Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways

// Xinyan Wu 1, 2, * , Muhammad Saddiq Zahari 1, 2, * , Binyun Ma 6 , Ren Liu 6 , Santosh Renuse 1, 2, 5 , Nandini A. Sahasrabuddhe 1, 2, 5 , Lily Chen 3 , Raghothama Chaerkady 1, 2 , Min-Sik Kim 1, 2 , Jun Zhong 1, 2 , Christine Jelinek 1, 2 , Mustafa A. Barbhuiya 1, 2, 5 , Pamela Leal-Rojas 1, 2, 7 , Yi Yang 1, 2 , Manoj Kumar Kashyap 1, 2, 5 , Arivusudar Marimuthu 1, 2, 5 , Min Ling 1 , Mary Jo Fackler 3 , Vanessa Merino 3 , Zhen Zhang 3 , Cynthia A. Zahnow 3 , Edward Gabrielson 3, 4 , Vered Stearns 3 , Juan Carlos Roa 8 , Saraswati Sukumar 3 , Parkash S. Gill 6 , Akhilesh Pandey 1, 2, 3, 4 1 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, USA 2 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, USA 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, USA 4 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, USA 5 Institute of Bioinformatics, International Technology Park, Bangalore, India 6 Department of Medicine, University of Southern California, Los Angeles, USA 7 Department of Pathology, Center of Genetic and Immunological Studies (CEGIN) and Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile 8 Advanced Center for Chronic Diseases (ACCDiS), Department of Pathology Pontificia Universidad Catolica de Chile, Santiago, Chile * These authors have contributed equally to this work Correspondence to: Akhilesh Pandey, e-mail: pandey@jhmi.edu Keywords: triple negative breast cancer, protein phosphorylation, kinase, AXL, proteomics Received: March 25, 2015 Accepted: August 24, 2015 Published: September 03, 2015 ABSTRACT Breast cancer is the most prevalent cancer in women worldwide. About 15–20% of all breast cancers are triple negative breast cancer(TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXLexpression. High levels of AXLexpression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXLwith a humanized AXLantibody inhibited cell proliferation and migration in vitro , and tumor growth in mice. Overall, our global phosphoproteomicanalysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXLin TNBC.