Invasiveness is a hallmark of aggressive cancer like malignant melanoma, and factors involved in acquisition or maintenance of an invasive phenotype are attractive targets for therapy. We investigated melanoma phenotype modulation induced by the metastasis-promoting microenvironmental protein S100A4, focusing on the relationship between enhanced cellular motility, dedifferentiation and metabolic changes. In poorly motile, well-differentiated Melmet 5 cells, S100A4 stimulated migration, invasion and simultaneously down-regulated differentiation genes and modulated expression of metabolism genes. Metabolic studies confirmed suppressed mitochondrial respiration and activated glycolytic flux in the S100A4 stimulated cells, indicating a metabolic switch toward aerobic glycolysis, known as the Warburg effect. Reversal of the glycolytic switch by dichloracetate induced apoptosis and reduced cell growth, particularly in the S100A4 stimulated cells. This implies that cells with stimulated invasiveness get survival benefit from the glycolytic switch and, therefore, become more vulnerable to glycolysis inhibition. In conclusion, our data indicate that transition to the invasive phenotype in melanoma involves dedifferentiation and metabolic reprogramming from mitochondrial oxidation to glycolysis, which facilitates survival of the invasive cancer cells. Therapeutic strategies targeting the metabolic reprogramming may therefore be effective against the invasive phenotype.

Tumor cells have the ability to exploit stromal cells to facilitate metastasis. By using malignant melanoma as a model, we show that the stroma adjacent to metastatic lesions is enriched in the known metastasis-promoting protein S100A4. S100A4 stimulates cancer cells to secrete paracrine factors, such as inflammatory cytokines IL8, CCL2 and SAA, which activate stromal cells (endothelial cells and monocytes) so that they acquire tumor-supportive properties. Our data establishes S100A4 as an inducer of a cytokine network enabling tumor cells to engage angiogenic and inflammatory stromal cells, which might contribute to pro-metastatic activity of S100A4.

Substantial evidence has linked the small calcium-binding protein S100A4 to metastatic progression. S100A4-mediated effects include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which the protein exerts these effects are incompletely elucidated. In the present study, we demonstrate that S100A4 induces NF-jB-dependent expression and secretion of osteopontin (OPN) in a selection of osteosarcoma cell lines. OPN is, as S100A4, known to result in a variety of cellular effects potentially leading to increased angiogenesis and metastasis, and several of the activated signaling pathways are common for the two proteins. In our study, extracellular S100A4 was found to upregulate enzymes of the plasminogen activator system and matrix metalloproteinase (MMP) family, especially urokinase plasminogen activator and MMP-13. Furthermore, increased motility and invasion was observed in vitro as a result of S100A4 treatment. OPN expression was inhibited by the use of siRNA molecules, and a partial blocking of S100A4-induced effects on protease expression and invasive capacity was detected. In conclusion, our results suggest regulation of OPN as a downstream molecular mechanism of S100A4 signaling. This novel finding adds more information to how S100A4 mediates tumor development and metastatic progression. The observation of a link between S100A4 and OPN, and also identification of common downstream effect molecules, highlights them, their receptors or downstream proteins, as targets for therapeutic approaches.The S100A4 protein belongs to the S100 protein family, a group of small, acidic, calcium-binding proteins with different expression patterns and distinct tissue distributions and biological functions. 1 Over the years, numerous reports have convincingly linked expression of S100A4 to the invasive and metastatic capacity of cancer cells. 2,3 The protein has been associated with more aggressive and invasive tumors and increased metastatic potential in transgenic animals. 4,5 In addition, clinical evidence has clearly indicated a correlation between augmented S100A4 expression and poor prognosis in a number of human cancers, and S100A4 has been proposed as a potential marker predicting metastasis and survival. 6,7 The biological function and molecular mechanisms by which S100A4 exerts its putative metastasis-promoting effects are largely unknown, and the protein is most likely involved in several facets of tumor progression. Moreover, both intracellular and extracellular S100A4 have been shown to promote cancer development. The protein has been demonstrated to interact with cytoskeletal proteins, suggesting a possible role in cell motility, 8 and extracellular S100A4 has been identified as a potent inducer of angiogenesis. 9 Importantly, an association between exposure to, or expression of, S100A4 and members of the matrix metalloproteinase (MMP) family is well documented, 10-13 and as a result, S100A4 is suggested to promote progression of c...

Malignant melanoma is highly aggressive cancer with poor prognosis and few therapeutic options. Interferon alpha (IFN-α) has been tested as adjuvant immunotherapy in high-risk melanoma patients in a number of studies, but its beneficial role is controversial. Although IFN-α treatment can prolong relapse-free survival, the effect on overall survival is not significant. However, a small subset of patients benefits from the treatment, signifying the need for biomarkers able to identify a responding subgroup. Here we evaluated whether serum osteopontin (OPN) could function as a biomarker identifying patients with poor prognosis that might benefit from IFN-α. The choice of osteopontin was based on the knowledge about the dual role of this protein in cancer and immune response, an apparent association between OPN and IFN signaling and a prognostic value of OPN in multiple other tumor types. Serum samples from 275 high-risk melanoma patients enrolled in the Nordic Adjuvant IFN Melanoma trial were analyzed for circulating OPN concentrations and OPN promoter polymorphisms in position -443. The potential relation between serum OPN levels, the genotypes and survival in non-treated patients and patients receiving adjuvant IFN-α was investigated. Although slightly better survival was observed in the treated patients that had high levels of OPN, the difference was not statistically significant. In conclusion, serum OPN (its level or the genotype) cannot distinguish melanoma patients with poor prognosis, or patients that might benefit from adjuvant treatment with IFN-α.

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