Dissecting the opposing roles of alpha-3 integrin in metastasis

描述（由申请人提供）：a3b1整合素在恶性肿瘤中的作用是复杂的：虽然一些研究表明a3b1具有肿瘤抑制作用，但许多其他研究表明a3b1是肿瘤细胞粘附、运动和侵袭的有效促进因子。这一悖论可能被以下事实所调和：除了a3b1介导层粘连蛋白-5快速迁移的能力外，它还可以转导促进基于e -钙粘蛋白的粘附连接稳定组织的信号。这些相反的a3b1功能之间的平衡可能由a3b1相关的四跨蛋白调节。特别是，在临床和实验研究中，肿瘤细胞中四联蛋白CD9的缺失与转移增强有关。尽管有这些数据，在肿瘤生物学的背景下，无论是a3b1功能丧失表型还是a3b1连接稳定活性的作用都没有被探索过。我们的长期目标是了解如何操纵肿瘤-宿主相互作用来抑制肿瘤细胞转移。本应用的目的是：(i)确定a3b1整合素在调节肿瘤细胞集体迁移、局部侵袭和转移定植中的作用，并结合或独立于其促进粘附连接稳定性的能力，以及（ii）确定a3b1信号促进癌细胞-细胞连接稳定性的机制。核心假设是a3b1促进肿瘤侵袭和转移定定的能力与a3b1促进粘附连接稳定性的能力相平衡，这种机制依赖于a3b1与四跨蛋白CD9的关联。这一假设将在三个具体目标中得到检验。第一个具体目的是确定a3b1整合素、四跨蛋白CD9和a3b1-CD9关联在促进癌细胞连接稳定性中的作用。使用表皮样细胞和乳腺癌细胞，我们已经操纵了(i) a3或CD9表达，（ii） a3b1-CD9关联，（iii） a3b1配体结合，我们将评估粘附连接的组织和稳定性，集体细胞迁移，以及完整细胞片内的细胞动力学。第二个具体目标是确定a3b1整合素表达及其与CD9在肿瘤侵袭和转移定殖中的作用。我们将使用一种新的表皮癌细胞原位侵袭试验和一种已建立的乳腺癌自发转移原位模型来测试分别操纵a3整合素或CD9表达、a3-CD9关联或a3配体结合的肿瘤细胞。第三个具体目的是确定a3b1信号促进粘附结稳定性的机制。我们将使用我们的肿瘤细胞变体以及在我们的初步实验中发现的特异性细胞质信号效应的选择性抑制剂和激活剂来确定a3b1信号传导与粘附连接的稳定性之间的联系。总的来说，本提案中的实验有望提供关于a3b1在转移性定植中的功能的关键数据，以及它与a3b1作为e -钙粘蛋白调节剂的作用之间的关系。这些结果可能为a3b1整合素、其相关蛋白和下游效应物作为恶性肿瘤治疗靶点的适应能力提供重要信息。公共卫生相关性：为了开发更有效和高效的癌症治疗方法，一个主要目标是确定在不损害健康组织的情况下控制转移的方法。所提出的研究与这一目标相关，因为它们有望阐明迄今尚不清楚的调节肿瘤细胞转移能力的机制。一旦我们对导致转移的因素有了更多的了解，我们可能就能确定肿瘤特异性的过程，从而成为治疗的目标。

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.