Dissecting the opposing roles of alpha-3 integrin in metastasis

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Project Summary. The role of ¿3¿1 integrin in malignancy is complex: while some studies have indicated a tumor suppressive role, many others have shown that ¿3¿1 is a potent promoter of tumor cell adhesion, motility and invasion. This paradox may be reconciled by the fact that, in addition to ¿3¿1's ability to mediate rapid mi- gration on laminin-5, it can also transduce signals that promote the stable organization of E-cadherin-based adherens junctions. The balance between these opposing ¿3¿1 functions may be regulated by ¿3¿1- 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 ¿3¿1 loss-of-function pheno- type nor the role of ¿3¿1'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 metasta- sis. The objectives of this application are to (i) determine the role of ¿3¿1 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 ¿3¿1 signals to promote the stabil- ity of carcinoma cell-cell junctions. The central hypothesis is that ¿3¿1's ability to promote tumor invasion and metastatic colonization is balanced by ¿3¿1's ability to promote adherens junction stability by a mechanism that depends on ¿3¿1 association with tetraspanin CD9. This hypothesis will be tested in three specific aims. The first specific aim is to determine the roles of ¿3¿1 integrin, tetraspanin CD9, and ¿3¿1-CD9 association in promoting carcinoma cell junctional stability. Using epidermoid and breast carcinoma cells in which we have manipulated (i) ¿3 or CD9 expression, (ii) ¿3¿1-CD9 association, and (iii) ¿3¿1 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 ¿3¿1 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 ¿3 integrin or CD9 expression, ¿3-CD9 association, or ¿3 ligand binding have been separately manipulated. The third specific aim is to determine the mechanism by which ¿3¿1 signals to promote adher- ens 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 ¿3¿1 signaling and the resulting stabilization of adherens junctions. Collectively, the experiments in this proposal are expected to provide critical data on the function of ¿3¿1 in metastatic colonization, and how it relates to ¿3¿1's role as a regulator of E-cadherin. Such results may yield important information on the suit- ability of ¿3¿1 integrin, its associated proteins, and downstream effectors as therapeutic targets in malignancy.