Abstract
HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO(2) chromatography utilizing a dual trap configuration, ~1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.
Highlights
HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases
The resistant cell lines retained a dependence on HER2 signaling and were growthinhibited by the HER2 tyrosine kinase inhibitor lapatinib (32) and by HER2 siRNA
The isoelectric focusing (IEF) fractions were loaded on the LC-MS system, and a second phospho-enrichment was performed using a dual-trapping method employing titanium dioxide (TiO2) and reversed phase packing materials (45)
Summary
HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases. Multiple trastuzumab-resistance mechanisms have been identified, and they alter signaling networks and protein phosphorylation patterns in either a direct or an indirect manner. These mechanisms can be grouped into three categories. Given that multiple trastuzumab-resistance mechanisms alter signaling networks and protein phosphorylation patterns, we reasoned that mapping phosphotyrosine signaling networks using quantitative proteomics would be a highly useful strategy for analyzing known mechanisms and identifying novel mechanisms of trastuzumab resistance. Multiple other tyrosine kinase signaling networks were analyzed using quantitative proteomics, including Ephrin receptor, EphB2 (26 –28), platelet-derived growth factor receptor (PDGFR) (21), insulin receptor (29, 30), and the receptor for hepatocyte growth factor, c-MET (31). This screen functionally tested the role of the identified proteins and identifies which proteins might have the largest effect on reversing trastuzumab resistance
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