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UC San Francisco Previously Published Works UCSF UC San Francisco Previously Published Works Title Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface. Permalink https://escholarship.org/uc/item/1291c90f Journal Oncotarget, 7(52) ISSN 1949-2553 Authors Ye, Xiaoying Chan, King C Waters, Andrew M et al. Publication Date 2016-12-01 DOI 10.18632/oncotarget.13566 Peer reviewed eScholarship.org Powered by the California Digital Library University of California www.impactjournals.com/oncotarget/ Oncotarget, 2016, Vol. 7, (No. 52), pp: 86948-86971 Research Paper Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface 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: [email protected] 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. www.impactjournals.com/oncotarget 86948 Oncotarget INTRODUCTION This approach resulted in the identification of cell- surface proteins that have not previously been linked to The causal role of KRas mutants in human cancers constitutive KRas activation, along with proteins already was established three decades ago [1]. Since then, our described in the context of cancer cell lines expressing understanding of the role of constitutively activated KRas KRas mutants. Results from this investigation provide signaling in tumorigenesis and cancer cell biology has further insights into KRas-mediated tumorigenesis and significantly increased. However, this knowledge has not offer potential novel targets residing at the surface of been translated into effective treatment of cancers driven cells bearing oncogenic Ras. In addition, this proteomic by oncogenic KRas, which includes approximately 95% platform permits direct quantitative measurements and of pancreatic and approximately 40% of colon and lung large-scale investigation of signaling pathways using carcinomas [2]. Notably, there are still no FDA-approved advanced bioinformatic tools to process data acquired drugs capable of directly targeting oncogenic KRas. [3] at the ultimate bio-effector (i.e., protein) level, including Thus, direct inhibition of the oncogenic KRas and/or any information related to subcellular location (e.g., cell of the downstream effectors remains a key goal in current surface) and post-translational modifications (e.g., cancer research. glycosylation). It is well accepted that proteins residing at the cell surface of cancerous cells are easily accessible targets for both biologics (e.g., trastuzumab) [4] and/or small RESULTS molecules (e.g., lapatinib) [5]. Notably, it has been shown that the differences in cell-surface protein expression are Scanning electron microscopy of KRasG12V- cancer- and/or cell-type specific and are reflective of their transfected MCF10A cells revealed phenotypic molecular signature/phenotype [6]. Hence, identification changes typical of transformed cells of differentially expressed proteins on the surface of cancer cells expressing oncogenic KRas may provide At the outset, we carried out a comparative scanning distinct opportunities for translation of these findings into electron microscopy (SEM) analysis of MCF10A- innovative treatments [7] aimed directly at proteins unique KRasG12V and control MCF10A cells virally transduced to or exceedingly upregulated on the KRas surface. with empty vector (MCF10A-EV) to examine the Recent strides in cancer immunotherapy, particularly extent and nature of changes in cell-surface morphology antibody-based treatments targeting proteins at the surface secondary to the oncogenic KRas activation. SEM has of cancerous cells [8, 9], further underscore the need for been frequently used to study the morphology of the a comprehensive map of the KRas surface. Likewise, surface of cultured cells [20, 21]. The SEM analysis successes in targeting MHC-I and/or MHC-II peptides revealed altered morphology of the MCF10A-KRasG12V via adoptive T-cell immune-therapy [10] or virus-related cells characterized by spindle-shaped bodies and multiple proteins via prophylactic cancer vaccines [11] accentuate cell-surface protrusions that are consistent with cellular the urgent need for developing technologies capable of in- protrusions formation (Figure 2A). These findings support depth profiling of the surface of in vitro cultured cancer increased mobility/invasion capabilities and are suggestive cells [12] and/or in their natural tissue microenvironment of epithelial-to-mesenchymal transformation (EMT) [22]. in vivo [13]. As a part of the NCI’s RAS initiative, one On the contrary, the surface of control MCF10A-EV project at the Frederick National Laboratory for Cancer cells showed flat “cobblestoned” surfaces and exhibited a Research (FNLCR) utilizes mass spectrometry (MS)- globule-shaped nucleus visible in the cell center, features based proteomics to identify/characterize proteins found of well-differentiated non-malignant epithelial cells on the surface of cancer cells bearing oncogenic KRas. (Figure 2A) [22]. In addition, we observed that MCF10A- FNLCR has pioneered methods for profiling cell-surface KRasG12V cells form spheres (Figure 2B) if grown in high proteins in cell lines and tissue specimens [14–18]. densities. This feature was absent during the culture of Here, we describe a liquid chromatography (LC) MCF10A-EV and parental MCF10A-ATCC cells, which MS-based proteomic approach for molecular phenotyping formed a monolayer (Figure 2B). of the KRasG12V surface using MCF10A-KRasG12V cells as a model of oncogenic KRas transformation. To obtain a Phenotypic cancer cell assays disclosed features detailed map of the KRasG12V surface, we applied targeted consistent with EMT-like driven malignant glycoprotein labeling using hydrazide-based cell surface transformation of MCF10A-KRasG12V cells capturing (CSC) technology [12] and global shotgun membrane (SGM) proteomics [19] to procure a broad Next, we carried out phenotypic cell assays to molecular profile of the surface of MCF10A-KRasG12V and investigate changes secondary to KRasG12V transfection MCF10A-EV cells (Figure 1). of epithelial MCF10A cells. Phenotypic cancer cell assay www.impactjournals.com/oncotarget 86949 Oncotarget screens are commonly used in the process of cancer drug Profiling the cell surface glyco-proteome of the discovery [23]. In comparison to MCF10A-EV cells, MCF10A-KRasG12V cells using MS-based cell the KRasG12V transfected cells showed an increase in surface proteomics invasion, migration, and anchorage independent growth (Supplementary Figure S1A–S1C). Amplified migration To identify
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