Critical determinants of cancer progression and death from carcinomas are the capacity of tumor cells to survive, grow, invade their matrix stroma, and metastasize. Invasion and metastasis depend on the coordinated and temporal expression of proteolytic enzymes necessary to degrade the surrounding extracellular matrix and of adhesion molecules to reorganize cell-cell and cell-matrix attachments. Strong circumstantial evidence indicates that the protease urokinase, its receptor (uPAR), and b1 integrins are important to lung cancer progression. And prior work indicates that uPAR is a b1 integrin ligand, modifying integrin matrix engagement and signaling, and promoting tumor cell migration in vitro and in vivo. This proposal will test the hypothesis that uPAR acts as a strong agent of tumor cell survival and metastasis, acting through interactions with b1 integrins to coordinate MMP upregulation and alter cytoskeletal organization favoring matrix invasion. First, extending prior work, critical amino acid residues in uPAR that mediate interactions with the b1 integrins a3b1 and a5b1 will be fully established. Based on this information, peptides which disrupt uPAR/b1 integrin interactions will be exploited as a diagnostic test for uPAR/integrin signaling in primary lung cancer cells. Secondly, using uPAR point mutants unable to bind specific b1 integrins, the molecular mechanisms of altered matrix engagement and MMP upregulation initiated by uPAR/integrin interactions will be defined. Finally, wild type or uPAR silenced, GFP tagged H1299 cells will be injected either orthotopically into lungs of athymic nude mice and lung cancer growth, node metastasis, and angiogenesis quantified and compared. Cells reconstituted with uPAR mutants unable to bind either a3b1 or a5b1 will be examined to determine if uPAR/integrin interactions rather than uPA binding alone is crucial to uPAR-dependent tumor progression in vivo. Together, these experiments should clarify the mechanistic basis for the promoting role of uPAR on tumor progression, establish whether uPAR/integrin interactions are a compelling therapeutic target, and determine whether a diagnostic test for lung cancers dependent on uPAR for their malignant potential can be defined. ? ? ? ?
