Irradiation-induced promigratory phenotype of melanoma cells: role of S100A4-RAGE interaction

Metastases are a devastating and debilitating complication of melanoma with a poor prognosis for the patient. The treatment of metastases would be either radiation only or surgery combined with adjunctive postoperative radiation therapy. S100A4 (metastasin) is known to play a direct role in these metastatic processes. We hypothesize that RAGE (receptor for advanced glycation endproducts) is a putative receptor for S100A4. However, the role of S100A4-RAGE interaction in melanoma metastasis is still unclear. The purpose of this study was to find out how mouse B16-F10 melanoma cells restrained to irradiation. Furthermore, we examined changes in the S100A4-RAGE interaction and the ability for migration of irradiated melanoma cells in the presence of tumor associated macrophages. B16-F10 cells were exposed to single dose irradiation (5 Gy, 20 Gy) and mouse RAW 264.7 cells were used as a model for tumor-associated macrophages. S100A4 and RAGE expression in these cells was quantified via real-time RT-PCR and Western-blot analysis three and six days after irradiation. Cell migration was investigated with B16-F10 cells six days after irradiation in a 24-transwell chamber for 16 h and 24 h. Furthermore, migration was influenced by seeding RAW cells as a chemoattracant into the lower compartments and recombinant S100A4 as a stimulus to the upper compartments. After labeling the cells with Calcein-AM the migratory cells were quantified in a standard fluorescence microplate reader. The total number of vital irradiated B16-F10 cells is significantly decreased with increasing dose up to 20 Gy and up to six days, thereby altering morphological appearance. Surprisingly, in surviving B16-F10 cells expression of S100A4 and RAGE significantly increased three and six days after 20 Gy (p<0.05). Furthermore, irradiated B16-F10 cells showed higher migratory activity supposed due to enhanced expression of S100A4 and RAGE. In the presence of RAW cells and/or recombinant S100A4 a further increasing migration activity of irradiated cells (20 Gy) was found. Our findings suggest an association of melanoma and macrophages with alterations of their migratory and invasive activity after irradiation due to a perpetual para-/autocrine expression mechanism of extracellular S100A4 and RAGE, and thereby changing functional properties of melanoma cells towards a promigratory phenotype. This study was supported in part by the Deutsche Forschungsgemeinschaft (grant Pi 304/1-1).