Furthermore, MMP9 is a confirmed target of MMP13, and it is also involved in the cleavage Sunitinib FDA of numerous substrates, includ ing integrin precursors and LIF. Whether these or yet unknown targets are responsible for proliferation in melanoma will be investigated in the future. Interestingly, protein expression of MMP13 is absent from nevi, but was noted in almost 50% of cutaneous melanoma. A functional role for stromal MMP13 in melanoma development was recently described in a MMP13 mouse model. In these mice, B16F1 melanoma grafts displayed reduced tumor growth and strongly decreased metastasis and angiogenesis com pared to wildtype mice. Together with our data, it appears that tumor cell or stroma derived Inhibitors,Modulators,Libraries MMP13 plays a role in several processes of melanoma develop ment.

This makes it a potentially attractive drug target. Selective MMP13 specific protease inhibitors are already developed and are currently used in mouse mod els for arthritis. In future Inhibitors,Modulators,Libraries studies, we will investigate the effect of specific MMP13 inhibitors in animal mela noma models. Conclusions Our data demonstrate that MMP13 links growth stimu latory signals such as EGF and FCS to cell cycle pro gression in melanocytes and melanoma cells and to dedifferentiation in melanocytes. The data indicate that the protease is important for migration independent processes of melanoma formation, possibly by releasing a yet unidentified growth factor. As MMP13 also plays a role for melanoma progression and specific inhibitors are already developed, it might be considered as a target for the treatment of MMP13 sensitive melanoma.

Inhibitors,Modulators,Libraries Methods Cell Culture A375 cells were maintained in DMEM, 10% FCS in the presence of penicillin streptomycin. Mouse melanocytes transgenic for the chimeric receptor HERmrk or human EGFR were cultivated in DMEM, 10% FCS in the presence of cholera toxin, TPA and penicillin streptomycin. Melan a cells are a non transformed cell line that are dependent on TPA for cell growth and proliferation. Starving of cells was per formed in DMEM medium containing no additives but 1. 5% dialyzed FCS, unless indicated other wise. EGF was used in the concentrations Inhibitors,Modulators,Libraries indicated in the text and figure legends. were annealed and cloned into pRe troSuper previously digested with BglII and HindIII. The resulting plasmid was retrovirally delivered into melan a Hm cells and selected by puromycin treatment to obtain stable cellular expression.

For human melanoma cells, commercially available control siRNA and siRNA against human MMP13 were used. Inhibitors,Modulators,Libraries siRNA was transfected using X treme gene transfection reagent, according to the manufacturers recom mendations. Downregulation was monitored by real time PCR. Cell proliferation assay Cells were read more starved for three days in DMEM containing 1. 5% dialyzed FCS and seeded at 3 104 cells per well of a 6 well plate.