title: Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface.
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.
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Ye
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: King C
authors.initials: KC
authors.lastname: Chan
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Waters
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bess
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Harned
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Bih-Rong
authors.initials: BR
authors.lastname: Wei
authors.affiliation: Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Jadranka
authors.initials: J
authors.lastname: Loncarek
authors.affiliation: Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Luke
authors.affiliation: Thermo Fisher Scientific, Waltham, MA 02451, USA.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Orsburn
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Hollinger
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stephens
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Bagni
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Martinko
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Dwight V
authors.initials: DV
authors.lastname: Nissley
authors.affiliation: UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158-9001, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: McCormick
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Gordon
authors.initials: G
authors.lastname: Whiteley
authors.affiliation: Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
authors.forename: Josip
authors.initials: J
authors.lastname: Blonder
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000951:Antigens, Neoplasm; D051926:Basigin / Q000032:analysis; D015815:Cell Adhesion Molecules / Q000032:analysis; D045744:Cell Line, Tumor; D002465:Cell Movement; D019295:Computational Biology; D058750:Epithelial-Mesenchymal Transition; D006023:Glycoproteins / Q000145:classification / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D008855:Microscopy, Electron, Scanning; D050505:Mutant Proteins / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis*
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; C497886:BSG protein, human; C432044:CDCP1 protein, human; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; C117307:KRAS protein, human; D008565:Membrane Proteins; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D051926:Basigin; D016283:Proto-Oncogene Proteins p21(ras)
keywords: KRasG12V; cell surface proteome; drug targets; mass spectrometry; proteomics