The Role and Inhibition of S100B in Melanoma Cell Signaling

Abstract

The calcium–binding protein S100B is an effective and extensively used prognostic marker for melanoma, with increasing S100B being predictive of disease stage, increased recurrence, and low survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate the significance of S100B in melanoma, knock–down and over–expression studies were conducted, finding a positive correlation between S100B expression and cell viability, as well as ERK phosphorylation. However, phosphorylation of RSK, a downstream ERK target, was determined to have an inverse relationship with S100B. Over–expression of a calcium–binding mutant S100B yields neither effect, indicating that each response is calcium–dependent. Pull–down experiments established the direct calcium–dependent binding of S100B to the C–terminus of RSK and kinase assays demonstrated that S100B prevents RSK phosphorylation at Thr573. Over–expression of S100B in melanoma cells reduces the phosphorylation of RSK, sequestering it in the cytosol. Conversely, cells with diminished S100B expression exhibited increased staining of phosphorylated RSK within the nucleus. Together these data are consistent with a mechanism in which elevated S100B binds RSK directly in a calcium–dependent manner, preventing ERK–mediated phosphorylation and subsequent nuclear translocation. Thus, S100B uniquely affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously preventing the phosphorylation of RSK. Capitalizing on this discovery, in addition to previously known S100B interactions such as with p53, we are searching for S100B inhibitors that will prevent cancer progression. To this end, in vitro FPCA was employed to rapidly screen 2,000 compounds, establishing whether they bind Ca<super>2+</super>–loaded S100B and inhibit S100B target complex formation. Building upon this, we developed a cell–based high throughput assay capable of screening an extensive library of 14,400 compounds, in addition to 26 putative S100B inhibitors identified through FPCA, by comparing their effects on cells expressing elevated S100B to cells where S100B has been significantly knocked–down. The desired endpoint of this research is the development of a drug with therapeutic activity for the treatment of malignant melanoma and/or other cancers with elevated S100B.