The functional role(s) of dual intermediate filament expression in tumor cell migration and invasion.

Abstract

Tumor cell invasion and metastasis are very complicated biological events which involve numerous classes of proteins that participate in controlling each step of the metastatic cascade. Therefore, identifying the key factor(s) that determine(s) how a tumor cell becomes more metastatic is a fascinating issue that challenges the field of cancer biology. Intermediate filaments are cytoskeletal proteins whose expression is highly regulated in a cell-type specific manner; however, recent evidence has indicated that coexpression of two different types of intermediate filaments--specifically keratin(s) (epithelial cell marker) and vimentin (mesenchymal cell marker), in the tumor cells correlates with their invasive and metastatic potential. The focus of this dissertation is to further elucidate the functional role(s) of this dual intermediate filament expression in tumor cells. A dominant negative mutant keratin cDNA was used to transfect a highly metastatic human melanoma cell line, C8161, which contains both vimentin and keratin filaments. The resulting transfected clones showed disrupted keratin filaments by immunofluorescence microscopy. Subsequently, their migratory and invasive ability were reduced, in addition to complete abrogation of metastatic potential. Another strategy involved the transfection of keratin 8 and 18 DNAs into keratin-negative cells (both mouse L fibroblasts and low invasive A375P melanoma), which resulted in clones expressing the dual intermediate filament phenotype, commensurate with increased migratory and invasive ability. Furthermore, these experimental clones have a retarded spreading ability on extracellular matrix compared to the control transfectants. In addition, they do not contain any detectable αᵥβ₃, α₃ or α₆ integrins in the focal contact sites by immunofluorescence staining. Hence, it is postulated that the mechanism responsible for differential spreading ability rests in the unique regulation of specific integrin(s) localized in focal contacts, acting either directly or indirectly, with the intermediate filaments and with extracellular matrix molecules. These results suggest that dual intermediate filament expression is important for the invasive phenotype, and their heretofore assigned role as general maintenance structural proteins has changed to that of dynamic cytoskeletal elements involved in active cellular function(s).