Cellular movement is required during normal embryonic development, tissue remodeling, wound repair and metastasis. During these biological processes, several different classes of molecules have been shown to affect cell motility and positioning. In particular, proteins of the extracellular matrix can provide directional clues for migrating cells. It would then follow that tumor cells which express two different phenotypes, both mesenchymal and epithelial, would be better able to respond to more diversified signals from the matrix. Our working hypothesis is that the """"""""anomalous"""""""" or unusual expression of cytokeratins (CKs) by certain human melanoma cells, in addition to their classical expression of vimentin, plays an important role in contributing to the invasive behavior of the highly metastatic tumor cells. Taking into consideration the hypothesis that intermediate filaments (the family to which cytokeratins and vimentin belong) can act as signal transducers (possibly via second messenger pathways), relaying information from the extracellular matrix to the nucleus, and that the matrix can regulate gene expression, we speculate that the ability to coexpress vimentin and CKs offers a selective advantage to tumor cells. Understanding the genetic regulation of this anomalous expression holds the key to more efficient diagnoses and more effective chemotherapeutic strategies. Toward this goal, our overall objective is to define a functional role for CKs (which have been thought of as general housekeeping proteins) in tumor cell invasion correlated with metastatic potential. In collaboration with Dr. Robert Oshima, an expert in intermediate filament cell and molecular biology, we will focus on the functional role of CKs 8 and 18 (based on preliminary data from our laboratories) in three well-characterized human melanoma cell lines of low, intermediate and high invasive and metastatic potentials.
Specific Aim #1 : To overexpress CKs 8 and 18 in CK-negative cells (of low metastatic potential) using transfection technology and subsequently measure invasive/metastatic potential, in addition to studying key mechanisms associated with changes in invasive/metastatic potential (e.g. adhesion, degradation and motility).
Specific Aim #2 : To select CK-positive cells from subpopulations of a cell line of intermediate metastatic potential (containing a minority of CK- positive cells) using an invasion model and cloning procedures, and then measure changes in invasive/metastatic properties associated with """"""""enriched"""""""" subpopulations.
Specific Aim #3 : To study alterations in the invasive/metastatic properties of stable clones of a highly metastatic cell line (containing 100% CK-positive cells) already transfected with a mutant CK18, resulting in disrupted CK filaments.
