1 Quantitative Chemical Proteomics Profiling Differentiates Erlotinib From

Author Manuscript Published OnlineFirst on January 31, 2013; DOI: 10.1158/1535-7163.MCT-12-0880 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from

Gefitinib in EGFR Wild-type Non-small Cell Lung Carcinoma Cell Lines

Angélique Augustin1, Jens Lamerz2, Hélène Meistermann1, Sabrina Golling1, Stefan

Scheiblich3, Johannes C. Hermann4, Guillemette Duchateau-Nguyen2, Manuel Tzouros1,

David W. Avila1, Hanno Langen1, Laurent Essioux 2, Barbara Klughammer5

1Protein and Metabolite Biomarkers, Translational Research Sciences, Pharmaceuticals

Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland; 2Bioinformatics and Exploratory

Data Analysis, Translational Research Sciences, Pharmaceuticals Division, F.

Hoffmann-La Roche Ltd, Basel, Switzerland; 3Discovery Research Oncology,

Pharmaceuticals Division, F. Hoffmann-La Roche Ltd, Penzberg, Germany; 4Discovery

Chemistry, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd, Nutley, USA; 5Tarceva

Clinical Biomarker Subteam, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd,

Basel, Switzerland

Running title: Chemical proteomics differentiates erlotinib from gefitinib

Keywords: non-small cell lung cancer (NSCLC); erlotinib; gefitinib; chemical proteomics;

epithelial–mesenchymal transition (EMT)

Corresponding Authors

Dr Angélique Augustin, Translational Research Sciences, Pharmaceuticals Division, F.

Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland. Phone: +41

61 688 4050; Fax: +41 61 688 1929; e-mail: [email protected]

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 31, 2013; DOI: 10.1158/1535-7163.MCT-12-0880 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Dr Barbara Klughammer, Tarceva Clinical Biomarker Subteam, Pharmaceuticals

Division, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland.

Phone: +41 61 688 63 86; Fax: +41 61 688 1370; e-mail:

[email protected]

Conflict of interest: All authors are employees of F. Hoffmann-la Roche Ltd.

Word count:

Number of figures: 6 (plus one supplementary figure, see separate document)

Number of tables: 2 supplementary tables (see separate document)

Abstract

Although both erlotinib and gefitinib target the epidermal growth factor 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

performed. 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), α-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.

Introduction

Over the past few years, several drugs targeting protein kinases have

demonstrated clinical efficacy as cancer therapy (1). Among these, erlotinib and gefitinib

are first generation tyrosine-kinase inhibitors (TKIs) that directly inhibit the epidermal

growth factor receptor (EGFR) (2–5). Recent clinical data have shown that patients with

non-small cell lung carcinoma (NSCLC) whose tumors harbor an activating EGFR

mutation respond especially well to treatment with these TKIs (3, 4, 6). Although both

TKIs target EGFR and are especially potent inhibitors of activating mutation-positive

EGFR, erlotinib has also shown efficacy versus placebo in EGFR wild-type NSCLC (2, 3,

7, 8).

To determine whether differences in clinical outcomes seen in patients with

EGFR wild-type tumors receiving erlotinib or gefitinib can be attributed to differences in

the protein interaction profiles of these two compounds, a quantitative chemical

proteomics study was performed. Chemical proteomics is a mass spectrometry (MS)-

based, affinity chromatography approach, which uses immobilized small molecules as

bait to capture and identify interacting protein complexes from an entire proteome. This

technique has been successfully applied to explore the mode of action of kinase

inhibitors, and to assess naturally-occurring small molecules to identify their targets (9,

10). To gain specificity by filtering out background binders generated by low-affinity

abundant proteins, the workflow is best accompanied with a competition-binding step

that involves free compound pre-treatment of the probed mixture. Here, competition-

binding experiments and label-free liquid chromatography (LC)-MS signal detection were

used in order to identify protein complexes that can be specifically displaced from an

affinity matrix by unmodified erlotinib or gefitinib. This reveals a potential complementary

mode of action for erlotinib in the context of epithelial to mesenchymal transition (EMT).

EMT is a known mechanism through which epithelial cells lose adhesion molecules like

E-cadherin and acquire a mesenchymal phenotype, which allows them to migrate into

the stroma and become invasive (11). The correlation between erlotinib sensitivity and

EMT has been demonstrated previously in vitro and in vivo (12, 13) and suggests that

patients with epithelial-type NSCLC tumors i.e. which had not undergone the transition,

could have a better outcome with an erlotinib-containing regimen (12). The complex

formed by integrin-linked kinase (ILK), α-parvin, and PINCH is bound to erlotinib but not

gefitinib in the chemical proteomics profiling of several EGFR wild-type NSCLC cell lines.

This complex, also known as IPP complex, forms a structural and signaling entity in the

integrin pathway (14) and has been shown to influence EMT. ILK and PINCH over-

expression are able to trigger EMT, by inducing loss of E-cadherin (15, 16). TGFβ-1-

induced EMT in mammary epithelial cells required functional ILK (17). The next step was

to investigate a potential structural explanation for this binding discrepancy between the

two compounds, using an EMT-inducible cellular model to assess their respective

functional effect on EMT. These findings were further correlated with gene expression

data from the MERIT open-label, multicentre, phase II clinical trial, which aimed to

identify genes with potential as biomarkers for clinical benefit from erlotinib therapy (18).

Materials and Methods

Reagents and cell lines

Anti-integrin linked kinase (ILK), E-cadherin and α-parvin were obtained from Cell

Signaling Technology, anti-PINCH1 and GAPDH-HRP from Santa Cruz Biotechnology,

and anti-vimentin from BD Biosciences. All cell lines were obtained from the American

Type Culture Collection (ATCC) without further authentication. Cell cultures H292, H226,

H322, H358, H441, H460, and H1299 were maintained in RPMI 1640, and Calu6 in

EMEM; both supplemented with 10% fetal bovine serum (FBS).

Chemical proteomics profiling in H292 cells

Erlotinib and gefitinib were modified with a 6-(3-aminopropyloxy) side chain for

covalent linking to epoxy-activated Sepharose® 6B beads (GE Healthcare, Stockholm,

Sweden) to generate affinity matrices, as described by Brehmer, et al. (19). Starting

material 4-[(3-ethynylphenyl)amino]-7-(2-methoxyethoxy)-6-quinazolinol was prepared

as described in WO9630347A1. Coupling of the derivatives to the epoxy-activated beads

was done in 50% dimethylformamide (DMF)/0.1 M Na2CO3 pH 11 in the presence of 10

mM NaOH, overnight at room temperature in the dark. After washing with 50% DMF/0.1

M Na2CO3, any remaining reactive groups were blocked with 1 M ethanolamine.

Subsequent washing steps were performed according to the manufacturer’s instructions.

Binding efficiency was estimated by comparing the optical densities of the compound

solutions before and after coupling at the maximal absorbance wavelengths 247 nm and

330 nm. Cells were lysed in 50 mM Hepes pH 7.3 buffer containing 150 mM NaCl, 1 mM

CaCl2, 1 mM MgCl2, 1% NP-40, protease inhibitors (Complete™ EDTA free, Roche

Applied Sciences), and 1 mM orthovanadate; samples were cleared by centrifugation at

12,000 g for 15 minutes at 4 °C. Cell lysates were pre-incubated with increasing

concentrations (0, 0.0856, 0.2862, 0.957, 3.2, 10.7, 35.78, 119.6 and 400 μM final

concentration) of unmodified erlotinib or gefitinib for 1 hour at 4 °C under rotation. The

pre-treated samples were loaded onto Sepharose® beads immobilized with the

respective compound (erlotinib or gefitinib) for 3 hours at 4 °C under rotation. Beads

were washed three times with lysis buffer, treated with sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) buffer for 5 minutes at 85 °C, and

eluted proteins separated on 4–20% Tris-Glycine SDS-PAGE. After protein fixation in

40% ethanol and 10% acetic acid, gels were stained overnight with Coomassie Brilliant

blue. For protein in-gel digestion, an adapted protocol of Shevchenko et al. (20) was

used. In brief, each gel lane was cut into five slices between the 20 and 150 kDa region.

Proteins were reduced with 50 mM dithioerythritol for 45 minutes at 56 °C, alkylated with

55 mM iodoacetamide for 1 hour at room temperature in the dark, and digested with

trypsin (Promega) overnight at room temperature. Peptides were extracted twice with 1:2

(v/v) acetonitrile/25 mM ammonium bicarbonate, and 1:2 (v/v) acetonitrile / 5% formic

acid, respectively, for 15 minutes at 37 °C, dried down using a speedvac, and stored at -

20 °C prior to analysis. Samples were re-constituted in 2% acetonitrile/5% formic acid

and run in triplicate by Liquid Chromatography-Mass Spectrometry (LC-MS). Nanoflow

LC-MS/MS was performed by coupling an Easy-nLC system (Proxeon, Odense,

Denmark) to an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany)

equipped with a Proxeon nanoelectrospray and an active background ion reduction

device (ABIRD; ESI Source Solutions, Woburn, MA). Peptides were loaded on a 10 mm

Aqua C18 trapping (packed in-house, 100 μm i.d.) and separated on a 200 mm Reprosil-

Pur C18-AQ analytical (Dr. Maisch GmbH, Ammerbuch, Germany) column (75 μm i.d.,

3 μm particle size, 120 Å). Individual raw data files were processed with SEQUEST

(version 27.0, revision 12, Thermo Fisher Scientific), and searches were performed

against a concatenated forward/reverse human UniProt/SwissProt database (June 2009

release, 34,275 entries, containing splice-variants) allowing for a spectrum false-

discovery rate (FDR) of 2.5%. A mass tolerance of 5 ppm for precursor and 1.0 Da for

fragment ions was set. Oxidized methionines (+15.9949 Da) and carbamidomethylated

cysteines (+57.0215 Da) were considered as differential and fixed modifications,

respectively. Only fully tryptic peptides with not more than one mis-cleavage were

considered for data analysis. Detailed LC-MS and data analysis procedure can be found

in supplemental text.

Chemical proteomics profiling of erlotinib and gefitinib in other NSCLC cell lines

H358, H322, H226 and Calu6 cells were lysed in 50 mM Hepes pH 7.3 buffer

containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% NP-40, protease inhibitors

(Complete™ EDTA free, Roche Applied Sciences), 1 mM orthovanadate, and samples

were cleared by centrifugation at 12,000g for 15 minutes at 4 °C. Cell lysates were

loaded on erlotinib-Sepharose® and gefitinib-Sepharose®, using the same experimental

conditions as in the profiling experiment described above but without competing with the

unmodified erlotinib or gefitinib. The elution fractions were analyzed by LC-MS. Spectral

counts corresponding to EGFR, GAK, LOK (Stk10), Abl2, SLK MEKK1 (M3K1), ILK, α-

parvin (PARVA) and PINCH1 (LIMS1) were extracted and normalized towards the sum

of all spectral counts corresponding to all proteins identified in the respective fractions.

Alignment of EGFR and ILK crystal protein structures

Publicly available crystal structure protein data bank (pdb) files of EGFR-bound

erlotinib or gefitinib and the crystal structure of ILK complexed with adenosine

triphosphate (ATP) were downloaded from the crystallographic database (21) (pdb

codes 1M17, 2ITY, 3KMW; [(22); (23); (24)]). The three kinase structures were aligned

using the coordinates from Cα atoms of a subset of the residues forming the ATP

binding site (residue numbers based on EGFR numbering 691–693, 701–703, 719–72

(20) 1, 751, 766–772, 820–823, 831–832). Alignment and distance measurements were

performed using the PyMOL visualization package (25).

Testing EMT on H358 NSCLC cell line

H358 cells were treated with recombinant 10 ng/mL TFGβ3 (Peprotech, New

York, NY, USA) and 5 μM erlotinib or gefitinib for 3, 6, 10 and 13 days. Cell culture

medium was renewed every 3 days. Cell lysis was performed in phosphate-buffered

saline (PBS) containing 1% NP-40 and protease inhibitors. After clearing by

centrifugation at 12,000g for 15 minutes at 4 °C, proteins were separated by SDS-PAGE

and processed for Western blot analysis. Images were acquired using a compact digital

camera (Luminescent Image Analyzer, Fujifilm, Tokyo, Japan) and quantified with

ImageQuant TL (GE Healthcare, Waukesha, WI, USA). Statistical analysis was

performed using JMP 8.0.2 (SAS Institute Inc., Cary, NC, USA). To determine the

influence of erlotinib and gefitinib on ILK and PINCH after TGFβ stimulation, the

following model was applied: ExpressionD13 ~ Treatment + ExpressionD3. For analysis of

IPP expression in NSCLC cell lines, simple t-tests were performed.

Analysis of MERIT data

Gene expression profiles were measured in tumor biopsy samples from 102

patients with stage IIIB/IV NSCLC, being treated with erlotinib (150 mg/day). Tumor

biopsies were analyzed using gene expression profiling with Affymetrix GeneChip

Human Genome U133A Array (Affymetrix, Santa Clara, CA, USA). All gene expression

results described here (fold changes and p values) are derived from the statistical

analysis already reported (18). We specifically looked at differences between gene

expression profiles of patients with and without clinical benefit from erlotinib. Clinical

benefit was defined as an objective response (complete responses or partial responses

were confirmed by repeated assessments ≥4 weeks apart at any time during the

treatment period) or maintenance of stable disease for ≥12 weeks after study entry.

The following 21 proteins, potentially involved in transcriptional regulation of EMT or in

IPP complex, were mapped to the corresponding genes and microarray probe sets:

Twist, Snail [Sma1], Zeb [TCF8], Lef-1, E12/E47 basic Loop helix [bHLH], Slug [Sma2],

E-cadherin [CDH1], N-cadherin [CDH2], Myc, Vimentin, Catenin beta, Catenin delta,

Fibronectin, Epimorphin, ILK, α-Parvin, β-Parvin, PINCH 1, PINCH 2, metastasis-

associated protein 3 [MTA3], and Y box binding protein 1 [YB-1].

Results

Interaction profile of erlotinib and gefitinib in H292 NSCLC cell line

A quantitative chemical proteomics method was applied to erlotinib and gefitinib

(Fig. 1A) using the same wild-type EGFR-expressing NSCLC cell line H292 as the

biological target to identify the interaction profiles of the compounds. Cell lysates were

pre-incubated with increasing concentrations of unmodified erlotinib or gefitinib before

fractionation on the respective affinity matrix, captured proteins were eluted, separated

by SDS-PAGE and identified by LC-MS and protein database mining (Supplementary

Fig. S1). A total of 1,285 and 1,303 protein groups were identified in the bound fractions

to erlotinib and gefitinib, respectively (Supplementary Table S1). A linear model (26, 27)

was applied on mass spectrometry (MS) signals to derive concentration-dependent

signal decrease of specific binders. Comparison of the binding profiles of the two

compounds was carried out using a robust, simple, statistical procedure adapted from

Bretz and Hothorn (28) on peptide signals, and extended to multiple testing of

corresponding protein groups. Nine proteins were found with an adjusted p value below

5% in the samples displaced with erlotinib (Fig. 1B, Regions A, B and x axis;

Supplementary Table S2), and 19 in those displaced with gefitinib (Fig. 1B; Regions A, C

and y axis; Supplementary Table S2), including EGFR, which was specifically displaced

by both compounds, confirming the validity of the approach. The presence of two other

proteins, GAK and Stk10, was also significant in both compound profiles (Fig. 1B;

Region A; Supplementary Table S2), whereas NUD12, K0564, SN1L1, KC1E and

EPHA1 were only identified in the gefitinib-bound fraction (Fig. 1B; y axis;

Supplementary Table S2); M3K1 and LIMS1 only in the erlotinib-bound fraction (Fig. 1B;

x axis; Supplementary Table S2).

While the majority of proteins displaced by either erlotinib or gefitinib were protein

kinases (seven for erlotinib i.e. EGFR, GAK, STK10, ILK, SLK, ABL2, M3K1; and 11 for

gefitinib i.e. EGFR, GAK, STK10, MKNK2, RIPK2, ADCK3; MK09; EPHB2, EPHB4,

KC1E, EPHA1), a notable difference between the two compounds was seen in the non-

protein kinase binding profile. In this subgroup, erlotinib only displaced two proteins

(PARVA and LIMS1) whereas gefitinib displaced eight (NUD12; K0564; SN1L1, AP1G2,

BLMH, HEAT3, SAAL1, DFNA5). Interestingly, LIMS1 (also known as PINCH1) forms an

active complex together with PARVA (α-Parvin) and ILK (integrin-linked kinase), another

protein displaced by erlotinib.

Interaction profile of erlotinib and gefitinib in other NSCLC cell lines

We next addressed the question of whether the chemical proteomics profile was

cell line-specific by testing four additional EGFR wild-type NSCLC cell lines (H358, H322,

H226 and Calu6) for the nine erlotinib-specific binders (EGFR, STK10, GAK, ILK, LIMS1,

PARVA, SLK, M3K1, ABL2). Figure 2 shows the normalized spectral counts of these

nine proteins, including EGFR, in the bound fractions. All nine proteins found in the

erlotinib profile in H292 were also identified in the protein purifications from all four of the

other cell lines. In addition, we observed that the spectral counts of Abl2, Slk, MEKK1

(M3K1), ILK, α-Parvin (PARVA) and PINCH1 (LIMS1) in the erlotinib-bound fraction

were higher than those in the gefitinib-bound fraction. This was particularly noticeable for

the ILK-PINCH-Parvin complex (IPP), which was enriched on the erlotinib but not the

gefitinib column from these EGFR wild-type NSCLC cell lysates.

Interaction profile of erlotinib and gefitinib in the ILK DVK-loop region

Erlotinib and gefitinib have a high degree of similarity in their chemical structure

(Fig. 1A). Knowing the resolved crystal structures of EGFR and ILK (23) (24), we

examined whether the binding of erlotinib to ILK was different from gefitinib, which might

explain the discrepancies in their binding profiles towards the IPP complex. As expected,

when erlotinib or gefitinib were in a complex with EGFR, they displayed the same mode

of binding to the ATP binding sites. The overall protein conformation of EGFR-erlotinib,

EGFR-gefitinib and the ATP binding pocket was very similar, resulting in an exact

overlay of the two structures after aligning (data not shown).

The pseudokinase ILK shows some crucial differences in fold and conformation

to other kinases, including EGFR (24). However, when ignoring sequence differences,

the buried part of the ILK ATP binding site is very similar to the ATP binding site of

EGFR and allows a reliable structural alignment of ILK with EGFR when in a complex

with either erlotinib or gefitinib (Fig. 3). A crucial difference in the ATP binding site is the

conformation of the DFG loop and Ala338, the residue upstream of the DVK loop in ILK

(residues 339–341, which form the typical DFG). This residue is part of a turn that is not

observed in EGFR and is close to the space occupied by the aniline portions of erlotinib

and gefitinib.

The different substitutions in the erlotinib and gefitinib aniline rings (the 3 position

is substituted with chlorine in gefitinib but with acetylene in erlotinib and the 4 position is

unsubstituted in erlotinib but substituted with fluorine in gefitinib) and the resulting

differences in atom proximities suggest that erlotinib is more likely than gefitinib to bind

favorably to the ATP binding site of ILK.

Potential influence of erlotinib on the integrin signaling pathway, connecting to

EMT

The IPP complex functions as a hub in the integrin signaling pathway (14). It

plays a structural role by connecting integrins to the cytoskeleton, and also transduces

signals from the extracellular matrix to various intracellular pathways involved in cell

proliferation, migration and survival. In addition, overexpression of both ILK and PINCH

seems not only to follow the occurrence of EMT (15, 16), but ILK activity is also required

for TGFβ1 mediated EMT in mammary epithelial cells (17). Based on the fact that

erlotinib binds to the IPP complex, as shown in our chemical proteomics approach, we

hypothesized that in NSCLC cell lines that have not undergone EMT, erlotinib could

potentially slow down the transition, by inhibiting the IPP-mediated E-cadherin pathway.

To test this hypothesis, we used a model NSCLC cell line, H358, which had been

previously described as switching from epithelial to mesenchymal state upon TGFβ3

treatment (29). The results show a clear decrease of E-cadherin after 13 days of TGFβ3

treatment, indicating that the cells are transitioning (Fig. 4A). Treatment with erlotinib

prevents this, while gefitinib fails to inhibit the TGFβ3-mediated decrease of E-cadherin

levels.

We also examined the effects of the transition on ILK and PINCH expression (α-

Parvin could not be tested because of low detection by Western blot). Fig. 4B and 4C

show the average quantified Western blot signals from both proteins, for 3 and 13 days

of treatment. The expression of both ILK and PINCH was increased by the transition.

Both proteins gave a lower signal when cells were treated with TGFβ3 and erlotinib.

Thus, this EMT model indicates that erlotinib reduces the extent of EMT by

inhibiting the loss of E-cadherin and also seems to prevent a TGFβ3-mediated increase

of both members of the IPP complex, ILK and PINCH.

Expression of IPP complex in NSCLC cell lines

ILK and PINCH over-expression has been correlated with tumor progression

and/or aggressiveness in several human malignancies (14). At present, the three

components of the complex are studied mainly on an individual basis, not addressing the

possibility of the expression of the entire complex being correlated with EMT status.

Eight EGFR wild-type NSCLC cell lines were selected based on this status (13, 29),

which was assessed by the expression of E-cadherin as an epithelial marker and

vimentin as a mesenchymal marker. All the IPP proteins were detected, with varying

intensities, in all eight cell lines except for α-Parvin in H358 (Fig. 5A). After quantification

and normalization, the overall sum of IPP complex intensities was almost two times

higher in mesenchymal versus epithelial cells (Fig. 5B).

ILK expression in erlotinib-treated patients – data from the MERIT trial

Taking into account the data from NSCLC cell lines, and considering that the

EMT status of biopsies from NSCLC patients has been previously correlated with clinical

benefit from erlotinib treatment (12), we evaluated the potential relationship between

IPP, a subset of genes involved in EMT, and response to erlotinib. Specifically, we

looked at the differences between their expression profiles in patients with and without

clinical benefit from erlotinib.

In total, 16 out of the 21 pre-selected genes were identified with detected probe

sets and statistically analyzed (Twist; Snail (Sma1); Zeb (TCF8); Lef-1; E12/E47 basic

Loop helix (bHLH); Slug (Sma2); E-cadherin; Myc; Vimentin; Catenin beta; Catenin

delta; Fibronectin; Epimorphin; ILK; α-Parvin; β-Parvin). Data for the following five

genes: PINCH 1; PINCH 2; Metastasis associated protein 3 (MTA3); Y box binding

protein 1 (YB-1) and N-cadherin were not available. Among the 31 probe sets

(corresponding to 16 unique genes), only two were differentially expressed in tumor

samples from patients with and without clinical benefit: ILK and CDH1 (E-cadherin),

which were downregulated (0.93-fold, p=0.011) and upregulated (1.38-fold, p=0.033),

respectively, in patients with clinical benefit. It should be noted that these genes

exhibited only small trends of regulation that were not significant when a procedure of p

value adjustment for multiple testing was applied, as in the initial work from Tan et al (18)

(Fig. 6).

Discussion

In tumors dependent on an EGFR-mutated receptor, the mutation triggers a

decreased affinity of the kinase for ATP, but its affinity to erlotinib is kept intact. This,

taken together, leads to an increased sensitivity to the TKI. The difference in efficacy

between erlotinib and gefitinib has been at least partially attributed to a difference in

pharmacokinetic properties, i.e. erlotinib at 150 mg has an area under the curve seven

times’ higher than gefitinib at 250 mg. In this study, we hypothesize that an additional

activity besides the inhibition of EGFR could explain the difference in clinical benefit

seen between erlotinib and gefitinib in EGFR wild-type tumors, e.g. inhibition of ILK.

Indeed, the chemical proteomics profiling of erlotinib and gefitinib has highlighted

differences in the binding profiles of the two compounds in at least five different EGFR

wild-type NSCLC cell lines. Many kinase inhibitors, which have been approved for

clinical use, have been profiled with this method leading to the discovery of new activities,

for example on DDR1 for imatinib (30), or CAMK2G for bosutinib (31). Given the

conservation of the ATP binding site among human protein kinases, the focus had been

placed on potential binding activity with other protein kinases. Indeed Abl2, Slk and

STK10 (LOK) have been confirmed to bind to erlotinib in vitro with at least 25-fold higher

affinity than gefitinib (32). However, these assays revealed also at least 30-fold lower

affinity of erlotinib for Abl2, Slk and STK10 by comparison with EGFR. A recent study

using native kinase binding profiling in HL60 and PC3 cell lines also reported inhibitory

activities of erlotinib on STK10 (LOK), MAP3K1 (M3K1), Slk and ILK, which correlates

well with our findings (33). Further experiments would be required to confirm the effect of

erlotinib on these kinases in the appropriate in vivo background. Our data show that

substantial information can also be deduced from the non-kinase proteins that form

complexes with the direct binders. Unlike gefinitib, erlotinib interacted with the ternary

complex formed by ILK, α-Parvin and PINCH in our assay.

We propose a structural explanation for the difference in binding to IPP between

erlotinib and gefitinib. The docking of erlotinib in the three-dimensional structure of ILK

shows that erlotinib is able to bind to the ATP binding site of the pseudokinase, as

opposed to gefitinib, which is less likely to bind with high affinity. Indeed, when the EGFR

crystal structures, complexed either with erlotinib or gefitinib, are aligned with the ILK

structure, the different substitutions of the aniline moieties of the compounds seem to

have crucial implications for their potential binding to ILK. Acknowledging that the

distance measured between overlaid structures depends on the alignment method used

and may vary slightly, a few observations can be made from our structural alignment.

Gefitinib has two substitutions on the aniline ring whereas erlotinib has only one, which

points inside the ATP binding site, close to the DVK loop. The 4-position is not

substituted in erlotinib, whereas in gefitinib it features fluorine. Compared with hydrogen,

the van der Waal’s radius of the fluorine atom is larger, and the carbon–fluorine bond is

significantly longer than the carbon–hydrogen bond (34). This hydrogen-to-fluorine

substitution appears to be important, since the fluorine sits in an environment where it

clashes and causes steric repulsions with the Cβ-methyl and the carbonyl oxygen of

Ala338 (~1.5Å and ~2Å respectively; Fig. 5). Furthermore, the fluorine also creates an

unfavorable electrostatic interaction with the carbonyl oxygen of Ala338. In contrast,

erlotinib projects a slightly polarized hydrogen atom from the aniline 4-position that can

establish an attractive interaction with the carbonyl oxygen of Ala338. The repulsion from

the fluorine atom in gefitinib could either prevent it from binding to ILK with reasonable

affinity or it could trigger a rearrangement of the loop containing the DVK motif upon

binding. Since this loop is connected to the activation loop of ILK that is crucial for

binding α-Parvin (24), such a rearrangement is likely to have an influence on the ability

of ILK to form a complex with α-Parvin. More sophisticated methods such as molecular

dynamics simulations could be applied to further elucidate the favorable and unfavorable

interactions between ligands and proteins. Measurements of in vitro binding affinity of

the compounds to the purified complex will be required to experimentally validate the

results of this in silico approach.

Our study demonstrates that erlotinib prevents EMT induced by TGFβ3 in a more

efficient way than gefitinib in the H358 cellular model. As expected, this does not

influence cellular proliferation in our in vitro assays, where the different cell lines used in

this study were growth inhibited to a varying extent, but equally by both compounds as

previously reported (35, 36). EMT will be more relevant for invasive tumor growth in vivo,

and clinical studies have already shown the link between E-cadherin (CDH1) levels and

response to erlotinib (12). The retrospective analysis presented here on data from the

MERIT trial confirms that patients with higher expression of E-cadherin (CDH1) have an

increased clinical benefit from treatment with erlotinib, as previously published (18). A

similar analysis has been done on gefitinib by Kook et al. and did not show any

correlation of E-cadherin expression with response rate, or with progression-free survival

and overall survival in inoperable stage IIIB/IV NSCLC patients treated with gefitinib (37).

Interestingly, a recent study has shown a causative relation between loss of E-cadherin

and overexpression of ILK in squamous-cell carcinoma and adenocarcinoma of the lung

(38).

Additionally, in our cellular model, erlotinib showed a tendency to prevent the

over-expression of ILK and PINCH induced by TGFβ3. It should be noted that neither

erlotinib nor gefitinib have been shown to bind the TGFβ receptors in vitro (32),

excluding the possibility of a direct effect in our model on TGFβ receptor signaling. Over-

expression of both ILK and PINCH has been shown to induce EMT by lowering E-

cadherin levels (16, 39, 40). Interestingly, whereas ILK is captured in our chemical

proteomics experiments to the same extent in all the cell lines, PINCH is most enriched

in H226 and Calu6 fractions (Fig. 2), which are mesenchymal cell lines (29). Overall,

these observations indicate that erlotinib may exert a long-term effect on the expression

of ILK and PINCH, which could explain the observed consequences on the EMT. Since

the expression of both proteins is regulated, at least in part, by proteasomal degradation

(41), we could hypothesize that erlotinib would accelerate this degradation and therefore

counteract the EMT process.

The respective over-expression of ILK and PINCH has been associated with poor

prognosis in NSCLC (42–44). Although PINCH could not be monitored in the MERIT trial,

low expression of ILK seems to indicate a better benefit with erlotinib. This strengthens

our theory that in patients with a low expression of IPP, the remaining free erlotinib can

still act on the complex, whereas in cases of over-expression this effect would not be

significant.

In conclusion, we propose that, in a wild-type EGFR context, unlike gefitinib,

erlotinib targets both EGFR and the IPP complex activities, resulting in the slowing down

of the metastatic process of epithelial tumors. Further investigations on additional

models will be necessary to confirm this observation and explore the early effects of

erlotinib on the activities of the IPP complex, for example in deciphering the details of the

signaling pathway leading to hindrance of EMT.

Acknowledgements

The authors would like to thank Nikos Berntenis for providing automated sequence

interpretation platform; Michel Petrovic for automating the quantitative interpretation

platform, Peter Berndt and Wolfgang von der Saal for seminal discussions.

Funding: Support for third-party writing assistance for this manuscript was provided by

F. Hoffmann-La Roche Ltd.

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Figure and table legends

Supplementary Tables 1 and 2 (See separate document).

Figure 1A. Chemical structures of erlotinib and gefitinib.

Figure 1B. Binding profiles of erlotinib and gefitinib in H292. p values of erlotinib

versus p values of gefitinib after log10 transformation. Lines indicate 5% probability of

error. Region A shows proteins significantly binding to erlotinib and gefitinib, Regions B

and C show proteins significantly binding to erlotinib or gefitinib, respectively. Proteins in

Region D did not show significant binding to either erlotinib or gefitinib. If a protein was

identified in one compound and not in the other, it could not be quantified and hence no

p-value is available (p-value = NA). These proteins are displayed outside the two-

dimensional plot for completeness (x axis and y axis). Note: all cell lines were obtained

from the American Type Culture Collection (ATCC) without further authentication.

Figure 2. Purification of erlotinib binders in other NSCLC cell lines. Additional

chemical proteomics experiments were performed using H226, Calu6, H358 and H322

cell lysates, loaded on erlotinib-Sepharose® and gefitinib-Sepharose®. Elution fractions

from each purification were analyzed by LC-MS. The averaged normalized spectral

counts out of two biological replicates, corresponding to the nine proteins previously

identified as binders to erlotinib, are represented from the erlotinib-Sepharose® and

gefitinib-Sepharose® eluted fractions. Note: all cell lines were obtained from the

American Type Culture Collection (ATCC) without further authentication.

Figure 3. ATP binding site of aligned and overlaid ILK and EGFR crystal protein

structures showing potential interactions of erlotinib and gefitinib with the area of

the ILK DFG-loop. Oxygen atoms are colored in red, nitrogen in blue, chlorine in green

and fluorine in light cyan. Carbon atoms of the erlotinib/EGFR complex (1M17) are

colored in salmon, carbon of the gefitinib/EGFR (2ITY) complex in yellow and carbon

atoms of ILK are colored in white. Picture was generated with PyMOL (24).

Figure 4. Erlotinib slows down the transition of H358 by stabilizing E-cadherin.

H358 cells were treated for 3, 6, 13 and 20 days with TGFβ3 alone or together with

erlotinib and gefitinib. The levels of expression of E-cadherin (fig4.A), ILK and PINCH

were analyzed by Western blot. For ILK (fig4.B) and PINCH (fig4.C), Western blot

signals were quantified and averaged from two biological replicates. The effect of 13

days of treatment by erlotinib and gefitinib was estimated on TGFβ-stimulated cells after

adjustment to the level at 3 days of respective treatment. Compared with TGFβ-

stimulated untreated cells, a downregulation (in the sense of a “non-upregulation”) by

gefitinib on ILK (one-sided p=0.085) and PINCH-1 (one-sided p=0.12) did not reach

significance. In contrast, there was a significant downregulation by erlotinib on ILK (one-

sided p=0.03) and PINCH-1 (one-sided p=0.02). Note: all cell lines were obtained from

the American Type Culture Collection (ATCC) without further authentication.

Figure 5. IPP expression in NSCLC cell lines. A) Western blots of ILK, α-parvin,

PINCH, E-cadherin, vimentin and GAPDH on EGFR wild-type NSCLC epithelial cell lines

H292, H441, H358, H322, and mesenchymal cell lines H460, H1299, H226 and Calu6.

B) Quantified Western blot signals normalized on GAPDH levels and averaged to

determine the level of expression of the IPP complex. A significant upregulation in the

GAPDH normalized expression was observed for α-Parvin (one-sided p=0.045), ILK

(one-sided p=0.007) and PINCH-1 (one-sided p=0.011) from epithelial to mesenchymal

cell lines. Note: all cell lines were obtained from the American Type Culture Collection

(ATCC) without further authentication.

Figure 6. E-cadherin (CDH1) and ILK expression in patients with versus without

clinical benefit. Each dot represents the gene signal (log2 transformed) measured in

one sample. For each group of patients (without or with clinical benefit), a boxplot is

displayed where the thick black horizontal line (within a box) represent the median gene

signal for that group. The top and bottom boundaries of the box correspond respectively

to the 25th percentile and 75th percentile of the gene signals. The whiskers extend to the

most extreme data point which is no more than 1.5 times the interquartile range from the

box.

Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-type Non-small Cell Lung Carcinoma Cell Lines

Angelique Augustin, Jenz Lamerz, Helene Meistermann, et al.

Mol Cancer Ther Published OnlineFirst January 31, 2013.

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