Tumor metastasis is the major killer of patients with cancer. Cells metastasizing via hematogenous and lymphatic routes must attach to endothelial cells and to subendothelial basement membrane (BM). Laminin is the major non-collagenous molecule in BM and has profound phenotypic effects on malignant cells. The laminin G domain is very important and among other things promotes tumor cell adhesion, migration, glycosaminoglycan binding, and neurite outgrowth. Invasive or metastatic cells appear to have increases or alterations in a number of receptors for laminin. These receptors are somewhat varied and this proposal will focus on two major classes-integrins and proteoglycans. The laminin binding integrins include among others: alpha-1, alpha-2, alpha-3, alpha- 6, alpha-7, and alpha-v; beta-1, beta-2, beta-3, beta-4. The A chain G domain of EHS laminin consists of 5 loop-like structures. We will continue the structural functional analysis of bioactive G domain sequences, using the human HT-1080 fibrosarcomas, the A375 highly metastatic melanoma or other tumors may be utilized if necessary; we will determine minimal sequences involved in receptor binding and modulation of cell function, and make constrained peptides that have greater affinity, avidity, or effects on metastatic cell adhesion and/or cell migration. We will do this by determining the smallest biologically active sequences and attempt to make various modifications by having cysteines at the amino and carboxy ends which could form disulfides between cysteines. Chemical crosslinking of amino and carboxy terminal amino acids will also be attempted. Next, we will pursue a further definition of receptors and specific binding sites on receptors for G domain peptide sequences that modulate adhesion and migration of human tumor cells. This will be performed by the use of a battery of MABs or polyclonal anti-integrin peptide antibodies, and random 15'mer or hexamer phage display libraries and biopanning. In """"""""biopanning"""""""" we select for phage that expression the putative receptor binding determinants for active G domain laminin peptides. We go through a series of rounds of selection under various conditions in an attempt to select the higher affinity phage """"""""receptor"""""""" mimetic determinants binding to laminin peptides. The phage display library should be helpful in defining the binding determinants of any class of receptors, integrins, proteoglycans or others. Next, we will define the role of cell surface GAGs/proteoglycans in modulating adhesion and migration of metastatic tumor cells in response to laminin and laminin peptides. Cells will be treated with beta-D xyloside, chondroitinase ABC or heparitinase to remove GAGs or block their synthesis and determine effects on tumor cell adhesion and migration. Next, we will discern if PGs bind to or can be isolated with active laminin peptides and how PGs may directly or indirectly influence metastatic cell adhesion and migration. Finally, we will pursue in vivo experimental and spontaneous metastasis assays using human tumors in SCID or nude mice and various protocols to attempt to inhibit metastasis. We will use peptides from #1 above, free, coupled to various carriers, or """"""""constrained"""""""" peptides we designed that may have greater effects and perhaps have greater half lives in vivo.
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