Abstract A7: Phosphoproteomics reveals novel targets in hemangiosarcoma
Authors: Helfand Stuart C. , Marley Kevin 
Published: June 2012

Abstract
Introduction: The purpose of this study was to elucidate the phosphotyrosine signature of malignant endothelial cells in order to identify novel activated tyrosine kinases. This information is important for developing rationally designed strategies intended to inhibit angiogenesis by targeting with tyrosine kinase inhibitors. It is also relevant for designing improved treatment of endothelial malignancies such as angiosarcoma that are highly resistant to conventional chemotherapy and carry a poor prognosis. Use of a phosphoproteomic approach facilitates broad screening of the kinome and may be able to identify activated kinases that can be targeted as new kinase inhibitors are developed. Hemangiosarcoma is an aggressive endothelial cell malignancy in the dog that arises from transformed bone marrow-derived hemangioblasts. Tumor cells have been validated to be similar to primitive, mitotically active endothelial cells and are therefore useful as a tool for angiogenesis research and development of novel therapies for malignancies with a similar histogenesis.
Methods: A validated canine hemangiosarcoma cell line, SB-HSA, was analyzed using a proteomic method to screen peptides containing phosphotyrosine. Cellular proteins were extracted and digested with trypsin. Peptides were purified and phosphotyrosine containing peptides were enriched using an immunoaffinity method. The collected peptides were dried and subjected to mass spectrometry analysis using electrospray ionization MS/MS. Up to 3 precursor ions per scan were chosen for MS/MS analysis. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.1.04) and X! Tandem (version 2006.04.01.2). Mascot was set up to search the SwissProt database (version 20060530, selected for mammals) assuming the digestion enzyme trypsin. X! Tandem used the same database without species limitation (222289 entries). Western blot was used to confirm identification of phosphorylated tyrosine kinases and kinase inhibition confirmed activity.
Results: Mass spectrometry analysis revealed the presence of at least 25 different peptides containing phosphotyrosine. Most shared identity with sequences derived from tyrosine kinases that affect cell cycle function, growth factor response, extracellular matrix interaction, motility, and others. Several adapter proteins, such as paxillin, which is phosphorylated by focal adhesion kinase (FAK) and Src upon integrin engagement or growth factor stimulation, were also identified. Phosphorylated FAK was detected. Consistent with its endothelial derivation, SB-HSA cells were found to contain phosphorylated forms of platelet endothelial cell adhesion molecule and vascular endothelial growth factor receptor 2. Western blot confirmed pY-FAK, and FAK inhibition by the inhibitor FAK-14 indicated a role for FAK in proliferation and migration of SB-HSA cells.
Conclusions: Phosphoproteomic screening can provide clues about proteins and pathways that may be activated in malignant endothelial cells such as hemangiosarcoma. It also provided information about proteins, such as FAK, that can be targeted in endothelial cells with tyrosine kinase inhibitors, through holistic analysis of phosphorylated signaling networks.
Citation Information: Clin Cancer Res 2010;16(14 Suppl):A7.