Dissecting the opposing roles of alpha-3 integrin in metastasis

描述（由申请人提供）：α 3 β 1整联蛋白在恶性肿瘤中的作用是复杂的：虽然一些研究表明具有肿瘤抑制作用，但许多其他研究表明α 3 β 1是肿瘤细胞粘附、运动和侵袭的有效促进剂。这种矛盾可能是由以下事实来调和的：除了a3 b1介导层粘连蛋白-5上的快速迁移的能力之外，它还可以抑制促进基于E-钙粘蛋白的粘附连接的稳定组织的信号。这些相反的a3 b1功能之间的平衡可能是由a3 b1相关的四跨膜蛋白调节。特别是，在临床和实验研究中，肿瘤细胞中四跨膜蛋白CD 9的丢失与增强的转移有关。尽管有这些数据，在肿瘤生物学的背景下，a3 b1功能丧失表型和a3 b1的连接稳定活性的作用都没有被探索。我们的长期目标是了解如何操纵肿瘤-宿主相互作用以抑制肿瘤细胞转移。本申请的目的是（i）确定α 3 β 1整联蛋白在调节集体肿瘤细胞迁移、局部侵袭和转移定植中的作用，以及其促进粘附连接稳定性的能力，和（ii）确定α 3 β 1信号传导促进癌细胞-细胞连接稳定性的机制。中心假设是a3 b1促进肿瘤侵袭和转移定植的能力通过a3 b1促进粘附连接稳定性的能力来平衡，该能力通过依赖于a3 b1与四跨膜蛋白CD 9的结合的机制来实现。这一假设将在三个具体目标中得到检验。第一个具体的目的是确定α 3 β 1整合素，四跨膜蛋白CD 9，和α 3 β 1-CD 9协会在促进癌细胞连接稳定性的作用。使用表皮样和乳腺癌细胞，我们已经操纵（i）α 3或CD 9表达，（ii）α 3b 1-CD 9缔合，和（iii）α 3b 1配体结合，我们将评估粘附连接组织和稳定性，集体细胞迁移，和完整细胞片层内的细胞动力学。第二个具体目标是确定α 3 β 1整合素表达的作用以及与CD 9在肿瘤侵袭和转移定植中的相关性。我们将使用一种新的表皮癌细胞原位侵袭测定法和一种已建立的自发性乳腺癌转移原位模型来测试其中α 3整联蛋白或CD 9表达、α 3-CD 9结合或α 3配体结合已被分别操纵的肿瘤细胞。第三个具体目标是确定a3 b1信号促进粘附连接稳定性的机制。我们将使用我们的肿瘤细胞变体与我们初步实验中鉴定的特异性胞质信号效应物的选择性抑制剂和激活剂一起，以确定a3 b1信号传导与粘附连接的稳定性之间的联系。总的来说，本提案中的实验预计将提供关于a3 b1在转移性定植中的功能的关键数据，以及它如何与a3 b1作为E-钙粘蛋白的调节剂的作用相关。这样的结果可以产生关于α 3 β 1整联蛋白、其相关蛋白和下游效应物作为恶性肿瘤治疗靶点的适合性的重要信息。公共卫生相关性：为了开发更有效的癌症治疗方法，一个主要目标是确定控制转移而不伤害健康组织的方法。拟议的研究与这一目标相关，因为它们有望阐明一种迄今知之甚少的调节肿瘤细胞转移能力的机制。一旦我们更多地了解了导致转移的因素，我们就可能能够确定可以治疗靶向的肿瘤特异性过程。

DESCRIPTION (provided by applicant): The role of a3b1 integrin in malignancy is complex: while some studies have indicated a tumor suppressive role, many others have shown that a3b1 is a potent promoter of tumor cell adhesion, motility and invasion. This paradox may be reconciled by the fact that, in addition to a3b1's ability to mediate rapid migration on laminin-5, it can also transduce signals that promote the stable organization of E-cadherin-based adherens junctions. The balance between these opposing a3b1 functions may be regulated by a3b1- associated tetraspanin proteins. In particular, the loss of tetraspanin CD9 in tumor cells is linked to enhanced metastasis in clinical and experimental studies. Despite these data, neither the a3b1 loss-of-function pheno- type nor the role of a3b1's junction-stabilizing activity has been explored in the context of tumor biology. Our long term goal is to understand how tumor-host interactions can be manipulated to inhibit tumor cell metastasis. The objectives of this application are to (i) determine the role of a3b1 integrin in regulating collective tumor cell migration, local invasion, and metastatic colonization in conjunction with, and independently of, its ability to promote adherens junction stability, and (ii) define the mechanism by which a3b1 signals to promote the stability of carcinoma cell-cell junctions. The central hypothesis is that a3b1's ability to promote tumor invasion and metastatic colonization is balanced by a3b1's ability to promote adherens junction stability by a mechanism that depends on a3b1 association with tetraspanin CD9. This hypothesis will be tested in three specific aims. The first specific aim is to determine the roles of a3b1 integrin, tetraspanin CD9, and a3b1-CD9 association in promoting carcinoma cell junctional stability. Using epidermoid and breast carcinoma cells in which we have manipulated (i) a3 or CD9 expression, (ii) a3b1-CD9 association, and (iii) a3b1 ligand binding, we will assess adherens junction organization and stability, collective cell migration, and the cellular dynamics within intact cell sheets. The second specific aim is to define the role of a3b1 integrin expression and association with CD9 in tumor invasion and metastatic colonization. We will use a novel orthotopic invasion assay for epidermal carcinoma cells and an established orthotopic model of spontaneous breast cancer metastasis to test tumor cells in which a3 integrin or CD9 expression, a3-CD9 association, or a3 ligand binding have been separately manipulated. The third specific aim is to determine the mechanism by which a3b1 signals to promote adherens junction stability. We will use our tumor cell variants together with selective inhibitors and activators of specific cytoplasmic signaling effectors identified in our preliminary experiments to determine the connection between a3b1 signaling and the resulting stabilization of adherens junctions. Collectively, the experiments in this proposal are expected to provide critical data on the function of a3b1 in metastatic colonization, and how it relates to a3b1's role as a regulator of E-cadherin. Such results may yield important information on the suit- ability of a3b1 integrin, its associated proteins, and downstream effectors as therapeutic targets in malignancy. PUBLIC HEALTH RELEVANCE: To develop more effective and efficient cancer treatments, a major goal is to identify methods for controlling metastasis without harming healthy tissue. The proposed studies are relevant to this goal because they are expected to illuminate an as yet poorly understood mechanism that regulates the ability of tumor cells to metastasize. Once we understand more about the factors that contribute to metastasis, we may be able to identify tumor-specific processes that could be therapeutically targeted.