A Disintegrin And Metalloproteases (ADAMs) represents a family of transmembrane proteins with a distinct multidomain structure including a metalloprotease, disintegrin, and cysteine-rich domain. These proteins function in adhesion, regulation of cell signaling by proteolysis of cell surface proteins and their receptors. ADAM-9 is a protease that cleaves membrane-bound proteins such as VCAM-1, TNF-α, as well as extracellular matrix proteins like fibronectin and laminin-111. ADAM-9 also contains adhesive domains that are involved in cell adhesion and migration. In human and murine melanoma, ADAM-9 is expressed by both tumor and stroma cells at the tumor-stroma border. The significance of this expression is however not clear. To explore the functional role of ADAM-9 produced by the host in melanoma progression, B16F1 murine melanoma cells were injected in the flank of Adam-9-/- and wild type animals. Tumors developed in Adam-9-/- mice were significantly larger compared to wild type animals and displayed significant increase in tumor cell proliferation accompanied by decrease in apoptosis. Using co-culture systems of primary fibroblasts and B16F1 melanoma cells, we could detect increased melanoma cell proliferation when cultured in the presence of supernatants from Adam-9-/- but not wild type fibroblasts. Among the proteins secreted in strongly enhanced amounts from Adam-9-/- fibroblasts, was tissue inhibitor of metalloproteinases-1 (TIMP-1). Neutralization of TIMP-1 in Adam-9-/- fibroblasts supernatant using specific antibodies abolished the induced B16F1 proliferation. Besides TIMP-1, soluble TNF-α and TNFR1 were also up-regulated in Adam-9-/- fibroblasts supernatants. Using various in vitro approaches, we could demonstrate that neutralization of TNF-α in Adam-9-/- fibroblast resulted in reduced B16F1 cell apoptosis. In addition, by RNA and proteomic analysis, we found altered expression of extracellular matrix proteins at the tumor-stroma border of tumors from Adam-9-/- animals compared to wild type, which likely contributes to the melanoma phenotype observed in Adam-9-/- animals. Interestingly, we identified collagen type I as a new substrate of ADAM-9. In summary, our results indicate that loss of ADAM-9 in stromal fibroblasts results in altered release of soluble factors, which in turn affect melanoma cell proliferation and apoptosis
