Melanoma inhibitory activity (MIA) protein - molecular relevance and functional characterization

Melanoma inhibitory activity (MIA), an 11 kDa protein expressed and secreted by malignant melanoma cells, plays a key role in development and progression of malignant melanoma. It facilitates the release of tumor cells from the primary tumor and thus strongly promotes formation of metastases. However, the processing of MIA protein during cell migration and the characterization of protein activity on a molecular level has not been elucidated so far. These studies detail the mechanism by which MIA protein contributes to the aggressive and invasive behaviour of malignant melanoma cells. After migratory stimuli of tumor cells a directed, microtubule based protein transport to the rear cell pole is induced. MIA protein secretion is a Ca2+-dependent process regulated by the calcium activated potassium channel KCNN4 (IKCa1). This channel type has previously been reported to be in the active state at the rear pole of migrating cells. Following secretion, MIA protein directly interacts with cell adhesion receptors, including integrin alpha4beta1 and alpha5beta1, and extracellular matrix molecules. MIA-integrin-complexes are subsequently internalized into the cell. After dissociation of these complexes, MIA protein is degraded in acidic vesicles while integrins are transported to the cell front to form new adhesion contacts. Since MIA protein secretion is restricted to the cell rear, it mediates localized tumor cell detachment from surrounding structures of the primary tumor and enables invasion into healthy tissues. At the molecular level, NMR spectroscopy revealed that MIA protein forms homodimers with head to tail linkages. As indicated by mutant analysis in Boyden Chamber invasion assays and further in vitro protein analyses, MIA protein reaches functional activity via self assembly. To inhibit MIA protein function by preventing protein dimerization, MIA binding peptides, which were previously identified in a page display experiment, were specifically screened for their ability to dissociate MIA protein dimers. In order to conduct this screening, a heterogeneous transition-metal based fluorescence polarization assay (HTFP assay) was developed. In this assay format, MIA protein was immobilized in a well plate and MIA protein interactions were detected when the addition of Ru(bpy)3-labeled MIA protein led to an increase in the fluorescence polarization signal. Dodecapeptide AR71 dissociated MIA protein assemblies, as evidenced by a strong decrease in the fluorescence polarization signal. In addition, Western blot analysis also confirmed monomerization of MIA protein after treatment with AR71, while NMR-spectroscopy revealed that AR71 directly binds to the dimerization domain. By performing Boyden chamber invasion assays functional inhibition of MIA protein by peptide AR71 was also demonstrated in vitro. Therefore, the MIA inhibitory effect of AR71 was analyzed in a murine melanoma mestastases in vivo model. It was clearly demonstrated that treatment with dodecapeptide AR71 strongly reduces the number of metastases in vivo. These studies may constitute a foundation for the development of a MIA protein inhibitor which could represent a new therapeutic strategy as an antimetastatic treatment for malignant melanoma.