A family of secreted proteins, which we term SIBLINGS (for Small Integrin-Binding LIgand, N-linked Glycoproteins), contain the integrin-binding tripeptide, ROD, are expressed in the skeleton and have genes clustered on human chromosome 4. Our work and that of others has shown that a variety of neoplasms frequently express one or more of the proteins and that expression correlates with disease severity. We have recently found that SIBLINGS can bind to and modulate the activity of matrix metalloproteinases (MMPs). SIBLING binding to latent pro-MMPs leads to catalytic activity, and SIBLING binding to MMPs inhibited by tissue inhibitors of metalloproteinases (TIMPs) or small molecular weight inhibitors leads to a restoration of enzymatic activity. We hypothesize that SIBLING expression by neoplasms promotes tumor progression through MMP modulation. To test this hypothesis, we will (a) characterize SIBLING and MMP interactions using purified components and standard sequence and kinetic analyses to characterize SIBLING effects of MMP and MMP inhibitor kinetics as well as identify SIBLING modifying reagents; (b) test SIBLING- modifying reagents (variants, blocking peptides, antibodies) in in vitro cell systems that yield a functional readout on angiogenesis (endothelial cells and tubule formation) and invasiveness (human cancer cells and modified Boyden chamber assays); and (c) employ SIBLINGS, SIBLING-modifying reagents to demonstrate the physiological relevance of SIBLING - MMP interactions in tumor progression (using two quantitative chick embryo chorioallantoic membrane (CAM) systems to study angiogenesis and metastasis). The CAM angiogenesis assay employs the implantation of SIBLING reagents in a collagen gel within a nylon mesh and quantifying vessel formation. The CAM metastasis assay uses a highly sensitive assay based on PCR amplification of human alu sequences for monitoring the metastatic dissemination of human tumor cells in the chick embryo. The molecular action of SIBLINGS in modulating MMPs may contribute to tumor progression. The alteration of MMP activity by SIBLINGS and their expression in human cancer may be a contributory factor to the failure of MMP inhibitors in clinical trials. It is the long term goal of this work to exploit knowledge of the basic biochemical and biological details involved in SIBLING binding to MMP to develop anti-tumor growth and progression therapy based on disrupting or altering SIBLING - MMP interactions. ? ? ?
