OCT 12, 2017 9:00 AM PDT

Profiling the cell surface of cancer cell lines for biomarker/drug target discovery using MS-based proteomics

Speaker
  • Senior Scientist, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research (FNLCR)
    Biography
      Dr. Blonder is the Clinical Proteomics Lead at the Cancer Research Technology Program at Frederick National Laboratory for Cancer Research (FNLCR), operated by Leidos Biomedical Research, Inc. For the National Cancer Institute. In 1978, Dr. Blonder received his M.D. at the Rijeka University School of Medicine, Croatia. In 2000, through Associated Western Universities, Dr. Blonder was awarded a post-doc fellowship in proteomics at the Pacific Northwest National Laboratory (PNNL), Richland, WA (Advisor: Dr. Richard D. Smith). In 2002, Dr. Blonder joined the Laboratory of Proteomics & Analytical Technology, at the FNLCR (formerly NCI-Frederick) where he continues to develop and apply MS-based proteomics to cancer research. Since 2006, he has led the Clinical Proteomics Group, extending his research to in-depth profiling of membrane proteome. In parallel, he worked on the methodology for cancer biomarker and drug target discovery using clinical specimens. His group was the first to optimize the immunodepletion of abundant proteins from tissue homogenates. In 2013, Dr. Blonder joined the Antibody Characterization Laboratory to lead the cell surface project within the Ras Initiative at the FNLCR/NCI focused on discovery of cell surface targets, enriched or unique to cancers driven by mutated RAS that could be targeted by drugs, antibodies, antibody-drug conjugates, or nanoparticles. This effort resulted in the first cell surface proteome map of a model cell line expressing oncogenic KRasG12V. Dr. Blonder brings unique combination of his expertise in clinical proteomics, medicine and systems biology, focusing his research on the development of innovative approaches for cancer biomarker and drug target discovery. He is an editor of the BMC Cancer and a lecturer at the Foundation for Advanced Education in the Sciences at NIH. Dr. Blonder has authored over 60 scientific publications in areas of biological mass spectrometry, clinical proteomics and cancer research.

    Abstract

    In order to expand the treatment options of cancers driven by oncogenic RAS, new cell surface targets need to be identified and characterized. Here, we describe mass spectrometry based phenotyping of the KRASG12V cell surface using MCF10A-KRASG12V as a cell line model of constitutively activated KRAS. Extensive view of the MCF10A-KRASG12V surface proteome was achieved by applying concurrently targeted hydrazide-based cell surface capturing (CSC) technology and global shotgun membrane (GSM) proteomics. Our combined approach revealed 666 plasma membrane proteins unique to the MCF10A-KRASG12V cell surface. Of these, 104 were cell surface glycoproteins identified by CSC technology while 562 cell surface proteins were identified using GSM proteomics. K-Ras was exclusively identified by GSM proteomics in the membrane fraction of MCF10A-KRASG12V cells and subsequently cross-validated by Western blot (WB) analysis. Subtractive proteomics, spectral counting-based based quantitation of changes in protein abundances, and Ingenuity Pathway Analysis showed that this investigation reliably revealed expected K-Ras induced changes at the surface of MCF10A-KRASG12V cells as well as alterations in cell surface protein expression with very little prior information. This analysis uncovered a subset of eight proteins identified exclusively at the cell surface of MCF10A-KRASG12V cells by both CSC and GSM technologies. Of these, two were further cross-validated using immunofluorescence and WB analysis. Furthermore, scanning electron microscopy and functional cell assays showed extensive changes at the KRASG12V cell surface consistent with widespread epithelial to mesenchymal transformation (EMT). Taken together, this dataset greatly extends the known molecular phenotype of the MCF10A-KRASG12V cell surface and reveals the important role of EMT in the pathophysiology of the KRASG12V driven malignant transformation in MCF10A- KRASG12V model cell line.


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    OCT 12, 2017 9:00 AM PDT

    Profiling the cell surface of cancer cell lines for biomarker/drug target discovery using MS-based proteomics


    Specialty

    Research

    Health

    Cancer

    University

    T-Cells

    Earth Science

    Gene

    Research And Development

    Cell

    Geography

    Asia100%

    Registration Source

    Website Visitors100%

    Job Title

    Research Scientist50%

    Medical Laboratory Technician50%

    Organization

    Clinical Laboratory50%

    Manufacturer - Other50%


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