1. Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface.
- Author
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Ye X, Chan KC, Waters AM, Bess M, Harned A, Wei BR, Loncarek J, Luke BT, Orsburn BC, Hollinger BD, Stephens RM, Bagni R, Martinko A, Wells JA, Nissley DV, McCormick F, Whiteley G, and Blonder J
- Subjects
- Antigens, CD analysis, Antigens, Neoplasm, Basigin analysis, Cell Adhesion Molecules analysis, Cell Line, Tumor, Cell Movement, Computational Biology, Epithelial-Mesenchymal Transition, Glycoproteins classification, Glycoproteins physiology, Humans, Mass Spectrometry, Microscopy, Electron, Scanning, Neoplasm Proteins analysis, Membrane Proteins analysis, Mutant Proteins analysis, Proteomics methods, Proto-Oncogene Proteins p21(ras) analysis
- Abstract
Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.
- Published
- 2016
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