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 Ca2+-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.
|Advisor:||Weber, David J.|
|Commitee:||Martin, Stuart S., Shapiro, Paul, Wilson, Gerald M., Winkles, Jeffrey A.|
|School:||University of Maryland, Baltimore|
|School Location:||United States -- Maryland|
|Source:||DAI-B 74/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Calcium, Cell signaling, Erk, Mapk, Melanoma, Rsk, S100b|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be