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Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-Type Non−Small Cell Lung Carcinoma Cell Lines

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Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-Type Non−Small Cell Lung Carcinoma Cell Lines Published OnlineFirst January 31, 2013; DOI: 10.1158/1535-7163.MCT-12-0880 Molecular Cancer Companion Diagnostics & Biomarkers Therapeutics Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-Type Non–Small Cell Lung Carcinoma Cell Lines Angelique Augustin1, Jens Lamerz2,Hel ene Meistermann1, Sabrina Golling1, Stefan Scheiblich4, Johannes C. Hermann5, Guillemette Duchateau-Nguyen2, Manuel Tzouros1, David W. Avila1, Hanno Langen1, Laurent Essioux2, and Barbara Klughammer3 Abstract Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), a-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATP- binding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors. Mol Cancer Ther; 12(4); 520–9. Ó2013 AACR. Introduction cially potent inhibitors of activating mutation-positive Over the past few years, several drugs targeting protein EGFR, erlotinib has also shown efficacy versus placebo kinases have shown clinical efficacy as cancer therapy (1). in EGFR wild-type NSCLC (2, 3, 7, 8). Among these, erlotinib and gefitinib are first-generation To determine whether differences in clinical outcomes tyrosine-kinase inhibitors (TKI) that directly inhibit the seen in patients with EGFR wild-type tumors receiving EGF receptor (EGFR; refs. 2–5). Recent clinical data have erlotinib or gefitinib can be attributed to differences in the shown that patients with non–small cell lung carcinoma protein interaction profiles of these 2 compounds, a quan- (NSCLC) whose tumors harbor an activating EGFR muta- titative chemical proteomics study was conducted. Chem- tion respond especially well to treatment with these TKIs ical proteomics is a mass spectrometry–based affinity (3, 4, 6). Although both TKIs target EGFR and are espe- chromatography approach, which uses immobilized small molecules as bait to capture and identify interacting protein complexes from an entire proteome. This tech- Authors' Affiliations: 1Protein and Metabolite Biomarkers, 2Bioinformat- nique has been successfully applied to explore the mode ics and Exploratory Data Analysis, Translational Research Sciences, and of action of kinase inhibitors and to assess naturally 3Tarceva Clinical Biomarker Subteam, Pharmaceuticals Division, F. Hoff- mann-La Roche Ltd., Basel, Switzerland; 4Discovery Research Oncology, occurring small molecules to identify their targets (9, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Penzberg, Ger- 10). To gain specificity by filtering out background binders many; and 5Discovery Chemistry, Pharmaceuticals Division, F. Hoff- generated by low-affinity abundant proteins, the work- mann-La Roche Ltd., Nutley, New Jersey flow is best accompanied with a competition-binding step Note: Supplementary data for this article are available at Molecular Cancer that involves free compound pretreatment of the probed Therapeutics Online (http://mct.aacrjournals.org/). mixture. Here, competition-binding experiments and Corresponding Authors: Angelique Augustin, Translational Research label-free liquid chromatography/mass spectrometry Sciences, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Grenza- cherstrasse 124, 4070 Basel, Switzerland. Phone: 41-61-688-4050; Fax: (LC/MS) signal detection were used to identify protein 41-61-688-1929; E-mail: [email protected]; and Barbara complexes that can be specifically displaced from an Klughammer, [email protected] affinity matrix by unmodified erlotinib or gefitinib. This doi: 10.1158/1535-7163.MCT-12-0880 reveals a potential complementary mode of action for Ó2013 American Association for Cancer Research. erlotinib in the context of epithelial-to-mesenchymal 520 Mol Cancer Ther; 12(4) April 2013 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst January 31, 2013; DOI: 10.1158/1535-7163.MCT-12-0880 Chemical Proteomics Differentiates Erlotinib from Gefitinib transition (EMT). EMT is a known mechanism through instructions. Binding efficiency was estimated by com- which epithelial cells lose adhesion molecules such as E- paring the optical densities of the compound solutions cadherin and acquire a mesenchymal phenotype, which before and after coupling at the maximal absorbance allows them to migrate into the stroma and become wavelengths 247 and 330 nm. Cells were lysed in 50 invasive (11). The correlation between erlotinib sensitivity mmol/L HEPES pH 7.3 buffer containing 150 mmol/L and EMT has been shown previously in vitro and in vivo NaCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 1% NP-40, (12, 13) and suggests that patients with epithelial-type protease inhibitors (Complete EDTA free; Roche Applied NSCLC tumors, that is, which had not undergone the Sciences), and 1 mmol/L orthovanadate; samples were transition, could have a better outcome with an erlotinib- cleared by centrifugation at 12,000 Â g for 15 minutes at containing regimen (12). The complex formed by integrin- 4C. Cell lysates were preincubated with increasing con- linked kinase (ILK), a-parvin, and PINCH is bound to centrations (0, 0.0856, 0.2862, 0.957, 3.2, 10.7, 35.78, 119.6, erlotinib but not gefitinib in the chemical proteomics and 400 mmol/L final concentration) of unmodified erlo- profiling of several EGFR wild-type NSCLC cell lines. tinib or gefitinib for 1 hour at 4C under rotation. The This complex, also known as ILK–PINCH–Parvin com- pretreated samples were loaded onto Sepharose beads plex (IPP) complex, forms a structural and signaling entity immobilized with the respective compound (erlotinib or in the integrin pathway (14) and has been shown to gefitinib) for 3 hours at 4C under rotation. Beads were influence EMT. ILK and PINCH overexpression are able washed 3 times with lysis buffer, treated with SDS-PAGE to trigger EMT, by inducing loss of E-cadherin (15, 16). buffer for 5 minutes at 85C, and eluted proteins separated TGF-b-1–induced EMT in mammary epithelial cells on 4% to 20% Tris–glycine SDS-PAGE. After protein required functional ILK (17). The next steps were to fixation in 40% ethanol and 10% acetic acid, gels were investigate a potential structural explanation for this stained overnight with Coomassie Brilliant blue. For pro- binding discrepancy between the 2 compounds, as well tein in-gel digestion, an adapted protocol of Shevchenko as using an EMT-inducible cellular model to assess their and colleagues (21) was used. In brief, each gel lane was respective functional effect on EMT. These findings were cut into 5 slices between the 20 and 150 kDa region. further correlated with gene expression data from the Proteins were reduced with 50 mmol/L dithioerythritol MERIT open-label, multicenter, phase II clinical trial, for 45 minutes at 56C, alkylated with 55 mmol/L iodoa- which aimed to identify genes with potential as biomar- cetamide for 1 hour at room temperature in the dark, and kers for clinical benefit from erlotinib therapy (18). digested with trypsin (Promega) overnight at room tem- perature. Peptides were extracted twice with 1:2 (v/v) Materials and Methods acetonitrile/25 mmol/L ammonium bicarbonate, and 1:2 (v/v) acetonitrile/5% formic acid, respectively, for 15 Reagents and cell lines a minutes at 37 C, dried down using a speedvac, and stored Anti-ILK, E-cadherin, and -parvin were obtained from at À20C before analysis. Samples were reconstituted in Cell Signaling Technology, anti-PINCH1 and glyceralde- 2% acetonitrile/5% formic acid and run in triplicate by hyde-3-phosphate dehydrogenase–horseradish peroxi- LC/MS. Nanoflow liquid chromatography/tandem mass dase (GAPDH-HRP) from Santa Cruz Biotechnology, and spectrometry (LC/MS-MS) was carried out by coupling anti-vimentin from BD Biosciences. All cell lines were an Easy-nLC system (Proxeon) to an LTQ Orbitrap Velos obtained from the American Type Culture Collection (Thermo Fisher Scientific) equipped with a Proxeon (ATCC) without further authentication. Cell cultures nanoelectrospray and an active background ion reduction H292, H226, H322, H358, H441, H460, and H1299 were device (ABIRD; ESI Source Solutions). Peptides were maintained in RPMI-1640, and Calu6 in EMEM; both loaded on a 10-mm Aqua C18 trapping (packed
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