CD44/Variant Cytoskeleton in Breast Cancer Progression

描述(申请人提供)：现在可以确定，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变异体(CD44v)亚型[透明质酸(HA)受体]的过度表达似乎赋予了乳腺上皮细胞生长、迁移和侵袭增加的恶性特性。在这项后续研究中，我们计划检验一种假设，即CD44v亚型(CD44v3、CD44v10和CD44v6/7)与特定的信号激活剂[如RhoGEFs/racgef(Tiam1)和c-Src Kinase]之间的相互作用在HA介导的致癌信号中发挥重要作用，如RhoA激活的韩国通路、Tiam1调节的rac1-PKNGamma激酶通路和c-Src诱导的Cortactin和Gab-1/PI3激酶-AKT通路。这些激活事件诱导多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)，从而导致乳腺癌的进展。为了验证这一假说，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特定下游效应器功能(例如，NHE1相关的酸性pH酶激活、锚蛋白/皮质肌动蛋白结合和PI3激酶/AKT介导的生物学活性)方面的功能分支，这些功能是细胞外基质降解、细胞骨架功能和转移肿瘤细胞特性所必需的。此外，我们计划用免疫细胞化学的方法分析CD44v亚型和不同的信号分子(如Rhogef、Tiam1、ROK、PKNGamma和c-Src)在乳腺癌组织中的共表达。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Cortactin)的显性-负性突变体来削弱HA-CD44v异构体介导的致癌信号。最后，我们将开发针对CD44的治疗性反义和小干扰RNA(SiRNAs)，以有效地阻断CD44的表达，抑制HA介导的下游致癌信号事件，并阻止乳腺肿瘤的进展。我们认为，从这一建议中获得的信息具有特别重要的临床实用价值，可以建立CD44v亚型和相关的信号分子(如Rhogef、Tiam1、韩国、PKNGamma和c-Src)作为重要的肿瘤标志物，用于早期发现和评估致癌潜力，并允许开发新的药物靶点来抑制乳腺癌的转移和癌症进展。

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.