Metastasis is the process by which blood-borne cancer cells establish new tumor colonies in select secondary organs. The selection of target organs for metastasis occurs in a nonrandom fashion and is dictated by compatible tumor cell and host cell characteristics. The initial step for arrest at the preferred, secondary location is adhesion to endothelium which is believed to be mediated by tumor cell surface molecules that recognize components on the endothelial cell lumenal surface of select vascular branches. This premise is supported by recent work in our laboratory detailing the isolation, cloning, and characterization of the 90 kD lung-specific, melanoma cell-binding endothelial cell adhesion molecule Lu-ECAM-1. The lectin-like adhesion molecule is constitutively expressed on endothelial of pleural and subpleural venules and, to a lesser extent, pulmonary venules and veins. Its expression in these blood vessels correlates closely with the topographical distribution of B16-F10 melanoma lung metastases. Anti-Lu- ECAM-1 mAbs inhibit colonization of the lungs by lung-metastatic B16 melanoma cells, but have no effect on the colonization of the lungs by other types of lung-metastatic cancers (e.g., KLN205 squamous carcinomas) or on the number of liver colonies produced by liver-metastatic B16-L8- F10 melanoma cells. Genomic Southern blot analyses show that the Lu- ECAM-1 gene is present in all mammalian species tested, usually as a single copy, but is absent in chicken and yeast. The present proposal is expanded to include biochemical, molecular, and functional studies on the human and mouse homologs of the Lu-ECAM-1 receptor/ligand pairs.
The specific aims are to first clone and sequence the human and mouse homologs of Lu-ECAM-1 [originally isolated from lung matrix-modulated bovine aortic endothelial cells] as well as the respective melanoma ligands. The cDNAs of these molecules and their purified recombinant proteins are then used in (a) the identification of the adhesion domains using oligonucleotide-directed mutagenesis and carbohydrate analysis (mapping and composition of oligosaccharides); (b) the determination of Lu-ECAM-1 ligand expression levels during melanoma progression in order to explore whether the Lu-ECAM-1 ligand might be useful as a prognostic indicator in melanoma disease; (c) the identification of cellular proteins that interact with the cytoplasmic domain of Lu-ECAM-1 in order to further define the function of this adhesion molecule, using glutathione-S-transferase/Lu-ECAM-1 fusion proteins and the yeast cell two-hybrid system; and (d) the generation of Lu-ECAM-1 knockout mice to provide ultimate information on the functional roles of the Lu-ECAM-1 gene. Clinically, these studies build the foundation for the identification of novel prognostic indicators of melanoma disease (Lu- ECAM-1 ligand expression levels) and for the design of new therapeutic strategies directed against melanoma dissemination based on anti-adhesion (e.g., anti-Lu-ECAM-1 monoclonal or polyclonal antibodies;synthetic peptides [or carbohydrates] directed against the binding domains; Lu- ECAM-1 anti-sense oligonucleotides).
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