信号分子(例如，Tiam1或Rok)的共表达，作为早期检测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相关的信号干扰，以及CD44V3/V10亚型的特定反义结构和某些致癌分子，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.