Proteomic Signatures of Epidermal Growth Factor Receptor and Survival Signal Pathways Correspond to...

Proteomic Signatures of Epidermal Growth Factor Receptor andSurvival Signal Pathways Correspond to GefitinibSensitivity in Head and Neck CancerFrancisco G. Pernas,1Clint T. Allen,1Mary E.Winters,2 BinYan,1Jay Friedman,1Bhavana Dabir,2

Kunal Saigal,1Gerhard S. Mundinger,2 Xiaojiang Xu,4 John C.Morris,3 Katherine R. Calvo,2

Carter Van Waes,1and Zhong Chen1

Abstract Purpose: Gefitinib targeting of the epidermal growth factor receptor (EGFR) has shown limitedactivity in clinical trials of head and neck squamous cell carcinoma (HNSCC). To investigate theunderlying molecular mechanism, the proteomic signatures and responses of EGFR anddownstream signals have been studied in a panel of HNSCC cell lines and tumor specimenspre- and post-gefitinib treatment.Experimental Design:The IC50 of gefitinib for HNSCC cell lines were determinedusing 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay.The effects of gefitinibon activation of EGFR and downstream signaling molecules were determined byWestern blot,ELISA, and reverse-phase protein microarray (RPMA). The biomarkers involved in the signalingpathways were examined in HNSCC tumor specimens from patients in a phase I gefitinib trial.Results: In vitro, gefitinib inhibited cell proliferationwith differing IC50, and suppressed activationof EGFR and downstream signaling molecules protein kinase B (AKT), extracellular signal-regulated kinase1/2, signal transducer and activator of transcription 3 (STAT3), andnuclear factornB. The drug sensitivity was statistically correlated with activation of phosphorylated AKT(p-AKT) and phosphorylated STAT3 (p-STAT3) detected by ELISA, and consistent with resultsmeasured by RPMA. In patient samples, a broad suppression of activation of EGFR and down-stream signaling molecules was observed in a molecular responder patient, in contrast to a lack ofinhibition or increased activation of biomarkers in different pathways in nonresponder patients.Conclusions: Gefitinib sensitivity is correlated with p-AKTand p-STAT3 activation in HNSCCcell lines and tumor specimens. p-AKTand p-STAT3 could serve as potentially useful biomarkersand drug targets for further development of novel therapeutic agents for HNSCC.

Increased understanding of molecular alterations in head andneck squamous cell carcinoma (HNSCC) and other cancers has

ushered in efforts to develop molecularly targeted therapies.Among these alterations, overexpression of the epidermalgrowth factor receptor (EGFR) has been identified in manycancers, including 80% to 100% of HNSCC (1–3), where it hasalso been implicated in a more aggressive phenotype, increasedresistance to treatment, and poorer clinical outcome (4, 5).These observations have led to interest in the development ofmolecularly targeted small-molecule inhibitors of EGFR for thetreatment of HNSCC. Gefitinib (Iressa), an EGFR tyrosinekinase inhibitor, has been tested in clinical trials in solidtumors, including HNSCC, as a single agent, or in thecombination with other chemotherapies or radiation, but hasshown limited clinical efficacy with response rates of 10% to15% (6–9). Molecular biomarkers that could identify patientsresponsive to gefitinib or other EGFR inhibitors would beuseful in the selection of patients for therapy.

The EGFR is a membrane-bound glycoprotein with anextracellular cysteine-rich ligand-binding domain linked by ashort single transmembrane sequence to an intracellular tyrosinekinase and carboxyl-terminal scaffolding domains (1). EGFR(ErbB1) is a member of the ErbB receptor family of tyrosinekinases, which also includes ErbB2 (HER-2/neu), ErbB3,and ErbB4 (1, 4). The activation of EGFR family members may

Cancer Therapy: Preclinical

Authors’Affiliations: 1Tumor Biology Section, Head and Neck Surgery Branch,National Institute on Deafness and Other Communication Disorders, 2Laboratoryof Pathology, National Cancer Institute, and 3Metabolism Branch, Center forCancer Research, National Cancer Institute, NIH, Bethesda, Maryland; and4Biochemistry andMolecular Genetics, University ofVirginia, Charlottesville,VirginiaReceived 4/18/08; revised 11/13/08; accepted 12/2/08; published OnlineFirst3/24/09.Grant support: Clinical ResearchTraining Program, a public-private partnershipsupported jointly by the NIH and a grant to the Foundation for the NIH from PfizerPharmaceuticals Group. Intramural Research Program of the NIH, NIDCD projectZ01-DC-00016, and NCI, Center for Cancer Research.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).Z. Chen and C.Van Waes contributed equally as senior authors.Requests for reprints: Zhong Chen, NIH-NIDCD Building 10, Room 5D5510 Center Dr., Bethesda, MD 20892. Phone: 301-435-2073; Fax: 301-594-4643; E-mail: [email protected] or Carter VanWaes, Phone: 301-402-4216;Fax: 301-402-1140; E-mail: [email protected].

F2009 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-1011

www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092361

be either ligand-dependent or independent as a result ofmutation or overexpression-induced homodimerization, orheterodimerization with other ErbB receptor family members(1). The binding by ligands such as epidermal growth factor(EGF), transforming growth factor a, amphiregulin, and heparinbinding-EGF results in autophosphorylation of multiple tyro-sine residues at the receptor carboxyl terminus of EGFR, whichacts as scaffolding for SRC and other adaptor proteins thatinteract with the receptor and transduce mitogenic signalsmediated by downstream molecular cascades (1, 4, 5). Ligand-independent activation of EGFR in HNSCC has been shown toresult from a truncation mutation, EGFR variant III (EGFRvIII;ref. 10). Ligand-dependent and independent activation play avital role in promoting cancer cell growth, cell migration,aberrant metabolism and differentiation, and enhanced cellsurvival (1, 4).

A number of pharmacologic agents that block EGFR signalinghave been developed and evaluated in clinical trials. Gefitinib isthe first of such molecules with oral bioavailability that inhibitsEGFR activation by competing with ATP binding at the catalytictyrosine kinase domain of the EGFR. Gefitinib was originallyapproved for the third-line treatment of non–small cell lungcancer, but has yielded limited results in several clinical trials ina variety of solid tumors (6–9). Gefitinib has been studied asmonotherapy for patients with recurrent HNSCC (11), and incombination with paclitaxel (Taxol) and radiation in patientswith local-regionally advanced HNSCC by our group.5 Consis-tent with the 10% to 15% response rates observed from largermonotherapy trials (11), only 1 of 7 patients whose tumorswere studied showed molecular responses to single-agentgefitinib as identified by immunohistochemistry. A lack ofunderstanding of downstream molecular mechanisms activatedby EGFR and suitable biomarkers for selection of molecular

responders has been a major hurdle for clinical trials and therational selection of patients for treatment with EGFRinhibitors.

Molecular heterogeneity in subsets of tumors and/or patientpopulations has been shown to contribute to variable responsesof non–small cell lung cancer to gefitinib and other EGFRinhibitors. It was previously shown that there is a beneficialclinical effect of gefitinib in Asian populations with non–smallcell lung cancer, who had specific EGFR gene mutations andamplifications (12–16). However, molecular analysis ofHNSCC tumor samples has not revealed the same spectrumof amplifications or mutations (17–19). Recently, an in-framedeletion in exon 19 of the EGFR gene was reported in 7.3% ofHNSCC in the Korean population (20), and a deletion of exons2 to 7 resulting in a truncated extracellular domain andconstitutive tyrosine kinase activation, designated as EGFRvIII ,was found in 42% of HNSCC (10). Because of the differentgenetic alterations of EGFR observed in HNSCC, the molecularmechanism(s) determining the therapeutic sensitivity togefitinib and other tyrosine kinase inhibitors may not be thesame as observed in lung cancer, and therefore need to be morethoroughly investigated.

In this study, we explored the effects of gefitinib on activationof the EGFR and downstream signal pathways, and determinedif there was a relationship between these biomarkers and theanticancer effects in HNSCC cell lines and patients’ specimens.In addition, we cross-validated the biomarkers identified byseveral methods, including Western blot analysis, ELISA, andreverse-phase protein microarray (RPMA), a newly developedproteomic platform that is capable of measuring numerousspecific phosphorylated proteins that are involved in importantsignaling pathways from a small tissue biopsy or a fewthousand cells (21, 22). We found that gefitinib inhibited cellproliferation in vitro with different IC50, and suppressedactivation of EGFR and downstream activation of proteinkinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3),and nuclear factor nB (NF-nB). Our observations indicate thatthese proteomic signatures to EGFR inhibitor could be useful inthe development of more accurate molecular diagnosis,selection of therapy, and determinations of HNSCC prognosis.Cross-validation of results from in vitro and in vivo using RPMAby Western blot, quantitative ELISA, and immunohistochem-istry provides an essential step for future application of thesetechnologies in clinical trials.

Materials andMethods

Cell lines and reagents. HNSCC cell lines UMSCC-6, -9, -11A,and -11B were kind gifts from Dr. T.E. Carey (University of Michigan,Ann Arbor, MI), and their characteristics were previously described(23). The UMSCC cell lines were cultured in MEM (Invitrogen) with10% fetal bovine serum and penicillin/streptomycin, and maintainedin a humidified incubator at 37jC with 5% CO2. Gefitinib wasprovided for studies by AstraZeneca, under a Materials CooperativeResearch and Development Agreement and Clinical Trials Agreementwith the National Cancer Institute (NCI), and was dissolved in DMSOwhen reconstituted. Recombinant human EGF was purchased fromR&D Systems.

3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cellular

proliferation assay and calculation of IC50. The cell lines UMSCC-6, -9,

Translational Relevance

Gefitinib, the first small molecule drug targeting epider-mal growth factor (EGF) receptor (EGFR), has not attainedthe efficacy anticipated in clinical trials. To address thisproblem, the molecular mechanism of gefitinib sensitivitywas investigated inhead andneck cancer lines and tumors.The differential sensitivity was associatedwith the basal orEGF-induced activation of downstream protein kinase Band signal transducer and activator of transcription 3,but not with the level of EGFR phosphorylation or knownEGFRmutations. A similar panel of biomarkers was studiedin tumor specimens from a phase I gefitinib trial, by immu-nohistochemistry and reverse-phase protein microarray(RPMA), and consistent results were observed. Our datasuggest that the activation status of EGFR down streamsignaling molecules, instead of EGFR itself, could serveas potentially useful biomarkers for predicting sensitivityof gefitinib, or other potential drugs targeting EGFR.The newly developed RPMA is a powerful and reliablemethod for detecting multiple biomarkers using clinicalbiopsy specimens.

5 Van Waes C, et al., IntJRad Onc Biol Phys, in press.


www.aacrjournals.orgClin Cancer Res 2009;15(7) April 1, 2009 2362

-11A, and -11B cells were plated in triplicate at 5 � 103 cells per wellonto 96-well plates. The cells were cultured overnight and thenexposed to gefitinib at varying concentrations. Cell proliferation wasmeasured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazoliumbromide (MTT) Cell Proliferation Kit (Roche Diagnostics). The opticaldensity was measured by a microplate reader at a wavelength of570 nm. The IC50 of the drug was calculated using MTT data at day 5comparing treatment samples versus control samples. The percentagechanges of MTT values between controls and treated samples werecalculated individually for each dose and cell line using the followingequation: (control-treated sample/control) �100. The percent changewas plotted on a graph against drug dose. A best fit logarithmiccurve was plotted and the IC50 was determined as the 50% inhibitionpoint.

Sequencing and reverse transcription-PCR analysis of mutations in

EGFR coding regions. RNA was isolated from UMSCC cells, and cDNA

was reverse-transcribed and sequenced for coding regions of EGFR exon

18 to 21 bidirectionally by SeqWright Inc., according to the hot spots

identified in the literature (24). The EGFRvIII deletion mutation was

analyzed by reverse transcription-PCR, using the primers described by

Dr. Grandis’ group (10).Whole cell extraction and Western blot analysis. Cultured UMSCC

cells at 75% confluence were treated with 1 or 10 Amol/L gefitinib for4 h and then 10 ng/mL recombinant EGF (R&D systems) were addedinto half of the dishes for each treatment condition for 1 h. Culturedcells were washed with ice-cold PBS, lysed, and harvested with ice-coldcell extraction buffer (Invitrogen). The whole cell lysates were incubatedon ice on a rocking platform for 30 min and vortexed every 10 min andthen spun at 13,000 g at 4jC for 10 min. The supernatants werecollected and the protein concentrations were quantified by BCAprotein assay kit (Pierce).

For Western blot analysis, whole cell lysate was mixed with an SDSprotein gel loading solution (Quality Biological, Inc.) and heated at85jC for 2 min. The samples were loaded onto Tris-glycine precast gels(Invitrogen), and electrophoresis was completed at 125V for 105 min.The Invitrogen iBlot apparatus was used to complete the transfer(Invitrogen). The membranes were preblocked in a preblockingsolution composed of 1% Tween-20 in TBS mixed with either 7.5%bovine serum albumin or 5% nonfat dry milk. Primary antibodies werediluted in either 5% nonfat powdered milk or 5% bovine serumalbumin prepared from Tween-20 in TBS for: phospho-EGFR (Tyr845)and phospho-EGFR (Tyr1068), 1:1000 (Cell Signaling Technology);and phospho-EGFR (Tyr1173), phospho-EGFR (Tyr1148), and totalEGFR, 1:1000 (Invitrogen). Each blot was incubated with PierceSuper Signal West Pico Substrate (Pierce) and exposed to KodakX-OMAT film.

ELISA assays. ELISA assays that were used to quantify total EGFR,

phospho-EGFR (Tyr 1173), total STAT3, phospho-STAT3 (Tyr 705),

total ERK1/2, phospho-ERK1/2 (Tyr185/187), total AKT, and phos-

phor-AKT (Ser 473), were done according to the manufacturer’s

protocol (Invitrogen). The ELISA assays used to detect phospho-EGFR

Tyr 1068, phospho-EGFR Tyr 845, and NF-nB phospho-p65 (Ser 536)

were done according to the manufacturer’s protocol (Cell Signaling

Technologies). The whole cell lysates were isolated as described for the

Western blot analysis for all ELISA assays, except that nuclear extracts

were used for testing NF-nB p-p65 (Ser 536; Active Motif). Samples were

measured for each condition against a respective standard provided by

the kits. The optical density was measured by a microplate reader at a

wavelength of 450 nm, and the data were calculated and presented as

mean plus SD from triplicate assays.Real time reverse transcription-PCR. RNA isolation and cDNA

synthesis were done as previously described (25). Real Time reversetranscription-PCR for EGFR and 18S rRNA was achieved with Assays-on-Demand Gene Expression Assay from Applied Biosystems. Ampli-fication conditions were as follows: activation of enzymes for 2 min at50jC and 10 min at 95jC, followed by 40 cycles at 15 sec at 95jC and1 min at 60jC. Thermal cycling and fluorescence detection was done

using an ABI Prism 7700 Sequence Detection System (AppliedBiosystems). EGFR values were normalized to 18S rRNA. Eachcondition was assayed in triplicate and data were presented as meanplus SD.

Patient treatment and tumor specimens. Patients with histologicallyproven HNSCC provided informed consent before being enrolled into aphase I study of gefitinib, paclitaxel, and radiation for patients withlocoregionally advanced HNSCC, in a protocol that was approved bythe Institutional Review Board of the NCI (NCI trial 04-C-0141).Gefitinib was produced by AstraZeneca Pharmaceuticals and suppliedthrough the NCI’s Cancer Treatment Evaluation Program. Gefitinib(250 mg orally) was administered daily for 1 wk prior to paclitaxel andradiation, so that tumor specimens pretreatment and after 1 wk oftreatment with gefitinib alone could be obtained from tumorsaccessible by transoral biopsy.

The patients received a total of 8 wk of gefitinib, including 7 wk incombination with weekly doses of paclitaxel and 35 fractions ofradiation. Biopsies of patients’ normal mucosa and tumor pretreatmentand 1 wk post-gefitinib treatment were obtained from 7 of 10 patients.Specimens were immediately embedded in optimum cutting temper-ature media and frozen at -80jC. Frozen tissues were sectioned at athickness of 10 um at the largest area and placed on silanated glassslides (Histoserv) for immunostaining. The complete clinical resultsand analysis of correlative immunohistochemical studies for EGFR,selected phospho-proteins, proliferation (Ki-67), and apoptosis [termi-nal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL)] markers have been described in a separate report.5

RPMA. The cell line lysates in SDS-based buffer were quantified

with BCA protein assay kit (Pierce), and adjusted to 1Ag/AL. The lysates

were printed onto Whatman FAST nitrocellulose coated slides

(Schleicher & Schuell) using a solid-pin 2470 Arrayer (Aushon

Biosystems). Lysates were printed in a serial dilution curve, in duplicate.Immunostaining and analysis of RPMAs. The antibodies used in

RPMA were first tested to ensure that a single band was generated inWestern blot on control HeLa cell line lysates that were treated oruntreated with EGF. The rabbit polyclonal antibodies from CellSignaling were affinity-purified against EGFR, phospho-EGFR(Tyr1068), phospho-EGFR(Tyr1148), AKT, phospho-AKT (Ser473),ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), phospho-ERK1/2(Tyr185/187), NF-nB p65, phospho-p65 (Ser536), STAT3 and phos-pho-STAT3 (Tyr705), mitogen-activated protein kinase kinase (MEK),and phospho-MEK (Ser 217/221).

Each array was incubated with a specific primary antibody, whichwas detected by using the catalyzed signal amplification system(DAKO). Briefly, each slide was blocked with I-block (Tropix)for 1 h. The slides were incubated with primary and biotin-labeled secondary antibodies, and then sequentially incubated withstreptavidin-biotin complex for 15 min, biotinyl tyramide (foramplification) for 15 min, streptavidin-peroxidase for 15 min, and3,3¶-diaminobenzidine tetrahydrochloride chromogen for 2 min.Between steps, the slide was washed with catalyzed signal amplificationbuffer. The signal was scanned with a Perfection 1200S scanner(Epson America) with 256-shade gray scale at 1,200 dots per inch. Fordetection of total protein, arrays were stained with colloidal goldsolution (Bio-Rad) and scanned similarly. Spot images were convertedto raw pixel values using ImageQuant software (v. 5.2, MolecularDynamics). Raw pixel values for the 1:2 dilutions of all samples wereused for subsequent analysis.

For RPMA study in human patient samples, frozen sections of

HNSCC or normal tissues were scraped, lysed, arrayed, and stained the

same ways as described above. Total protein of each spotted lysate was

determined using Sypro Ruby Protein Blot Stain (Molecular Probes)

and a fluorescence imaging system (Kodak). Stained arrays were

scanned on a flatbed scanner and spot images were converted to raw

pixel values by ProteinScan/P-SCAN software [Peak quantification with

Statistical Comparative Analysis, (http://abs.cit.nih.gov/pscan)], and

were normalized to total protein.

Proteomic Response to Gefitinib in Head and Neck Cancer


Results

UMSCC cells exhibited differential sensitivity to gefitinib. Aphase I clinical trial combining gefitinib with paclitaxel andradiation in HNSCC patients was carried out at the NCI, andheterogeneous clinical responses were observed in 10 patientsenrolled.5 To investigate the possible mechanism(s) forheterogeneous clinical responses observed in this and otherclinical studies of HNSCC, we selected a panel of HNSCC celllines, UMSCC-6, -9, -11A, and -11B. The selection criteria forthese cell lines were based on our previous studies, in which wefound that UMSCC-11A and -11B exhibited relatively higherEGFR expression and activation, and UMSCC-6 and -9exhibited intermediate and lower levels (26, 27). In addition,multiple signal transduction pathways downstream from EGFRinvolving AKT, ERK, STAT3, and NF-nB have been extensivelystudied and characterized in these cell lines by our laboratory(23, 25–31). The effects of gefitinib on the activation of EGFRand downstream molecules were investigated in vitro. First, wetested gefitinib drug sensitivity of these cell lines using MTTassay (Fig. 1). UMSCC-6, -9, and -11A showed similar in vitrosensitivity to gefitinib with the IC50 range of f1 to 2.5 Amol/L(Fig. 1). However, the UMSCC-11B cell line was less sensitive,with an IC50 of f17 Amol/L, or about 10-fold more than thatobserved in the other cell lines (Fig. 1). In addition, a lowerdose of gefitinib (0.5-1.0 Amol/L) showed a paradoxicalstimulation of UMSCC-11B cell growth (Fig. 1).

EGFR mutations have been found to be rare among HNSCCfrom the United States or Europe (17–19), but to rule outthe possibility that the gefitinib sensitivity is due to EGFRmutation, we sequenced EGFR exon 18 to 21, which code forthe hot spots of EGFR mutations of ATP domains identified inlung cancer (24). We found a polymorphism in EGFR exon 18,which substitutes A for T at nucleotide 2133. This is the thirdcodon for a threonine (aa# 629), where the T to A transversionis a silent change (Supplemental Fig. S1). The transversion wasfound to be homozygous in control HEKA and UMSCC-9 cells,T/A heterozygosity was found in UMSCC-6 and -11A cells,whereas the wild-type T residue presented in GENBANK wasfound only in UMSCC-11B cells. There was no sequenceevidence for the previously identified in-frame deletion in exon19 of the EGFR gene, which was reported in HNSCC of theKorean population (20). In addition, we did not find thedeletion of EGFR exon 2 to 7 (EGFR vIII , PCR data not shown),which results in a truncated extracellular domain and consti-tutive tyrosine kinase activation (10).Gefitinib significantly inhibited EGF-induced EGFR phosphor-

ylation. Gefitinib was designed to inhibit tyrosine kinaseactivity of EGFR. To test the specific phosphorylation sites ofEGFR that could be affected by gefitinib in the HNSCC celllines, we did a series of experiments to identify the status ofknown EGFR phosphorylation sites and elucidate the drugeffects by ELISA (Fig. 2A) and Western blot analysis (Fig. 2B).The cultured cells exhibited relatively low basal levels of

Fig. 1. A dose-dependent antiproliferative effect of gefitinib in HNSCC cell lines. Cultured UMSCC cells were plated in 96-well microplates in quadruplicates, and the nextday the cells were treated with different doses of gefitinib. The cell density was determined on days1, 2, 3, and 5 byMTT assay. The optical density (570 nm) was measuredand presented as the mean plus SD from quadruplicates. The IC50 values were calculated based on the day 5 MTTdata.The vehicle control was included as the NoTxcondition.



activated p-EGFR sites tested by both ELISA and Western blot,and gefitinib had little if any significant effect on theconstitutive EGFR activation (Fig. 2). Upon EGF stimulation,the cell lines showed different responses in terms of phosphor-ylation of EGFR at different sites. UMSCC-6 and -11B exhibitedrelatively high levels of phosphorylation of p-EGFR at Tyr854;UMSCC-9 exhibited the highest level of phosphorylation ofp-EGFR at Tyr1068; and UMSCC-11B exhibited the highestlevels of p-EGFR at Tyr1173 (Fig. 2). Gefitinib at 1 to 10 timesthe pharmacologic serum concentration achieved with standarddaily dosages of 250 to 500 mg orally (f1 and 10 Amol/L;

ref. 32) showed completely inhibitory effects on each of thedifferent EGFR phosphorylation sites in all cell lines, regardlessof the drug sensitivity detected by MTT assay (Fig. 1). For totalEGFR protein expression, both UMSCC-11A and -11B cellsshowed a relatively high level of expression when comparedwith UMSCC-6 and -9 cells (Fig. 2A). Neither EGF nor gefitinibsignificantly affected the total EGFR protein expression levels inany of the cell lines (Fig. 2).Gefitinib significantly inhibited EGFR-regulated downstream

pathways. Next, we tested the effects of gefitinib on down-stream pathways potentially regulated by EGFR, such as AKT,

Fig. 2. Gefitinib effects on phosphorylated and total EGFR expressed by HNSCC cell lines. UMSCC cell lines were cultured in the log growth phase and treated with differentdoses of gefitinib for 4 h.Then the cells were treated with10 ng/mL of recombinant EGF for1h followed by harvesting of the cell lysates. Phosphorylated and total EGFRwere quantified by ELISA (A) or tested byWestern blot analysis (B).The data for ELISAwere calculated from triplicates from one of repeated experiments, and presented asthe mean plus SD.The statistical differences are shown for comparisons with NoTx control (*), with EGF treatment (**), and with1 Amol/L gefitinib and EGF (#, P < 0.05,Student’s t-test).



ERK, and STAT3, by assessing the level of the total andphosphorylated forms of these proteins (Fig. 3). These path-ways were constitutively activated, with varying levels of basalphosphorylated and total proteins observed in the different celllines. Gefitinib sensitivity of cell lines UMSCC-6, -9, and -11Acorresponded with elevated basal levels of phospho- and totalAKT relative to gefitinib-resistant UMSCC-11B cells. Bycontrast, UMSCC-11B cells showed the highest basal levels ofphosphorylated STAT3. UMSCC-9 cells showed the highestbasal phosphorylated and total ERK proteins (Fig. 3). UnderEGF stimulation, UMSCC-9 cells showed the strongestinduction in p-AKT (S473) and ERK1/2 (TYR185/187), andUMSCC-11B cells exhibited the largest induction in phospho-STAT3 (Y705; Fig. 3). Gefitinib significantly inhibited the basalp-AKT, as well as EGF inducible phosphorylation of AKT, ERK1/2, and STAT3 in the cell lines, in a dose-dependent fashion

(Fig. 3). The levels of total proteins were not affected by eitherEGF stimulation or by gefitinib (Fig. 3).

Next we tested if there were correlations between IC50 valuesand EGFR downstream molecules tested. The IC50 werestatistically and negatively correlated with levels of basalp-AKT (R 2 = -0.93; P = 0.022) and EGF-inducedp-AKT (R2 = -0.89; P = 0.037), and positively correlated withEGF-induced p-STAT3 (R2 = 0.98; P = 0.008). The othercorrelations, such as basal and EGF-induced p-ERK, as well asbasal p-STAT3, did not reach statistical significance, possiblydue to the smaller sample sizes.Gefitinib significantly inhibits the EGFR-regulated NF-kB

pathway. In addition, we also tested the effects of the drugon the NF-nB pathway, because our laboratory has shown thatNF-nB promotes HNSCC pathogenesis, and EGFR can contrib-ute to aberrant activation of NF-nB in HNSCC cell lines (26),

Fig. 3. Gefitinib effects on phosphorylated and total proteins of signal pathways downstream of EGFR. UMSCC cells were treated and the whole cell lysates were harvestedthe same way as indicated in Fig. 2.The phosphorylated and total proteins were quantified using ELISA and the data were calculated from triplicates from one of repeatedexperiments with the mean plus SD.The statistical differences are shown for comparisons with NoTx controls (*), with1 Amol/L gefitinib (H ), EGF treated with NoTxconditions (**), EGF treated with1 Amol/L gefitinib and EGF-treated conditions (#), and between different doses of gefitinib when both treated with EGF (##), P < 0.05(Student’s t-test).



which was inhibited in the tumor specimen of the molecularresponder to gefitinib.5 We tested the effects of EGF andgefitinib on the NF-nB subunit p65 (S536), implicated as themost important site for p65 transactivation by InB kinase h ofthe canonical InB kinase a/h/g complex (33). EGF inducedsignificant activation of NF-nB p65 (S536) in 3 of 4 cell lines(Fig. 4). Gefitinib partially inhibited constitutive and signifi-cantly inhibited EGF-induced activation of NF-nB p65 (S536)in 2 of 3 of the gefitinib-sensitive lines, UMSCC-6 and -11A(Fig. 4). In general, EGF and gefitinib did not show inhibitoryeffects on total p65 protein expression levels.Cross-comparison of the results from RPMA with those detected

by ELISA and Western blot analyses. RPMA is a recentlydeveloped proteomic microarray platform that is capable ofsimultaneously quantifying concentrations of numerous pro-teins and posttranslational modifications such as phosphory-lation from small amounts of tissue or cells using validatedantibodies (21, 22). To compare the results obtained by RPMAwith results previously obtained using the classic Western blotand ELISA methods, RPMA was done with the same whole celland nuclear lysates as used in Figs. 2–4. We observed similartrends of EGF induction and gefitinib inhibition of theactivation of EGFR and downstream molecules in UMSCC-9and -11B cells (Fig. 5), although there was some variability inresults among RPMA, Western blot, and ELISA. More significantalteration in phosphorylation of signaling proteins than inabundance of total signaling proteins was observed, consistentwith the findings observed by the classic methods.Identification of potential biomarkers in HNSCC patients’

samples from a phase I clinical trial using RPMA. A phase Iclinical trial combining gefitinib with paclitaxel and radiationin HNSCC patients was concluded at the NCI, and heteroge-neous clinical responses were observed in 10 patients enrolled(Fig. 6A and B). The details of clinical responses, and theselection and results of the immunohistochemistry biomarker

studies will be presented elsewhere.5 In biopsies obtainedfrom patients on treatment day 8 of gefitinib alone, significantinhibition of EGFR, multiple-signal phospho-proteins, andproliferation marker Ki-67 was observed, which was accom-panied with increased apoptosis (TUNEL) staining in 1 of 7subjects. This patient was one of the five patients who had acomplete response to the combination (patient 3; Fig. 6Aand B) and was identified as a molecular responder. Ofthe remaining six patients defined as molecular nonrespondersto gefitinib based on the biomarkers detected by immunohis-tochemistry, most of the patients responded to standardmodalities included in the combined therapy of gefitinib,paclitaxel, and radiation. As expected for a chemotherapy andradiation therapy combination of known activity for previous-ly untreated patients, the 6 of 10 (60%) complete clinicalresponse observed is close to that seen with paclitaxel andradiation without gefitinib in our previous trial (34). It isexpected that the response to paclitaxel and radiation exceededthe molecular response to gefitinib, because clinical respond-ers received 8 weeks of gefitinib, including 7 weeks incombination with weekly doses of paclitaxel and 35 fractionsof radiation, whereas the patient samples collected for thebiomarker study to define the molecular responders wereobtained after receiving gefitinib alone for a run-in period ofonly 7 days, as indicated in Fig. 6A and B.

We did RPMA from tumor lysates obtained pre-gefitinib andafter 7 days of gefitinib alone and compared the results with theimmunohistochemistry biomarker analysis done on the samebiopsy specimen.5 Protein microarrays were generated fromtissue lysates of patient biopsy samples, and 13 biomarkersrelated to EGFR and its downstream pathways were tested. Inpatient 3 (Fig. 6B), who was determined to be a molecularresponder based on effects on EGFR signaling, apoptosis, andproliferation biomarkers by immunohistochemistry, we alsoobserved consistent molecular responses by RPMA (Fig. 6C, left

Fig. 4. EGF and gefitinib effects on NF-nB activation in HNSCC. UMSCC cells were treated the same way as indicated in Fig. 3, and nuclear extracts were harvested.Thephosphorylated and total NF-nB p65 proteins were quantified in triplicates, and the data were calculated as mean plus SD.The statistical significances are shown forcomparisons between gefitinib-treated samples with untreated controls (*), EGF-treated samples with controls (InB), and EGF and gefitinib-treated samples with EGF-treatedsamples (##), P < 0.05 (Student’s t-test).



panel). Consistent with immunohistochemistry data, a broaddecrease in RPMA staining of EGFR and downstream signalmolecules (10 of 13 biomarkers) was observed in this patientafter gefitinib treatment, including the molecules involved inthe AKT, ERK, STAT3, and NF-nB pathways (Fig. 6C, leftpanel). In addition, the only molecular responder identified inthis study did show significantly higher levels of total AKT, p-AKT (Ser473), and total STAT3 in the pretreated samples, butdid not show higher levels of total and p-EGFR, p-AKT(Ser308), and total and p-ERKs and MEKs (data not shown).An increased staining in cleaved caspase 3 was only observed in

specimen from this patient, consistent with the increase inapoptosis detected by TUNEL assay in the same tumorspecimen.5 In contrast, in a representative sample of amolecular nonresponder (Fig. 6C, right panel), there was onlya f40% decrease of total EGFR and p-AKT, a 10% to 20%decrease of total AKT and MEK, but f60% increase of p-MEK,f100% increase of STAT3, f40% increase of p-STAT3, and aslight increase of p-NF-nB. No increase in cleaved caspase 3 wasobserved in this or other RPMA nonresponder samples. Inaddition, an increase of either total or phospho-proteinsinvolved in each of the ERK/MEK, STAT3, or NF-nB pathways

Fig. 5. Reverse phase protein array detection of phosphrylated and total proteins in UMSCC cells lines. Reverse phase protein array was done using the same whole celland nuclear extracts as used in the previous experiments.The images of immunohistochemical staining were scanned, and data were extracted by ImageQuant5.2 softwarefrom replicated slides. * indicates the statistical significance when compared with NoTx conditions; ** indicates the difference when compared with EGF-treated conditions(P < 0.05, Student’s t-test).



was observed in 4 of the 5 patients as the molecularnonresponders with evaluable RPMA data. For each of themolecular nonresponders, increased staining of >2 moleculesinvolved in different pathways was observed. Thus, the data areconsistent between RPMA and immunohistochemistry, show-ing that lack of response to gefitinib may have been due toactivation of the ERK, STAT3, and NF-nB pathways, either by orin compensatory response to gefitinib, or other mechanismsindependent of gefitinib or EGFR pathway.

Discussion

We investigated the effect of gefitinib on EGFR and relatedmolecular signaling pathways in HNSCC cell lines and patienttumor samples. We showed that gefitinib suppressed EGF-induced EGFR phosphorylation to basal levels at threephosphorylation sites studied, and it inhibited the activation-specific phosphorylation of the downstream signal pathwaycomponents AKT, ERK, STAT3, and NF-nB to various degrees indifferent HNSCC cell lines and tumors. In vitro, a dose-dependent inhibition of tumor cell proliferation was observedthat corresponded to higher basal AKT phosphorylation, andwas associated with EGF-dependent phosphorylation of AKTand STAT3. In a clinical trial using gefitinib in HNSCC patients,consistent results were observed from tumor specimens ofseven patients where the phosphorylation of coactivateddownstream pathways and a marker of apoptosis werecompared by immunohistochemistry and RPMA. Thus, ourstudy identified alterations in a panel of specific EGFR anddownstream signal pathway components of known biologicaland clinical significance in HNSCC, which could be used asearly biomarkers for molecular responsiveness in clinical trialsof EGFR-targeted therapeutic agents alone, or used in combi-nation with other agents targeting these multiple pathways.

Using a panel of four UMSCC cell lines, we examined EGFRand downstream molecular pathways activated in HNSCC thatare potentially related to EGFR sensitivity. The four cell linesexhibited various levels of total EGFR and EGF-inducedreceptor phosphorylation (Fig. 2), which are consistent withthe reported overexpression of EGFR in HNSCC (1–5, 35). TheUMSCC-11A and -11B cell lines exhibited the higher total EGFRthan UMSCC-6 and -9 by ELISA (Fig. 2A), which is consistentwith our previous reports (26, 27). The constitutive activationof EGFR was low among the sites tested, and variabilityin inducible EGFR phosphorylation was observed (Fig. 2).However, drug sensitivity of the cell lines (IC50) could not besimply explained by the level of total EGFR, basal or EGF-induced EGFR phosphorylation, in that gefitinib inducedstrong suppression of all EGFR phosphorylation sites in all ofthe cell lines tested (Figs. 1 and 2).

The gefitinib sensitivity in HNSCC lines (IC50) seemed tocorrelate most closely with the extent of phosphorylation of theindividual EGFR sites and their mediated AKT, ERK, and STAT3signal molecules (Figs. 2 and 3). Phosphorylation at EGFRTyr845 of the kinase domain is involved in c-SRC activationand regulated by both EGF- and integrin-mediated EGFRactivation (4, 5, 36). Tyr1068 serves as docking site for PIP3Kand growth factor binding protein 2 (Grb2), and thesemolecules mediate activation of the AKT and ERK pathways(8, 37–39). The pair of phosphorylated residues, Tyr1148 andTyr1173, provides a docking site for the Shc scaffold protein,

and both sites are involved in mitogen-activated protein (MAP)kinase signaling activation (38, 39). In addition, Tyr1173 is thefavored autophosphorylation site in ligand-activated wild-typeEGFR and seems to be the major site of autophosphorylation inthe mutant EGFRvIII (10, 40). Among these sites, UMSCC-9cells exhibited the greatest level of activation of Tyr1068 afterEGF stimulation, and also showed the highest basal ERK, aswell as strong EGF-induced AKT and ERK phosphorylation(Figs. 2 and 3), consistent with the role of this EGFR site inphosphoinositide 3-kinase, AKT, and ERK pathway activation.UMSCC-9 was also the most sensitive cell line to gefitinib, withan IC50 of f 1.09Amol/L. In addition, the statisticalcorrelations were observed between gefitinib sensitivity withbasal and EGF-induced phosphorylation of AKT across the celllines. The data from cell lines are consistent with the data fromthe molecular responder in the clinical study, who retained thehighest total AKT and p-AKT Ser 473 prior to treatment (datanot shown). Although we did not find known mutationspreviously detected in lung cancer, our data are consistent withprevious preclinical studies in lung cancer cells that harborEGFR mutations (12–16, 41), in which the drug sensitivity togefitinib is closely correlated with EGFR-dependent AKTactivation. However, the overexpression and activation of ERKobserved did not reach statistical significance in the cell linestudy, and was not associated with the molecular responder inthis small clinical trial. The significance of ERK activation as thebiomarker for gefitinib sensitivity in HNSCC needs to bevalidated in a larger clinical correlative biomarker trial. Ourresults suggest that phosphorylation of EGFR and AKT mayserve as useful early markers of EGFR activation and drugresponse, which may be due to different etiology.

In addition, a truncation mutation of EGFR, EGFRvIII , hasbeen reported, which harbors an in-frame deletion that resultsin a truncated 150 kDa protein with constitutive phosphory-lation (10, 42–44). These HNSCC with the EGFRvIII deletionshowed increased proliferation and resistance to cisplatin andcetuximab treatment, but the response to gefitinib in the cellswith such a mutation has not been well studied (10). In thisstudy, however, the EGFR protein from the four UMSCC celllines comigrate with EGFR of normal keratinocytes andrecombinant EGFR protein on Western blot analysis,6 andsequencing and PCR analyses did not show such deletionmutation. Our data suggest that these truncated forms areunlikely to account for EGFR activation, as well as gefitinibsensitivity and resistance in these HNSCC cells. We sequencedthe EGFR genes of these lines for ATP domain mutationsidentified in gefitinib-responsive lung cancers from Asianpopulations (17–19), but we did not find the mutations inthe exon 18 to 21 of EGFR coding regions. We did find apolymorphism at the EGFR exon 18, but the T to A transversionis a silent sequence change which did not alter the coded aminoacid (Supplemental Fig. S1). In addition, the constitutive EGFRtyrosine phosphorylations in UMSCC-9 cells were at thecomparable or lower levels compared with those found inother UMSCC cells, suggesting that the gefitinib sensitivityobserved in UMSCC-9 and other responsive HNSCC linescould be due to different mechanisms.

6 Pernas F, unpublished observation.



Gefitinib significantly suppressed basal AKT and ERKphosphorylation but not the basal EGFR phosphorylation,which suggests that the basal AKT and ERK activation could bedue to other sites of EGFR phosphorylations or other receptortyrosine kinases, such as hepatocyte growth factor receptor/c-Met (25). The observation is consistent with the fact thatgefitinib may not be strictly specific for EGFR, but also be acompetitor for the ATP site of other tyrosine kinases as well(45). We previously reported that the hepatocyte growth factor/c-Met pathway is constitutively activated in UMSCC cells,which modulates downstream ERK and PI3K pathways (28). Inaddition, the relatively high IC50 in UMSCC-11B correlated

with the higher level of constitutive and EGF-induced activationof STAT3, in which gefitinib did not affect basal and onlypartially suppressed EGF-induced STAT3 activation (Figs. 3and 5). As previously reported, STAT3 activation could bemediated by EGFR and interleukin-6R signaling in HNSCC(27, 46, 47), in which gefitinib does not affect interleukin-6R–activated STAT3 signal pathway (27). Consistent with thisobservation, the rest of the cell lines in the panel exhibited onlya modest basal and increase in p-STAT3 when treated with EGF,indicating that EGF is not the major signal to induce STAT3activation in these cells (Figs. 3 and 5). The data provided bythe UMSCC cell lines are in good agreement with the data from

Fig. 6. Clinical information and reverse phase protein array detection of phosphorylated and total proteins in HNSCC patient tumor samples.Ten patients with HNSCC wereenrolled in the phase I clinical trial combining gefitinib, radiation, and paclitaxel. A, biopsy was done prior to treatment and1wk after 7 doses of gefitinib treatment. Gefitinibwas given weeks 2-8 concurrent with weekly paclitaxel on Monday and radiation Monday through Friday, as indicated. B, molecular response and clinical responses ofHNSCC patients.TNM, tumor-node-metastasis staging. Molecular response (with gefitinib treatment alone, a), indicated by inhibition of phospho-signaling and increasedapoptosis after treatment (yes); no consistent inhibition of signaling or apoptosis (no); not available (NA). Clinical response, determined 3 mo after treatment (gefitinib +paclitaxel + radiation, b). Complete response (CR); Partial response (PR); progressive disease (PD). C, reverse phase protein array was done using the lysates from tumortissues of this clinical trial. Left panel, data frompatient 3, who was identified as the molecular responder and a complete responder clinically. Right panel, data frompatient 6,who was identified as the molecular nonresponder. However, clinically, after the combination treatment, the patient showed partial response.The data presented are thepercentage changes in intensity, which were calculated from the intensity ratio between the stained samples before versus after treatment.



RPMA from the HNSCC patient specimens, in that themolecular responder showed highest basal total STAT3, as wellas suppressed total and phosphorylated STAT3 proteins aftergefitinib treatment; however, in the molecular nonresponders,4 of 5 patients showed increased total and phosphorylatedSTAT3 proteins after treatment (Fig. 1 and data not shown).These observations suggest that supplementing inhibition ofEGFR with agents blocking interleukin-6 or STAT3 may havecombinatorial or broader activity in patients with HNSCC.

Furthermore, we found evidence of inhibition and stimula-tion by EGFR of the NF-nB pathway. Previously we showed thatEGFR signaling partially contributes to NF-nB activation (28),which is also inducible and important for cell survival inresponse to tumor necrosis factor-a, radiation, and chemother-apy (48), consistent with its known functions modulating thecritical cellular processes involved in proliferation and apopto-sis (48). In this study, EGF induced and gefitinib suppressedNF-nB p65 Ser536 phosphorylation in 2 of 3 of the gefitinib-sensitive UMSCC cell lines (Fig. 4). Our data are consistent withour previous report (26), showing that EGFR signals partiallycontribute to NF-nB activation, and are in good agreement withthe studies from other laboratories that targeting EGF cansensitize renal cell carcinoma cell lines to NF-nB inhibition bybortezomib (49).

We compared the results from this study using thetraditional ELISA and Western blot (Figs. 2–4) with newlydeveloped RPMA technology (Fig. 5). The development andimprovement of RPMA technology provide a new platform forproteomic mapping of signaling pathways in tumor specimensthat can help us to simultaneously identify and characterizemultiple protein biomarkers that are critical for diagnosis,prognosis, and selection of targeted agents for personalizedcancer treatment (21, 50). The advantages of this technologyare the small sample size required, as well as the semiquan-titative and sensitive protein measurements permitted with useof high-quality validated antibodies. A 1-cm long core orpunch biopsy can yield up to 100 RPMA slides, which makesit possible to screen a panel of biomarkers (21, 22, 50), incontrast to the extensive usage of tissue specimen by classicimmunohistochemistry staining or other testing methods.Secondly, this method is able to generate the quantitativemeasurement of the specific biomarkers using a fitting curvewith serial dilution of the samples, whereas classic immuno-histochemistry is primarily qualitative. The successful cross-comparison of biomarkers by RPMA technology identified

in vitro and in small pilot clinical studies herein shows thefeasibility of applying the newly developed technology inclinical trials in which a major obstacle includes the limitedclinical samples from patients. In this study, we obtained dataconsistent with the results using classic immunohistochemis-try5 and the studies from UMSCC cell lines (Figs. 2–4). Weobserved the best molecular response was associated with thehighest level of pretreatment p-AKT Ser 473, total AKT, andtotal STAT3 (data not shown), as well as with the stronggefitinib-suppressed p-EGFR Tyr1148, p-AKT Ser473, p-ERKSer185/187, and NF-nB p-65 Ser536 in the only molecularand clinical responder (Fig. 6). In addition, an increase ofcleaved caspase 3 activation was also observed in theresponder, which is consistent with the tumor apoptosisdata.5 In contrast, gefitinib failed to inhibit but rather inducedactivation of the MEK, STAT3, and/or NF-nB pathways inthe rest of nonresponder patients. Our study revealed theheterogeneity of cancer as a major hurdle that limits theeffective treatment. Such heterogeneity usually remainsundetected by standard histologic pathologic classificationand clinical grading systems. To address this problem, werecently have identified such subgroup-specific gene expres-sion signatures in HNSCC cell lines with increased frequencyof promoter binding sites for transcription factors NF-nB,AP-1, and STAT3 or p53 (23, 30), that support conclusionsdrawn from this study. Our study suggests that by utilizingRPMA and other proteomic technologies, it may be feasible toidentify clinically useful biomarkers associated with the higherrisk for HNSCC recurrence and metastasis, and provide moreaccurate prognosis and selection of therapy based on theproteomic signatures.

Disclosure of Potential Conflicts of Interest

The authors have received gefitinib for research and clinical trials fromAstraZeneca Inc., under a cooperative research development and clinical trialsagreement with the National Cancer Institute.

Acknowledgments

We thankJustin L. Ricker, MD, PhD, medical director at Abbott, Abbott Park, IL,and John Sunwoo, MD, assistant professor in the Department of Otolaryngology,Washington University School of Medicine, for reading and providing useful com-ments on themanuscript.



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