Transmembrane Proteins Involved in Human Tumor Expansion

 描述（由申请人提供）：肿瘤细胞通过其跨膜蛋白（TmPs）与宿主细胞外因子（如可溶性配体或基质蛋白）之间的相互作用与其周围环境进行通信：长期以来与癌症进展和转移有关的通信。为了鉴定促进肿瘤细胞侵袭行为的TmPs，我们选择了几对在自发转移能力方面不同的同源人肿瘤细胞变体，并通过在消减方法中使用这些变体，鉴定了许多与转移密切相关的TmPs。重要的是，这些TmPs之一，CDCP 1的功能，已被链接到其胞外域切割肿瘤细胞表面，导致一个新的假设，特定的TmPs的蛋白水解修饰决定逃逸的原发性肿瘤细胞的生存。为了研究这一假设并测试其诊断和翻译潜力，我们在目标1中提出：阐明一种可靶向的机制，以控制肿瘤细胞的生存和转移定植增强纤溶酶介导的跨膜蛋白，CDCP 1裂解。我们已经证明，70 kDa的膜保留的CDCP 1片段变得磷酸化，并诱导生存信号，使逃逸的原发性肿瘤细胞，以避免凋亡，并成功地完成殖民。我们还开发了一种小鼠肺滞留试验，该试验可同时定量体内裂解的CDCP 1、从头产生的纤溶酶、表达CDCP 1的转移细胞的存活率以及肺滞留肿瘤细胞中蛋白水解诱导的信号级联。因此，我们将：（1）鉴定调节体内纤溶酶原活化和协调纤溶酶介导的肿瘤细胞CDCP 1裂解的分子;（2）确定与CDCP 1裂解特异性相关的纤溶酶产生的组织位置和起始事件;（3）阐明CDCP 1的细胞表面裂解如何启动增强肿瘤细胞存活的体内信号转导;以及（4）检查在人前列腺癌的组织病理学中检测CDCP 1及其切割片段的翻译潜力。我们在目标2中提出：建立一种通用机制，由此可靶向宿主蛋白酶对不同细胞表面TmPs的有限切割决定了组织保留的肿瘤细胞的体内存活。通过使用缺乏特异性宿主蛋白酶表达的基因确定的小鼠，我们将：（1）将组织保留的肿瘤细胞的体内存活与其转移能力直接联系起来;（2）鉴定增加高度恶性肿瘤细胞存活率的特异性宿主蛋白酶;（3）鉴定新的TmPs，其细胞表面切割增强肿瘤细胞存活;（4）研究MMP-9介导的CD 44裂解在肿瘤细胞血管存活和转移定植中的体内功能作用。拟议的研究将最终证明不同TmPs的不可逆修饰决定其体内功能，从而影响整体转移结果，并代表最终旨在开发诊断或/或风险分层工具以帮助癌症患者治疗的重要翻译方面。

 DESCRIPTION (provided by applicant): Tumor cells communicate with their surroundings via interactions between their transmembrane proteins (TmPs) and host extracellular factors such as soluble ligands or matrix proteins: communications that have long been linked to cancer progression and metastasis. To identify TmPs that foster aggressive behavior of tumor cells we selected several pairs of congenic human tumor cell variants differing in their capacity of spontaneous metastasis and by using these variants in subtractive approaches, identified a number of TmPs that have been strongly linked to metastasis. Importantly, the function of one of these TmPs, CDCP1, has been linked to its ectodomain cleavage on the tumor cell surface, leading to a novel hypothesis that specific proteolytic modifications of certain TmPs determine survival of escaping primary tumor cells. To investigate this hypothesis and test its diagnostic and translational potential, we propose in Aim 1 to: Elucidate a targetable mechanism to control tumor cell survival and metastatic colonization enhanced by plasmin-mediated cleavage of the transmembrane protein, CDCP1. We have demonstrated that the 70-kDa membrane-retained CDCP1 fragment becomes phosphorylated and induces survival signaling allowing escaping primary tumor cells to avoid apoptosis and successfully complete colonization. We also have developed a mouse lung retention assay that uniquely allows for simultaneous quantification of in vivo cleaved CDCP1, de novo generated plasmin, survival of CDCP1-expressing metastatic cells, and proteolysis-induced signaling cascades in lung-retained tumor cells. Therefore we will: (1) identify the molecules that regulate in vivo plasminogen activation and coordinated plasmin-mediated cleavage of tumor cell CDCP1; (2) determine the tissue locale and initiating events for plasmin generation linked specifically to CDCP1 cleavage; (3) elucidate how cell surface cleavage of CDCP1 initiates in vivo signal transduction enhancing tumor cell survival; and (4) examine the translational potential of detecting CDCP1 and its cleaved fragments in the histopathology of human prostate cancer. We propose in Aim 2 to: Establish a general mechanism whereby limited cleavage of distinct cell surface TmPs by targetable host proteases determines the in vivo survival of tissue-retained tumor cells. By employing genetically-defined mice lacking the expression of specific host proteases, we will: (1) directly link the in vivo survival of tissue-retained tumor cells to their metastatic capability; (2) identify specific host proteases that increase survival rates of highly malignant tumor cells; (3) identify new TmPs, cell surface cleavage of which enhances tumor cell survival; and (4) investigate the in vivo functional role of MMP-9-mediated CD44 cleavage in tumor cell vascular survival and metastatic colonization. The proposed research will ultimately prove that irreversible modifications of distinct TmPs determine their in vivo functionality, thereby affecting overall metastatic outcome and representing an important translational aspect ultimately aimed for development of diagnostic or and/or risk stratification tools aiding cancer patient therapy.