Oncotarget

Research Papers:

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

Xiaoying Ye _, King C. Chan, Andrew M. Waters, Matthew Bess, Adam Harned, Bih-Rong Wei, Jadranka Loncarek, Brian T. Luke, Benjamin C. Orsburn, Bradley D. Hollinger, Robert M. Stephens, Rachel Bagni, Alex Martinko, James A. Wells, Dwight V. Nissley, Frank McCormick, Gordon Whiteley, Josip Blonder

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Abstract

Xiaoying Ye1, King C. Chan1, Andrew M. Waters1, Matthew Bess1, Adam Harned1, Bih-Rong Wei2, Jadranka Loncarek3, Brian T. Luke4, Benjamin C. Orsburn5, Bradley D. Hollinger1, Robert M. Stephens1, Rachel Bagni1, Alex Martinko6, James A. Wells6, Dwight V. Nissley1, Frank McCormick7, Gordon Whiteley1, Josip Blonder1

1Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA

2Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA

3Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA

4Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA

5Thermo Fisher Scientific, Waltham, MA 02451, USA

6Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517, USA

7UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158-9001, USA

Correspondence to:

Josip Blonder, email: blonderj@mail.nih.gov

Keywords: KRasG12V, 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 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.

Author Information

Xiaoying Ye
Primary Contact  _

King C. Chan

Andrew M. Waters

Matthew Bess

Adam Harned

Bih-Rong Wei

Jadranka Loncarek

Brian T. Luke

Benjamin C. Orsburn

Bradley D. Hollinger

Robert M. Stephens

Rachel Bagni

Alex Martinko

James A. Wells

Dwight V. Nissley

Frank McCormick

Gordon Whiteley

Josip Blonder


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