The long-term objectives of Project I are to understand the role of integrin receptors in mediating colorectal carcinoma adhesion, migration and growth on laminin, and to assess how such receptors influence tumor behavior. During the last granting period, the alpha6beta4 and alpha2beta1 integrins were identified as colon carcinoma laminin receptors. In addition, a novel mode of cell-laminin interaction was discovered that is characterized by rapidly formed but transient adhesive contacts mediated solely by the alpha6beta4 integrin. The properties of this """"""""dynamic adhesion"""""""" would facilitate tumor cell migration and suggest that alpha6beta4-mediated migration is distinct from beta1-integrin-mediated migration. More recently, we have found that the beta4 cytoplasmic domain regulates the growth and expression of the cyclin kinase inhibitor p21 in cells that express wild type p53. The alpha6beta4-mediated mechanisms that regulate dynamic adhesion, migration and growth will be examined in detail. Specific sequences in the beta4 cytoplasmic domain that regulate these processes will be identified by functional analysis of beta4- deficient colon carcinoma cell lines transfected with mutant forms of the beta4 cDNA. Time-lapse video microscopy will be used to compare the mechanics of migration of colon carcinoma cells that express both alpha6beta4 and alpha2beta1 laminin receptors and cells that express only beta1 integrin laminin receptors. Also, the possibility that differences in the localization and function of the alpha6beta4 and alpha2beta1 integrins results from their association with distinct cytoskeletal proteins will be examined using light and electron microscopy. These studies will include the use of beta4 cytoplasmic domain mutants to define specific sequences required for spatial localization and cytoskeletal associations. The ligand binding and signaling functions of the alpha6beta4 integrin associated with migration and dynamic adhesion most likely involve the interaction of specific proteins with the beta4 cytoplasmic domain. Such proteins will be identified by several approaches that involve capturing their association with beta4-specific immune complexes. The important issue of how alpha6beta4 integrin expression affects tumor behavior will be addressed by using wild-type and dominant negative beta4 constructs to modulate alpha6beta4 expression in colon carcinoma cell lines and by assessing the effect of this modulation on their tumorigenicity, invasive potential, and metastatic potential. Finally, we will assess the expression of the alpha6beta4 integrin and its structural variants, as well as E-cadherin and alpha-catenin, in tumor specimens in collaboration with the Pathology and Molecular Biology Cores.
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