Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition - PubMed (original) (raw)

. 2012 May;11(5):1071-81.

doi: 10.1158/1535-7163.MCT-11-0852. Epub 2012 Mar 12.

Vitor M Faca, Lindsey D Hughes, Wenxuan Zhang, Qiaojun Fang, Babak Shahbaba, Roland Luethy, Jonathan Erde, Joanna Schmidt, Sharon J Pitteri, Qing Zhang, Jonathan E Katz, Mitchell E Gross, Sylvia K Plevritis, Martin W McIntosh, Anjali Jain, Samir Hanash, David B Agus, Parag Mallick

Affiliations

Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition

Kian Kani et al. Mol Cancer Ther. 2012 May.

Abstract

Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response.

©2012 AACR

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Figures

Figure 1

Figure 1. Sub-proteome analysis of A431 cells with and without gefitinib treatment

A431 cells were maintained in SILAC media and treated with 100 nM gefitinib for either 2 or 16 hours and analyzed by LC-MS/MS. Cell-surface proteins were isolated by biotin labeling and subsequently captured by avidin chromatography. Solid-phase extraction of glycoproteins was used to enrich in shed proteins. A) Chemical structure of gefitinib (top) and erlotinib (bottom). B) Total protein identifications based on compartment with a PeptideProphet score ≥ 0.9 and ProteinProphet score of ≥ 0.90. C) Histogram of (log2) fold change versus unique protein identifications for the 2 hr and 16 hr treatments shows quantitative changes in A431 proteomes were greater after 16 hours.

Figure 2

Figure 2. In vitro verification of SILAC ratios

A) Fold change of candidate biomarkers is given as a function of compartment of identification with 16 hour gefitinib treatment. Proteins identified by cell-surface capture are depicted by rounded rectangles while proteins identified in the whole cell lysate are ellipses. Normalized fold change for each protein is given numerically and by the grayscale heat-map. (B) A431 cells were treated with vehicle control, 100nM, 500nM, and 1,000nM gefitinib for 16 hours. Densitometry analysis for three independent biological replicate experiments are listed below each immunoblot.

Figure 3

Figure 3. A431 tumors are responsive to gefitinib in vivo

A) Tumor growth curve for A431 xenografts in animals treated with 50 mg/kg gefitinib or left untreated and tumor growth curve for gefitinib-resistant tumors (A431-ZDR) in animals treated with 50 mg/kg gefitinib or left untreated. B) Tumor lysates were generated from A431 xenografts and analyzed by immunoblot for the 16 proteins in the panel. Lysates were fractionated to enrich for soluble and insoluble proteins.

Figure 4

Figure 4. Analysis of panel proteins from serum of animals bearing gefitinib-sensitive and gefitinib-resistant tumors

Sera were collected from naïve mice and mice implanted with A431 cells and A431-ZDR tumors and analyzed by immunoblot for the 16 proteins in our panel. Seven of the 16 proteins were detectable in sera.

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