Autocrine regulation of receptor for advanced glycation endproducts (RAGE) by S100A4 promotes migration and invasion in A375 melanoma cells

Background
The calcium-binding protein S100A4 is associated with metastasis of different cancer entities, including melanoma. The multiligand receptor for advanced glycation endproducts (RAGE) has been suggested to interact with extracellular S100A4 protein. We hypothesized that the interaction between RAGE and S100A4 plays an important role in activation of growth, adhesion, motility and migration in a human melanoma cell line with high metastatic potential.

Materials and methods
In order to investigate the cellular role of the RAGE-S100A4 interaction in vitro, we produced recombinant S100A4 and soluble RAGE (sRAGE). Furthermore, we established A375 melanoma cells stably transfected with S100A4 using vector pIRES2-AcGFP1 (A375-S100A4). The overexpression of S100A4 has been verified by western blot and flow cytometry. Assays for determination of migratory, invasive and adhesive behaviour of A375-S100A4 cells were performed. Furthermore, specific interaction of S100A4 with RAGE was characterized by surface plasmon resonance spectroscopy using immobilized sRAGE.

Results
The overexpression of S100A4 did not influence growth properties and adhesive behaviour of the A375-S100A4 cells; however, it affects their motility and migratory activity in comparison to mock-transfected cells. A375-S100A4 cells show an increased secretion of S100A4 into the extracellular space and, in consequence, an enhanced RAGE protein expression. Molecular interaction studies revealed high affinity (lower micromolar range) of S100A4 towards immobilized sRAGE, suggesting a biochemical rationale for the observed effects.

Conclusion
This investigation shows that overexpression of S100A4 influences the metastatic behavior of A375 melanoma cells. The enhanced secretion of S100A4 leads to an autocrine upregulation of RAGE expression and synthesis in A375-S100A4 cells. The findings support the supposed functional role of RAGE-S100A4 interaction in promoting a metastatic phenotype of human melanoma.