Abstract
Introduction: Recent years have seen great progress in the development of targeted therapy strategies for melanoma, with impressive therapeutic responses now being observed in patients whose melanomas harbor either activating mutations in BRAFor c-KIT. In contrast, few therapeutic options exist for the 15-20% of melanomas with mutations in NRAS. Previous attempts to define the biology of NRAS mutant melanomas have focused upon microarray analysis-an approach that failed to distinguish NRAS from BRAF-mutated melanomas. Despite apparently overlapping RNA expression profiles between the two sub-groups, BRAFand NRAS mutated melanomas were found to respond differently to small molecule signal transduction inhibitors, suggesting important differences in cell signaling. The aim of the current study was to perform comparative phosphoproteomic analysis on a panel of NRAS and BRAF mutated melanoma cell lines and to build intracellular signaling networks of the two groups.
Methods: Phosphotyrosine enrichment was performed using the PhosphoScan protocol (Cell Signaling Technology) where phosphotyrosine-containing peptides were immunoprecipitated with immobilized anti-phosphotyrosine antibodies before being analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These data yielded a catalog of tyrosine phosphorylations across a wide range of signaling proteins and enabled the relative quantification of each site based on exctracted ion chromatograms from high resolution accurate mass measurements of the peptides. Analysis of the phosphorylated proteins and the quantitative data was performed using GeneGO software, so that signaling maps of BRAFand NRAS mutated melanoma cell lines could be constructed.
Results: A preliminary analysis of the data revealed BRAFmutated melanomas to be less diverse than the NRAS group, which tended to have constitutive phosphorylation in a wide-range of receptor tyrosine kinases (RTKs), such as c-MET, EGFR, and HER2, as well as non-receptor tyrosine kinases, such as c-Abl. Studies are ongoing to validate the role of these RTKs in the oncogenic behavior of the NRAS-mutated melanoma cell line panel and address whether any of the identified RTKs would be suitable therapeutic targets for this group of melanomas.
Conclusions: Phosphoproteomics has proven useful in elucidating cancer biology of melanomas that harbor mutations in BRAF and NRAS. These differences in kinase activation and protein phosphorylation may prove to be useful in selection of targeted therapy regimens for melanoma patients and serve as candidate biomarkers for patient assessment.
∗This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics Project LINK (Leaders In New Knowledge-Emerging Technologies) that also emphasizes education in health disparities and community outreach.
Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A66.
RSS Feeds