Melanoma inhibitory activity in melanoma diagnostics and therapy - a small protein is looming large.

Malignant melanoma is a highly aggressive cancer with a very poor prognosis after the onset of metastasis. We have previously demonstrated that the protein melanoma inhibitory activity (MIA) is involved in the metastasis of and immunosuppression in malignant melanoma. Recently, we further established MIA as a therapeutic target to inhibit metastatic spread in malignant melanoma. We could show that an inhibition of MIA by a synthetic peptide decreased both the number of metastases as well as immunosuppression in a murine model of malignant melanoma. To control recurrence after surgical resection of a primary lesion, it is paramount to have diagnostic tools available that can detect a relapse due to the strong metastatic potential of melanoma. This follow-up is maintained with periodic re-examinations. Due to high cost and the associated radiation exposure, radiology examinations are avoided if possible. The analysis of prognostic markers in patient serum is therefore attractive. In this review, we focus on the quantitative analysis of the MIA protein as a prognostic tool because it has proven to be a useful serum marker for documenting disease progression of malignant melanoma. The MIA quantification assay itself is readily performed using an ELISA kit and common laboratory equipment. Because analysing MIA serum levels in combination with other established markers such as S100B improves their prognostic value, we feel that the quantification of MIA in the serum, among other markers, should be performed as a general standard of care in patients at risk of developing metastatic melanoma.

MIA/CD-RAP Regulates MMP13 and Is a Potential New Disease-Modifying Target for Osteoarthritis Therapy.

Melanoma inhibitory activity/cartilage-derived retinoicacid-sensitive protein (MIA/CD-RAP) is a protein expressed and secreted by chondrocytes and cartilaginous tissues. MIA/CD-RAP-deficient mice develop milder osteoarthritis than wildtype mice. In this study, we investigated MIA/CD-RAP downstream targets to explain this reduced disease development. As a possible mediator, we could detect matrix metalloproteinase 13 (MMP13), and the influence of MIA/CD-RAP on MMP13 regulation was analyzed in vitro using SW1353 chondrosarcoma cells and primary chondrocytes. The femoral head cartilage of WT and MIA/CD-RAP -/- mice were cultured ex vivo to further investigate MMP13 activity. Finally, osteoarthritis was surgically induced via DMM in C57BL/6 mice, and the animals were treated with an MIA/CD-RAP inhibitory peptide by subcutaneously implanted pellets. MMP13 was regulated by MIA/CD-RAP in SW1353 cells, and MIA/CD-RAP -/- murine chondrocytes showed less expression of MMP13. Further, IL-1ß-treated MIA/CD-RAP -/- chondrocytes displayed less MMP13 expression and activity. Additionally, MIA/CD-RAP-deficient ex vivo cultured cartilage explants showed less MMP13 activity as well as reduced cartilage degradation. The mice treated with the MIA/CD-RAP inhibitory peptide showed less osteoarthritis development. Our findings revealed MIA/CD-RAP as a new regulator of MMP13 and highlighted its role as a potential new target for osteoarthritis therapy.

Binding of phosphorylated peptides and inhibition of their interaction with disease-relevant human proteins by synthetic metal-chelate receptors.

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal-chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal-chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon-gamma receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding.

Detection of protein phosphorylation on SDS-PAGE using probes with a phosphate-sensitive emission response.

Fluorescent probes for the detection of protein phosphorylation on SDS-PAGE are presented. The probes were designed using a dinuclear metal-chelate phosphate recognition unit and an environmentally sensitive fluorophore. The specificity of the probes is determined by their binding site selectivity toward phosphate ions and the emission response induced by the change in the electrostatic environment of the fluorophore upon binding to a phosphorylated amino acid residue. The staining is fully reversible due to the noncovalent binding of the probe. Gel bands with less than 100 ng of phosphorylated alpha-casein are detectable with the new probes on a normal UV-table without specialized equipment like a laser-based gel scanner or a cooled camera detector.

MIA--a new target protein for malignant melanoma therapy.

Malignant melanoma, a malignancy of pigment-producing cells, causes the greatest number of skin cancer-related deaths worldwide. The tumor is characterized by its aggressive phenotype and can metastasize at very early stages of the disease. Since metastatic lesions are usually characterized by an intrinsic resistance to standard radiation and chemotherapy, the prognosis of this tumor remains very poor in advanced stages. Melanoma inhibitory activity (MIA), an 11 kDa protein expressed and secreted by melanoma cells after their malignant transformation, is known to play a key role in melanoma development, progression and tumor cell invasion. After its secretion, which is restricted to the rear pole of migrating cells, MIA protein directly interacts with cell adhesion receptors and extracellular matrix molecules. By this mechanism, MIA protein actively facilitates focal cell detachment from surrounding structures at the cell rear and strongly promotes tumor cell invasion and formation of metastases. It has further been demonstrated that MIA contributes to immunosuppression frequently seen in malignant melanomas by binding to integrin α(4)ß(1) expressed by leukocytes and thus inhibiting cellular antitumor immune response. Analyses at the molecular level revealed that MIA protein reaches functional activity by self assembly. Functional inactivation of MIA protein by dodecapeptides that directly bind to the dimerization interface leads to a strongly reduced tumor cell invasion in an in vivo mouse melanoma model. The molecular understanding of the contribution of MIA protein to formation of metastases provides an excellent starting point for the development of a new strategy in malignant melanoma therapy.

Inducing anti-tumor cytokines and an immune response in melanoma by inhibition of MIA using the peptide AR71.

BACKGROUND: Malignant melanoma is known for its aggressive metastatic spread and suppression of the host immune system. Immunosuppression in melanoma is mediated in part by the protein melanoma inhibitory activity (MIA). OBJECTIVES: In this study, we assessed the in vitro and in vivo efficacy of the MIA-inhibitory peptide AR71 in the inhibition of MIA-induced immunosuppression. This follows a previous study that revealed an increase in CD3-positive cells and cleaved caspase-3 in an in vivo model of hepatic metastasis after MIA inhibition. MATERIALS AND METHODS: We used Multiplex-ELISAs and qRT-PCR for determining changes in cytokine expression in vitro and in vivo and calcein release assays for determining immune cell response in vitro. RESULTS: By evaluating the serum levels of tumor-associated cytokines of the melanoma-bearing mice, we found beneficial decreases of several cytokines, including TNF-alpha, after AR71 treatment. Additionally, we demonstrated an increase of anti-tumor lymphokine-activated killer (LAK) cell cytotoxicity in the presence of the MIA inhibitor AR71. Stimulation of anti-tumor immune responses by AR71 could be observed via increased numbers of NK cells in the metastases-bearing murine livers in vivo. CONCLUSION: In summary, inhibition of MIA activity results in reduced immunosuppression in vitro and in vivo.

Pitfalls in immunohistochemistry--a recent example.

Immunohistochemistry is an important and valuable technique in many fields of research, although several common pitfalls can lead to wrong or misinterpreted results. A recently published study [1] claims that the protein MIA (melanoma inhibitory activity) is expressed in Purkinje cells in the cerebellum. Careful re-analysis resulted in negative results. Due to these results of our group we feel that this analysis could serve as example for the potential problems in immunohistochemistry caused by the combination of an unspecific antibody and the omission of evaluating control tissue samples.

Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition.

Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.

DNA staining in agarose gels with Zn²+-cyclen-pyrene.

A pyrene-labeled Zn²+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (∼300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn²+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.

Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions.

Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein-binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high- or low-molecular weight interaction partners. Our new approach eliminates this limitation by immobilizing a known binding partner of MIA protein to a well plate and by labeling the target protein using luminescent transition metal labels such as Ru(bpy)3 for binding studies with both high- and low-molecular weight interaction partners. Due to the use of a functionalized surface, we termed our concept heterogeneous transition metal-based fluorescence polarization (HTFP) assay. The assay's independence from the molecular weight of potential binding partners should make the technique amenable to investigations on subjects as diverse as multimerization, interactions with pharmacophores, or binding affinity determination.

Synthetic receptors for the differentiation of phosphorylated peptides with nanomolar affinities.

Artificial ditopic receptors for the differentiation of phosphorylated peptides varying in i+3 amino acid side chains were synthesized, and their binding affinities and selectivities were determined. The synthetic receptors show the highest binding affinities to phosphorylated peptides under physiological conditions (HEPES, pH 7.5, 154 mM NaCl) reported thus far for artificial systems. The tight and selective binding was achieved by high cooperativity of the two binding moieties in the receptor molecules. All receptors interact with phosphorylated serine by bis(ZnII-cyclen) complex coordination and a second binding site recognizing a carboxylate or imidazole amino acid side chain functionality.

Utilizing reversible copper(II) peptide coordination in a sequence-selective luminescent receptor.

Although vast information about the coordination ability of amino acids and peptides to metal ions is available, little use of this has been made in the rational design of selective peptide receptors. We have combined a copper(II) nitrilotriacetato (NTA) complex with an ammonium-ion-sensitive and luminescent benzocrown ether. This compound revealed micromolar affinities and selectivities for glycine- and histidine-containing sequences, which closely resembles those of copper(II) ion peptide binding: the two free coordination sites of the copper(II) NTA complex bind to imidazole and amido nitrogen atoms, replicating the initial coordination steps of non-complexed copper(II) ions. The benzocrown ether recognizes the N-terminal amino moiety intramolecularly, and the significantly increased emission intensity signals the binding event, because only if prior coordination of the peptide has taken place is the intramolecular ammonium ion-benzocrown ether interaction of sufficient strength in water to trigger an emission signal. Intermolecular ammonium ion-benzocrown ether binding is not observed. Isothermal titration calorimetry confirmed the binding constants derived from emission titrations. Thus, as deduced from peptide coordination studies, the combination of a truncated copper(II) coordination sphere and a luminescent benzocrown ether allows for the more rational design of sequence-selective peptide receptors.

Recognition of the helix-loop-helix domain of the Id proteins by an artificial luminescent metal complex receptor.

Synthetic agents specifically interacting with a protein interface are important not only for the better understanding of protein dimer or complex formation but also for medical applications. Here we describe the recognition of the helix-loop-helix (HLH) dimerization domain of the Id proteins by an artificial luminescent receptor containing two binding sites for a Lewis acid and a Lewis base, respectively. The Id proteins are inhibitors of bHLH transcription factors and play key roles during development of cancer. We show that a receptor/Id-HLH-domain complex was formed cooperatively (K(0.5) approximately 2 microM under physiological conditions) and with moderate specificity, as compared to the related MyoD and Max HLH domains. Accordingly, a preferred receptor binding motif, CYSR(K), was identified within the Id HLH domains. These results are promising and may be exploited to design highly selective synthetic receptors for the Id HLH domain.