CD44/Variant Cytoskeleton in Breast Cancer Progression

描述（由申请人提供）：现在确定致癌信号和细胞骨架功能直接参与乳腺癌的进展。我们的实验室已经确定，CD44变体（CD44v）异构体[透明质酸（HA）受体]的过表达似乎赋予乳腺上皮细胞生长、迁移和侵袭增加的恶性特性。在本继续研究计划中，我们计划验证CD44v亚型（CD44v3、CD44v10和CD44v6/7）与特异性信号激活因子（如RhoGEFs/RacGEF （Tiam1）和c-Src激酶）之间的相互作用在ha介导的致癌信号传导中发挥重要作用，如rhoa激活的ROK通路、Tiam1调节的Rac1-PKNgamma激酶通路以及c-Src诱导的接触和gab1 /PI3激酶- akt通路。这些激活事件诱导各种肿瘤细胞特异性行为（如细胞酸化、ECM降解、细胞骨架功能、肿瘤细胞生长/存活、迁移和侵袭）导致乳腺癌进展。为了验证这一假设，我们计划使用各种生化，分子生物学技术和免疫组织化学染色来阐明HA-CD44v异构体与各种信号分子的相互作用。我们将评估这些相互作用在ECM降解、细胞骨架功能和转移性肿瘤细胞特性所需的特定下游效应功能（例如nhe1相关的酸性pH酶激活、锚蛋白/接触蛋白-肌动蛋白结合和PI3激酶/ akt介导的生物活性）方面的功能后果。此外，我们计划利用免疫细胞化学方法分析CD44v亚型和各种信号分子（如RhoGEF、Tiam1、ROK、PKNgamma和c-Src）在乳腺癌患者乳腺癌组织中的共表达情况。我们还将采用新的信号扰动策略，通过构建某些信号分子（如ROK、PKNgamma、c-Src和cortnn）的显性负突变体，来破坏HA-CD44v异构体介导的致癌信号。最后，我们将开发特异性靶向CD44的治疗性反义rna和小干扰rna (sirna)，以有效阻断CD44的表达，抑制ha介导的下游致癌信号事件并阻断乳腺肿瘤的进展。我们认为，本研究获得的信息具有特别重要的临床应用价值，可以建立CD44v亚型和相关信号分子（如RhoGEF、Tiam1、ROK、PKNgamma和c-Src）作为早期发现和评估致癌潜力的重要肿瘤标志物，并允许开发新的药物靶点来抑制乳腺癌转移和癌症进展。

DESCRIPTION (provided by applicant): It is now certain that oncogenic signaling and cytoskeleton function are directly involved in breast cancer progression. Our laboratory has determined that overexpression of CD44 variant (CD44v) isoforms [hyaluronan (HA) receptors] appears to confer the malignant properties of increased growth, migration and invasion on breast epithelial cells. In this continuation research proposal, we plan to test the hypothesis that the interaction between CD44v isoforms (CD44v3, CD44v10 and CD44v6/7) and specific signaling activators [e.g. RhoGEFs/RacGEF (Tiam1) and c-Src kinase] plays an important role in HA-mediated oncogenic signaling such as the RhoA-activated ROK pathway, Tiam1-regulated Rac1-PKNgamma kinase pathway and c-Src-induced cortactin and Gab-1/PI3 kinase-AKT pathways. These activation events induce various tumor cell-specific behaviors (e.g. cellular acidification, ECM degradation, cytoskeleton function, tumor cell growth/survival, migration and invasion) leading to breast cancer progression. To test this hypothesis, we plan to use a variety of biochemical, molecular biological techniques and immunohistochemical staining to elucidate HA-CD44v isoform interaction with the various signaling molecules. We will evaluate the functional ramifications of these interactions with respect to specific downstream effector functions (e.g. NHE1-related acidic pH enzyme activation, ankyrin/cortactin-actin binding and PI3 kinase/AKT-mediated biological activities) required for ECM degradation, cytoskeleton function and metastatic tumor cell properties. In addition, we plan to analyze the co-expression of CD44v isoforms and various signaling molecules (e.g. RhoGEF, Tiam1, ROK, PKNgamma and c-Src) in human breast carcinoma tissues obtained from breast cancer patients using immunocytochemistry. We will also employ novel signaling perturbation strategies to impair HA-CD44v isoform-mediated oncogenic signaling by constructing dominant-negative mutants of certain signaling molecules (e.g. ROK, PKNgamma, c-Src, and cortactin). Finally, we will develop therapeutic antisense and small interference RNAs (siRNAs) specifically targeting CD44 in order to effectively block CD44 expression, inhibit HA-mediated downstream oncogenic signaling events and block breast tumor progression. We believe that the information obtained from this proposal has particularly important clinical utility and could establish CD44v isoforms and associated signaling molecules (e.g. RhoGEF, Tiam1, ROK, PKNgamma and c-Src) as important tumor markers for early detection and evaluation of oncogenic potential, as well as allow the development of new drug targets to inhibit breast tumor metastasis and cancer progression.