Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface

// Xiaoying Ye 1 , King C. Chan 1 , Andrew M. Waters 1 , Matthew Bess 1 , Adam Harned 1 , Bih-Rong Wei 2 , Jadranka Loncarek 3 , Brian T. Luke 4 , Benjamin C. Orsburn 5 , Bradley D. Hollinger 1 , Robert M. Stephens 1 , Rachel Bagni 1 , Alex Martinko 6 , James A. Wells 6 , Dwight V. Nissley 1 , Frank McCormick 7 , Gordon Whiteley 1 , Josip Blonder 1 1 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA 2 Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA 3 Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA 4 Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA 5 Thermo Fisher Scientific, Waltham, MA 02451, USA 6 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517, USA 7 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158-9001, USA Correspondence to: Josip Blonder, email: blonderj@mail.nih.gov Keywords: KRas G12V , cell surface proteome, drug targets, proteomics, mass spectrometry Received: September 06, 2016      Accepted: November 07, 2016      Published: November 24, 2016 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 KRas G12V transfected MCF10A (MCF10A-KRas G12V ) 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 KRas G12V 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-KRas G12V 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 KRas G12V activation. Taken together, this dataset significantly expands the map of the KRas G12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.