CD44/VARIANT CYTOSKELETON IN BREAST CANCER PROGRESSION

在这项继续研究提案中，我们计划验证以下假设：CD 44变体（CD 44 V）与细胞骨架蛋白锚蛋白之间的跨膜相互作用在促进肿瘤相关性[例如Tiam 1催化的RhoA激活和Rho-Kinase（ROK）介导的细胞骨架功能]中发挥重要作用，导致肿瘤细胞粘附生长。乳腺癌进展过程中的侵袭和迁移。 这项研究的结果肯定会提供一个更好地了解参与人类乳腺癌转移和进展的细胞和分子机制。 具体而言，我们计划使用各种生物化学，细胞生物学和分子生物学技术来阐明的结合位点的多样性的细胞骨架蛋白，锚蛋白，所需的CD 44变体（CD 44 V）协会。 重点将放在转移性乳腺肿瘤细胞中CD 44 V亚型和锚蛋白重复结构域（ARD）之间的结构和功能关系上。 我们还将探索一种新的CD 44 V和锚定相关的致癌信号通路，特别关注转移性乳腺肿瘤细胞中的鸟嘌呤核苷酸交换因子、Tiam 1（T淋巴瘤侵袭和转移）催化的RhoA信号传导和Rho激酶（ROK）激活。 此外，我们将采用一种新的信号转导干扰策略，通过首先构建各种Tiam 1和/或ROK缺失和缺乏特定功能结构域的定点突变体（例如显性失活突变体）来损害Tiam 1-RhoA信号转导和/或ROK特异性细胞骨架功能。 然后，这些显性阴性Tiam 1和/或ROK突变多肽将在转移性乳腺上皮细胞中表达，以测试它们下调CD 44 V同种型介导的转移性肿瘤细胞行为（例如肿瘤细胞粘附生长、迁移和侵袭）的能力。在第二部分的建议，我们将使用免疫组织化学分析的CD 44变体（如CD 44 V3和CD 44 V10的异构体）与锚蛋白和某些致癌信号分子（如Tiam 1或ROK）在人类乳腺癌的共表达，以建立有用的结构/功能相关的标志物乳腺癌的检测和预后。 最后，我们计划建立一种治疗性反义策略，以特异性抑制编码某些CD 44亚型（例如CD 44 V3/V10）的mRNA，从而有效阻断CD 44 V亚型的表达及其导致转移性乳腺肿瘤进展的下游致癌信号传导事件。我们相信这些拟议实验的结果将使我们更好地了解CD 44 V亚型与锚蛋白的相互作用，锚蛋白调节Tiam 1-RhoA信号传导和细胞骨架功能和各种转移性肿瘤细胞特性（例如）所需的ROK激活（例如肿瘤细胞粘附、生长、侵袭和迁移）。 从这一提议中获得的信息可能建立CD 44 V（例如，含CD 44 V3/CD 44 V10的同种型）与锚蛋白和某些致癌信号分子（例如Tiam 1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新的Tiam 1/ROK相关的信号扰动，以及CD 44 V3/V10亚型与某些致癌分子的特异性反义构建体，可能会发现抑制乳腺肿瘤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.