Differential Effects of Tyrosine Kinase Inhibitors on ... · Signal Transduction Differential Effects of Tyrosine Kinase Inhibitors on Normal and Oncogenic EGFR Signaling and Downstream

Signal Transduction

Differential Effects of Tyrosine Kinase Inhibitorson Normal and Oncogenic EGFR Signaling andDownstream EffectorsYoungjooKim1, Mihaela Apetri1, BeiBei Luo1, Jeffrey E. Settleman2, andKaren S. Anderson1

Abstract

Constitutive activation of EGFR due to overexpression or muta-tion in tumor cells leads to dysregulated downstream cellularsignaling pathways. Therefore, EGFR as well as its downstreameffectors have been identified as important therapeutic targets. TheFDA-approved small-molecule inhibitors of EGFR, gefitinib(Iressa) and erlotinib (Tarceva), are clinically effective in a subsetof patients with non–small cell lung cancer (NSCLC) whosetumors harbor activating mutations within the kinase domain ofEGFR. The current study examined effects of these drugs in 32Dcells expressing native (WT) or oncogenic (L858R) EGFR as well asin cancer cell lines A431 andH3255. Distinct patterns for gefitiniband erlotinib inhibition of EGFR autophosphorylation at individ-ual tyrosines were revealed for wild-type (WT) and L858R EGFR.Phosphorylation of Y845 has been shown to be important incancer cells and Y1045 phosphorylation is linked to Cbl-mediated

ubiquitination and degradation. Dramatic differences wereobserved by greater potency of these drugs for inhibiting down-stream effectors for L858R EGFR including Cbl and STAT5. Selec-tive targeting of Cbl may play a role in oncogene addiction andeffects on STAT5 identify features of signaling circuitry for L858REGFR that contribute to drug sensitivity and clinical efficacy. Thesedata provide new understanding of the EGFR signaling environ-ment and suggest useful paradigms for predicting patient responseto EGFR-targeted therapy as well as combination treatments.

Implications: This study offers fundamental insights for under-standing molecular mechanisms of drug sensitivity on oncogenicforms of EGFR and downstream signaling components as well asconsiderations for further drug optimization and design of com-bination therapy. Mol Cancer Res; 13(4); 765–74. �2015 AACR.

IntroductionCancer is the leading cause of death worldwide, and there are

limited options for treatment, which typically includes surgery,irradiation, and/or chemotherapy. Since the successful develop-ment of the first kinase-targeted drug, imatinib (Gleevec), whichinhibits the Abelson tyrosine kinase that promotes chronic mye-logenous leukemia (1), many drugs have been subsequentlydeveloped that target specific proteins that are mutationallyactivated or overexpressed in various forms of cancers. BecauseEGFR is overexpressed or constitutively active due tomutations invarious cancers, it is an important target for drug development.Erlotinib and gefitinib are 2 of the first FDA-approved drugsspecifically targeting EGFR in non–small cell lung cancer

(NSCLC). These compounds are small ATP mimetics that inhibitautophosphorylation of EGFR at its cytoplasmic tyrosine residuesby binding to the catalytic site. Notably, these 2 drugs are moreeffective in patients with activating mutations in the kinasedomain of EGFR. The most common activating EGFR mutationsare an in-frame deletion affecting exon 19 (delE746-A750) and apoint mutation at residue 858 in exon 21 (L858R; L858R is alsoknown as L834R; the difference in numbering is based upon the24 amino acid nuclear localization sequence that is absent inthe mature form of EGFR; ref. 2). The underlying mechanisms forenhanced potency are unclear. Previous structural studies showsimilar inhibitor binding in the kinase domain for native (wild-type, WT) and L858R-mutant forms of EGFR (3), and subsequentwork has suggested altered ATP affinity, dimerization propensity,and conformational enzyme dynamics as possible factors govern-ing increased drug sensitivity (4–6).

Phosphorylated tyrosine residues in EGFR serve as specificrecruitment sites for downstream signaling molecules that areinvolved in various cellular processes such as proliferation,migra-tion, and apoptosis (7). Although studies have shown that dif-ferent signaling molecules in pathways downstream of EGFR areactivated in many cancers, few global studies address linkingautophosphorylation of a tyrosine residue in EGFR to the acti-vation of specific downstream signaling molecules.

Studies byWu and colleagues show thatmany tyrosine residuesin EGFR are differentially phosphorylated in lung cancer com-pared with normal lung tissue and provide a phosphosignaturethat is strongly correlated with the lung cancer phenotype (8).Similarly, Klammer and colleagues looked at the phosphopro-teome profile from NSCLC cell lines identifying predictive

1Department of Pharmacology, Yale University School of Medicine,New Haven, Connecticut. 2Center for Molecular Therapeutics, Massa-chusetts General Hospital Cancer Center and HarvardMedical School,Charlestown, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Current address for J.E. Settleman: Genentech Inc. 1 DNA Way, South SanFrancisco, CA 94080; and current address for Y. Kim: Department of Chemistryand Physics, SUNY College at OldWestbury, 223 Store Hill Road, OldWestbury,NY 11568.

Corresponding Author: Karen S. Anderson, Yale University School of Medicine,333 Cedar Street, New Haven, CT 06520. Phone: 203-785-4526; Fax: 203-785-7670; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0326

�2015 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 765

on April 13, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 8, 2015; DOI: 10.1158/1541-7786.MCR-14-0326

http://mcr.aacrjournals.org/

phosphosignatures that differ between sensitive and resistant celllines (9). In another study, Chen and colleagues report distinctiveactivation patterns in constitutively active and gefitinib-sensitiveEGFRmutants comparedwithWTEGFR (10). It was observed thatgefitinib has variable growth-inhibitory effects in cells expressingdifferent EGFRmutants. These studies show that various cell lineswithdistinct phosphorylated EGFRdue tomutations have specificphosphosignatures that respond differently to the drugs. There-fore, correlating specific phosphosignatures to drug responsive-ness may be a useful strategy for individualized therapy.

Previously, our biochemical studies examined the temporalresolution of autophosphorylation for WT and oncogenic L858REGFR using the recombinant cytoplasmic domain of EGFRexpressed in Sf9 cells (5, 11). Differences in autophosphorylationkinetics and the unique signature patterns of drug sensitivity wereobserved between WT and L858R EGFR. With these biochemicalstudies as a foundation, we extended our studies at a cellular levelusing 32D cells, a myeloid cell line lacking endogenous EGFR.Isogenic 32D cells overexpressing either native (WT) or oncogenicL858R-mutant forms of EGFR allowed the study of normal andaberrant EGFR signaling and drug responsiveness without con-cern for cell line heterogeneity. Additional studies examined WTand L858R-mutant forms of EGFR in the setting of cancer cells.A431 is a human epidermoid carcinoma cell line overexpressingEGFR and H3255 is a human lung cancer cell line expressingL858R EGFR. These cell lines were included as part of an earlierstudy to understand the effects of the EGFR antibody cetuximab inlung cancer cells and xenografts expressing oncogenic forms ofEGFR (12). The current study was designed to address the fol-lowing mechanistic questions related to the clinical efficacy ofgefitinib and erlotinib: (i) Are differences in drug responsivenessobserved in EGFR autophosphorylation patterns for individualtyrosines in 32D cells expressing WT and L858R forms of EGFR?;(ii) Are some downstream pathways more significant than otherswhen comparing normal and oncogenic EGFR signaling?; and(iii) Can we identify key tyrosines in EGFR or downstreamsignaling molecules that may play prominent roles in determin-ing drug sensitivity in the context of oncogenic EGFR signaling?

The current study establishes that gefitinib and erlotinib havedifferential effects at a cellular level as assessed by examiningautophosphorylation of individual tyrosines in 32D cells expres-sing WT or L858R-mutant forms of EGFR, consistent with ourprevious biochemical studies. In addition, it was observed thatthere were marked differences in drug sensitivity with respect toinhibition of downstream signaling proteins. By examining nor-mal and oncogenic EGFR signaling in 32D cells, it was found thatboth drugs significantly impacted the activation of the Y845residue in L858R EGFR compared with WT EGFR. Among down-stream signaling proteins, STAT5 activation was substantiallydiminished by erlotinib (288-fold) and Cbl activation was mostaffected by gefitinib (267-fold) in L858R EGFR signaling relativeto WT EGFR signaling. Our results suggest that L858R EGFRsignaling may be mediated through activation of EGFR by autop-hosphorylation or Src phosphorylation of Y845 followed bySTAT5 activation. Inhibition of this pathway for L858R EGFRmaybe linked to the effectiveness of gefitinib. Likewise, the potentinhibition of Cbl activation in L858R signaling by erlotinibrelative to WT EGFR may circumvent receptor degradation andcontribute to an oncogene-addicted cellular phenotype. This in-depth analysis of receptor activation, downstream signaling, anddifferential effects of clinically important drugs aids in under-

standing mechanistic differences in normal and oncogenic EGFRsignaling. These major findings in 32D cell lines were well-translated to cancer cell lines. These results provide insights intothe complexity of the EGFR signaling network in human tumorsand pinpoint key features of downstream signaling that may beessential for designing even more selective therapies.

Materials and MethodsCell lines and culture conditions

The cell lines used in this study (32D, A431, and H3255cells) were kindly provided by Dr. Carlos Arteaga at VanderbiltUniversity School of Medicine (Nashville, TN; refs. 12, 13). The32D and A431 cells were originally obtained from ATCC. TheNCI H3255 cells have been previously characterized (14–16);they are heterozygous for the L858R missense mutation in exon21 of the EGFR gene. The IL3-dependent hematopoietic 32Dcell lines stably transfected with EGFR-WT and EGFR-L858Rwere maintained in RPMI-1640 containing 10% FBS and 10%WEHI conditioned medium (as a source of IL3) and supple-mented with antibiotics (penicillin/streptomycin, 100 U/mL).The human lung cancer cell line NCI H3255 was grown inRPMI-1640 supplemented with 10% FBS plus antibiotics (pen-icillin/streptomycin, 100 U/mL; gentamicin, 50 U/mL). Thehuman epidermoid carcinoma cell line A431 was maintainedin Improved MEM Znþ2 Option (Richter Modification) sup-plemented with 10% FBS and antibiotics (penicillin/strepto-mycin, 100 U/mL). All cells were grown at 37�C in a humidifiedatmosphere with 5% CO2.

Reagents and antibodiesEGF was purchased from R&D Systems. We used the following

antibodies: anti-phospho-EGFR (to phosphotyrosines 1045,1068, 1148, and 1173), total EGFR, Akt (#4691), phospho-Akt(Thr308, #9275), MAPK (#4695), phospho-MAPK (Thr202/Tyr204, #4370), PI3K (#11889), phospho-PI3K (Tyr458,#4228), PLCg1 (#5690), phospho-PLCg1 (Tyr783, #2821), Src(#2123), phospho-Src (Tyr416, #2101), Stat3 (#4904), phospho-Stat3 (Tyr705, #9145), phospho-Stat5 (Tyr694, #4322), Cbl(#2747), and horseradish peroxidase (HRP)-linked rabbit IgGsecondary antibody from Cell Signaling Technology; anti-phos-pho-EGFR (phosphotyrosines 845 and 992) from Millipore; andStat5 (sc-835), phospho-Cbl (Tyr700, sc-377571), and goat anti-mouse IgM-HRP (sc-2064) from Santa Cruz Biotechnology.

Rapid cell stimulation with EGFBefore EGF stimulation, the 32D nonadherent cells grown to a

density of approximately 2 � 106 cells/mL were starved for 3hours in serum-free RPMI-1640. After starvation, equal volumesof the cell suspension and EGF ligand in RPMI-1640 medium(200 ng/mL) were mixed using a KinTek RQF-3 rapid-quenchinstrument. The reactions were quenched at different time pointswith lysis buffer [50mmol/LHEPES (pH 7.5), 150mmol/L NaCl,1mmol/L EDTA, 1mmol/L EGTA, 10%glycerol, 1%TritonX-100,25 mmol/L NaF, protease inhibitors (Complete tablets, RocheDiagnostics), and 1 mmol/L sodium vanadate]. The zero timepoint was collected using RPMI-1640media alone in the absenceof EGF ligand and represented the unstimulated state. Lysateswere incubated on ice for 30minutes, cleared by centrifugation at13,000� g for 10minutes and the supernatants were collected forfurther analysis or stored at �80�C.

Kim et al.

Mol Cancer Res; 13(4) April 2015 Molecular Cancer Research766




Inhibition of EGFR phosphorylation32D-EGFR WT and 32D-EGFR L858R stable cell lines were

serum-starved as described previously (17). A431 and H3255cells grown to 80% confluence were starved overnight in serum-free medium. Before stimulation, cells were preincubated with arange of concentrations of gefitinib or erlotinib (0–10 mmol/L forWT and 0–0.1 mmol/L for L858R EGFR) for 30 minutes and thenstimulated with 100 ng/mL EGF for 2 minutes. 32D cells werelysed directly with lysis buffer, whereas A431 and H3255 cellswere washed with ice-cold PBS and scraped immediately afteradding lysis buffer. All lysates were cleared by centrifugationand supernatants were collected. Receptor phosphorylationwas detected by Western blotting with specific phospho-EGFRantibodies.

Western blot analysesEqual amounts of protein extracts (10 ng) were resolved by

SDS-PAGE (BioRad, 7.5% Tris-HCl gels) and then transferred topolyvinylidene difluoride (PVDF) membrane (Millipore). Themembrane was first incubated in blocking buffer (3% BSA inTBST-Tris-buffered saline with 0.05% Tween-20), then incubatedwith a primary antibody (dilution of antibodies were determinedas described previously; ref. 5), washed, and blotted with sec-ondary antibody conjugated with HRP (1:2,000). Blots weredeveloped by enhanced chemiluminescence (Amersham Phar-macia Biotech) and exposed to X-ray films. The level of receptorphosphorylation was evaluated from theWestern blotting using aMolecular Imager FX. Standard curves for each antibody weregenerated to determine the dilution factor necessary to obtain asignal that is linear in response to the amount of protein loaded.Adjusted antibody dilution was as follows: 1:2,000 for 845,1:5,000 for 992, 1:200 for 1045, 1:1,000 for 1068, 1:250 for1148, 1:5,000 for 1173, and1:2,000 for EGFR. To estimate the rateof EGFR phosphorylation, the intensity of bands was plottedagainst increasing times and the data were fitted to equation 1. Todetermine the concentration of kinase inhibitor (gefitinib orerlotinib) that resulted in 50% EGFR activity (IC50), the datawere fitted to the equation B.

Intensity ¼ Intensitymax � 1� exp ð�time � rate ofðautophosphorylationÞ þ constant ðAÞ

Percent activity ¼ activitymax � IC50ð Þ= IC50 þðkinase inhibitor½ �Þ þ constant ðBÞ

ResultsDifferential effect of gefitinib and erlotinib onautophosphorylation of EGFR

Previously, we showed that the pattern of gefitinib sensitivitytoward tyrosine residues determined at a biochemical level isunique inWT and L858R EGFR (5, 11). Using recombinant EGFRexpressed in Sf9 insect cells, we observed that gefitinib was morepotent for L858R EGFR thanWT and the ranges of drug sensitivityrequired to inhibit individual tyrosine residues were very differ-ent. To further examinedrug sensitivity in a cell culture context, weperformed similar experiments using 32D cell lines. 32D cells arean IL3-dependent myeloid cell line and EGFR-expressing 32Dcells become EGF-dependent in the absence of IL3. Because there

is no endogenous expression of EGFR, 32D cell lines with stableexpression of EGFR are widely used as an isogenic model systemfor studying EGFR signaling. To obtain IC50 values of gefitinib anderlotinib for EGFR inhibition, serum-starved 32D cells expressingWT or L858R EGFR were incubated with increasing concentra-tions of inhibitors for 30 minutes and then stimulated with EGFfor 2 minutes as described in Materials and Methods. EGF stim-ulation of 32D cell lines expressing WT and L858R-mutant formsof EGFR and kinetic analysis of EGFR phosphorylation estab-lished that a maximum level of EGFR activation was achievedafter 2 minutes (data not shown). Levels of phosphorylation ofindividual tyrosine residues for WT and L858R-mutant EGFR inthe presence of gefitinib or erlotinib were followed by Westernblot analysis using phospho-specific EGFR antibodies (Figs. 1and 2). Similar to the biochemical data obtained previously, IC50

values for drugs inhibiting the various tyrosine residues of EGFRvaried widely at a cellular level (Supplementary Table S1). Forexample, IC50 values for gefitinib forWT EGFR ranged from0.019to 0.083 mmol/L and those for erlotinib ranged from 0.015 to0.079 mmol/L. L858R EGFR was more sensitive to both gefitiniband erlotinib than WT. In addition to the overall differences inIC50 values, with erlotinib beingmore potent than gefitinib, therewere differential effects on the inhibition of tyrosine autopho-sphorylation in EGFR. For WT EGFR, gefitinib was most selective

Wild-type L858R

Gefitinib (μmol/L) Gefitinib (μmol/L)

0 0.04 0.1 0.5 1 10

Anti-pY845

Anti-pY992

Anti-pY1045

Anti-pY1068

Anti-pY1148

Anti-pY1173

Anti-EGFR

0 0.005 0.01 0.02 0.04 0.1

32D WT

32D L858R

(6)(1) (4)

(1) (2)

(4)

Tyrosine residue

0.09

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

0845 992 1045 1068 1148 1173

IC50

(μm

ol/L

)

A B

C

Figure 1.A and B, effect of gefitinib on WT (A) and L858R EGFR (B)autophosphorylation in 32D cells. C, bar graph representation of IC50 values ofgefitinib forWTandL858REGFR in 32Dcells. Parentheses indicate the ratio ofIC50 values of gefitinib for WT relative to those of L858R EGFR. After serumstarvation, cells were preincubated with the indicated concentrations ofgefitinib for 30 minutes and then stimulated with 100 ng/mL EGF for 2minutes. Cell lysates were run on SDS-PAGE and receptor phosphorylation atdifferent tyrosine residues was detected by Western blotting with specificphospho-EGFR antibodies.

Effect of Gefitinib and Erlotinib on Normal and Oncogenic EGFR Signaling

www.aacrjournals.org Mol Cancer Res; 13(4) April 2015 767




for Y1045 and Y992 (�0.02 mmol/L), followed by Y1173 (0.037mmol/L), and Y1148, Y1068, and Y845 (0.07–0.083 mmol/L).Erlotinib inhibited phosphorylation of tyrosine residues in asimilar order of potency as gefitinib. For L858R EGFR, the patternin drug sensitivity was altered relative to the WT. All tyrosineresidues except Y1068 were similarly sensitive to gefitinib withIC50 of 0.008 to 0.018 mmol/L versus 0.054 mmol/L for Y1068. Aswith WT EGFR, erlotinib showed a similar pattern of sensitivity,but with greater potency. Interestingly, gefitinib and erlotinibwere most potent for inhibition of Y1045 in both WT andL858R EGFR. This is consistent with our observations at a bio-chemical level (5). Y1045 of EGFR is a docking site for Cblproteins, which are E3 ligases responsible for ubiquitination anddegradation of EGFR (18, 19). Therefore, these results suggest thatdrugs inhibit phosphorylation at Y1045 and prevent EGFR fromubiquitination.

To quantitatively assess differential drug effects on tyrosineresidues of WT and L858R EGFR, the ratios of IC50 values (IC50

values of WT EGFR/IC50 values of L858R EGFR) were compared.

These values are represented as fold differences in parenthesesin Figs. 1C and 2C. Even though gefitinib and erlotinib weremorepotent toward L858REGFR thanWTEGFR in general (up to6-foldfor gefitinib and up to 30-fold for erlotinib), the most strikingdifference observed was in potency of the 2 drugs on Y845phosphorylation. As shown in Figs. 1C and 2C, the IC50 ofgefitinib in inhibiting Y845 phosphorylation in L858R EGFR wasabout 6-fold lower than that seen for WT EGFR and erlotinib hadabout 30-fold lower IC50 in inhibiting Y845 of L858R EGFR thanWT. Y845 is located in the activation loop and has been suggestedto be phosphorylated by the Src kinase, which is required for EGF-induced tumorigenesis (20–22). The Y845 residue could also beautophosphorylated, but this is not required for EGFR activation,unlike formany other receptor tyrosine kinases (22). These resultssuggest that the 2 EGFR inhibitors are more effective towardL858R EGFR by specifically inhibiting phosphorylation of theY845 residue, and that this effect is greaterwith erlotinib thanwithgefitinib.

Effect of gefitinib and erlotinib on phosphorylation ofdownstream signaling proteins

Autophosphorylation of EGFR leads to activation of down-stream signaling (23). Gefitinib and erlotinib have been shownto target EGFR by inhibiting autophosphorylation of EGFRtyrosine residues; however, further effects of these drugs ondownstream signaling proteins have not been previously stud-ied in isogenic cells in a comprehensive manner. Here, weexamined the effect of gefitinib and erlotinib on inhibition ofsignaling proteins downstream of WT and L858R EGFR bymeasuring phosphorylation levels using Western blot analysis.Briefly, serum-starved 32D cells expressing WT or L858R EGFRwere incubated with increasing concentrations of drugs for 30minutes and stimulated with EGF for 2 minutes. Phosphory-lation levels of downstream signaling proteins including Src,PI3K, Akt, STAT3, STAT5, ERK, PLCg , and Cbl were followed byWestern blot analysis using total and phosphospecific antibo-dies (Supplementary Fig. S1). These particular signaling pro-teins were selected because their overexpression and activationhave been linked to EGFR activation and are associated withcancer (24–27). Supplementary Table S2 summarizes IC50

values of gefitinib and erlotinib with respect to 8 downstreamsignaling proteins. These IC50 values do not represent directinhibition of signaling proteins by drugs, rather they examinehow the upstream inhibition of EGFR phosphorylation bythese drugs is manifested downstream and whether inhibitionof phosphorylation is amplified or dampened in the context ofWT and L858R EGFR signaling.

As shown in Supplementary Table S2A, in 32D cells expressingWT EGFR, the IC50 values of gefitinib for downstream EGFRsignaling proteins indicate that STAT3 and STAT5 are the mostsensitive (0.017–0.068 mmol/L), followed by PLCg and PI3K(0.13–0.16 mmol/L), then Akt and ERK (0.92–1.08 mmol/L), andfinally, Cbl and Src (3.74–4.12 mmol/L). Also in SupplementaryTable S2A are results for the 32D cells expressing L858R EGFR,showing that gefitinib was much more potent in exerting effectson downstream EGFR signaling proteins. Distinct drug sensitivitypatterns were observed. The IC50 values of gefitinib revealed thatthe downstream proteins most sensitive were STAT5 and PLCg(0.0038–0.008 mmol/L), followed by ERK, Cbl, STAT3, and AKT(0.012–0.028 mmol/L), whereas Src and PI3K were not inhibitedby as much as the highest concentration of gefitinib tested

Wild-type L858R

Erlotinib (μmol/L) Erlotinib (μmol/L)

A

C

B

0 0.04 0.1 0.5 1 10 0 0.005 0.01 0.02 0.04 0.1

Anti-pY845

Anti-pY992

Anti-pY1045

Anti-pY1068

Anti-pY1148

Anti-pY1173

Anti-EGFR

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

0845 992 1045 1068 1148 1173

Tyrosine residue

IC50

(μm

ol/L

)

32D WT

32D L858R

(30)

(2)

(2)

(4)

(5)(1)

Figure 2.A and B, effect of erlotinib on WT (A) and L858R EGFR (B)autophosphorylation in 32D cells. C, bar graph representation of IC50 values oferlotinib for WT and L858R EGFR in 32D cells. Parentheses indicate the ratioof IC50 values of erlotinib for WT to that of L858R EGFR. After serumstarvation, cells were preincubated with the indicated concentrations oferlotinib for 30 minutes and then stimulated with 100 ng/mL EGF for 2minutes. Cell lysateswere run on SDS-PAGE and receptor phosphorylation atdifferent tyrosine residues was detected by Western blotting with specificphospho-EGFR antibodies.

Kim et al.





(0.5 mmol/L). A comparison of fold differences for WT/L858Rcells is shown in the right hand column.

Similar to gefitinib, erlotinib was more potent toward L858REGFR than WT EGFR, with the exception of Src and PI3K phos-phorylation, as shown in Supplementary Table S2B.However, thepattern of erlotinib inhibition of downstream signaling proteinswas not the same as for gefitinib. In this case, the IC50 values forerlotinib toward WT EGFR signaling proteins showed that STAT3was the most sensitive (0.0061 mmol/L), followed by Src and Cbl(0.042 and 0.05 mmol/L, respectively), and PI3K, Akt, STAT5,PLCg , and ERK (0.46–0.84 mmol/L). For the L858R-mutant EGFRsignaling, the IC50 values of erlotinib for inhibition of down-stream phosphorylation of signaling proteins showed that STAT3was the most sensitive (0.00087 mmol/L), followed by Cbl,STAT5, and PLCg (0.0015–0.0048 mmol/L), and ERK and Akt

(0.019 and 0.023 mmol/L, respectively). Src and PI3K phosphor-ylationwas not inhibited by asmuch as 0.5mmol/L of erlotinib, aswas seen with gefitinib.

A summary of the ratio of IC50 values for each drug in thecontext of signaling proteins in WT and L858R EGFR-expressingcells is shown as bar graphs in Fig. 3A (gefitinib) and B (erlotinib).The data reveal that these drugs exhibit distinct patterns ofinhibition for downstream signaling pathways in WT andL858R EGFR-expressing cells. Those values having >5-fold differ-ence are shown. One of the most striking results was the effect ofgefitinib on Cbl phosphorylation, where the drug was 288-foldmore potent on L858R EGFR relative to WT (Fig. 3A). Cbl is anubiquitin ligase and is responsible for ubiquitination and ulti-mate targeting of EGFR for receptor degradation. On the otherhand, erlotinib showed the greatest fold difference in IC50 values

300

250

200

150

100

50

0

300

250

200

150

100

50

0

pCbl pERK pAKT pPLCγ pSTAT5


Impact of gefitinib

on downstream signaling proteins

Impact of erlotinibon downstream signaling proteins

Fold

diff

eren

ce b

etw

een

IC50

for

Fold

diff

eren

ce b

etw

een

IC50

for

WT

and

L858

R E

GFR

in 3

2D c

ells

WT

and

L858

R E

GFR

in 3

2D c

ells

A

B

Figure 3.A, bar graph representation of fold difference inratio of IC50 values of gefitinib inhibition ofdownstream signaling partners for WT andL858R EGFR in 32D cells. B, bar graphrepresentation of fold difference in ratio of IC50

values of erlotinib inhibition of downstreamsignaling partners for WT and L858R EGFR in32D cells. In each case, only those values having>5-fold difference are shown. In each case, afterserum starvation, cells were preincubated withthe indicated concentrations of gefitinib orerlotinib for 30minutes and then stimulatedwith100 ng/mL EGF for 2 minutes. Cell lysates wererun on SDS-PAGE and various EGFR signalingproteinswere detected byWestern blottingwithtotal and phospho-antibodies.






between WT and L858R EGFR-expressing cells in the context ofinhibition of downstream signaling protein STAT which in thiscase was 267-fold (Fig. 3B). These results suggest that the down-stream pathway effects of gefitinib and erlotinib are achieved inunique ways despite their similar chemical properties and directinhibition on EGFR.

To examine whether the effect of drugs on phosphorylation ofdownstream EGFR signaling proteins seen in 32D cells is alsoobserved in cancer cell lines, these studies were extended toevaluate A431 and H3255 cell lines. The A431 cancer cells over-express WT EGFR, whereas the H3255 cells overexpress themissense oncogenic L858R EGFR. The data for the effects ofgefitinib and erlotinib on A431 and H3255 are found in Supple-mentary Table S3A and S3B and Supplementary Fig. S2A and S2B,respectively. Figure 4 summarizes in bar graphs the fold differencein the ratio of IC50 values for WT (A431) and L858R (H3255)inhibition of downstream EGFR proteins for gefitinib (Fig. 4A)

and erlotinib (Fig. 4B). Those values showing >5-fold inhibitionare shown. Consistent with the results in 32D cells, in cancer cellsexpressing WT or L858R EGFR, the largest effect on downstreamEGFR signaling observed for gefitinib was on Cbl (100-fold) asshown in Fig. 4A. Also in accordwith 32D cells as illustrated in Fig.4B, for erlotinib, the most notable effect on EGFR-mediateddownstream signaling partners for WT versus L858R was STAT5(404-fold).

DiscussionCellular signaling via EGFR is initiated by ligand-stimulated

autophosphorylation of key tyrosine residues of EGFR that havebeen shown to recruit a host of downstream signaling proteinsleading to activation of various cellular pathways responsible forcontrolling growth and survival (28). Our previous study withFGFR1 (fibroblast growth factor receptor 1) have shown that the

100

90

80

70

60

50

40

30

20

10

0pCbl pERK pAKT pPLCγ pSTAT5


Impact of gefitinib



Impact of erlotinib

400

350

300

250

200

150

100

50

0

Fold

diff

eren

ce b

etw

een

IC50

Fold

diff

eren

ce b

etw

een

IC50

for

A43

1 and

H32

55 E

GFR

for

A43

1 and

H32

55 E

GFR

A

B

Figure 4.A, bar graph representation of fold difference forratio of IC50 values of gefitinib for WT versusL858R EGFR in A431 and H3255 cell lines. B, bargraph representation of fold difference for ratioof IC50 values of erlotinib for WT versus L858REGFR in A431 and H3255 cells lines. In each case,after serum starvation, cells were preincubatedwith the indicated concentrations of gefitinib orerlotinib for 30 minutes and then stimulatedwith 100 ng/mL EGF for 2 minutes. Cell lysateswere run on SDS-PAGE and various EGFRsignaling proteins were detected by Westernblotting with total and phospho-antibodies.

Kim et al.





order of autophosphorylation is important in providing bothtemporal and spatial resolutions to receptor signaling and thisorder is perturbed in an oncogenic form of FGFR1 (29, 30).Therefore, the pattern and timing of phosphorylation events onEGFR may be important for proper orchestration of downstreampartner recruitment and activation of relevant pathways in adefined manner for appropriate regulation of cell growth. Iden-tifying specific phosphosignatures of global EGFR signaling mayoffer insights thatmay be useful for predicting patient response toEGFR-targeted therapy as well as combination treatments. Ourprevious biochemical studies using a recombinant form of thecytoplasmic domain for EGFR expressed in Sf9 cells have shownthat there is a unique signature pattern of drug sensitivity betweenWT and L858R EGFR, indicating that the activation of specifictyrosine residues determines drug sensitivity (5). The currentstudy was designed to extend those findings in a cellular contextto determine how inhibition of EGFR autophosphorylation bygefinitib and erlotinib may differentially affect specific down-stream signaling proteins. EGFR autophosphorylation patternsmay emerge that predict drug sensitivity and explain the effec-tiveness of gefitinib and erlotinb in cells expressing L858R onco-genic form of EGFR relative to WT. Downstream signaling part-ners and pathways may be identified that are also incongruentlyinfluenced in oncogenic signaling and define essential features ofdrug efficacy.

Similar to our findings at the biochemical level, tyrosine 1045within the C-terminal tail of EGFR was identified as being one ofthe most sensitive to drug inhibition (Supplementary Table S1and Figs. S1 and S2; ref. 5). Phosphorylation of Y1045 has beenestablished as a mechanism to recruit Cbl, an ubiquitin ligase, forEGFR ubiquitination followed by degradation in the lysosome(31, 32). In another study by Gui and colleagues, hypopho-sphorylation at Y1045 was shown to cause impaired degradationand enhanced recycling of EGFR (33). Our results with gefitinibsuggest that this drug may prevent the oncogenic L858R form ofEGFR from degradation by inhibiting phosphorylation at Y1045.If thismutant form of EGFR is less susceptible to degradation, thisin turn might render cell growth more dependent on EGFRactivation, thereby enhancing oncogene "addiction" (34).Accordingly, the mode of action for gefitinib in cells expressingL858R EGFR may, in part, be linked to inhibition of Y1045 andsubsequent prevention of Cbl activation, as Cbl is downstream ofY1045. However, relative to L858R-expressing cells, gefitinib is aless potent inhibitor of Cbl phosphorylation in cells expressingWT EGFR (Figs. 3A and 4A). In this case, Cbl might be activatedthrough a different pathway. These observations may explain themolecular features of inhibiting key tyrosines on EGFR anddownstream effectors responsible for the clinical effectiveness ofgefitinib in treating patients having EGFR mutations. Recentstudies have suggested that Cbl inhibitors may be effective insome cancers (35).

Our findings with erlotinib imply that additional mechanismsmay by operative in patients with tumors harboring EGFR muta-tions such as L858R. The most significant difference in terms ofEGFR activation in comparing cells expressing WT and oncogenicEGFR was found to be erlotinib sensitivity toward inhibition ofphosphorylation of Y845 in EGFR, with an observed 30-folddifference in IC50 WT/ IC50 L858R as shown in SupplementaryTable S1B. Further examination of downstream signaling in thepresence of erlotinib revealed differences in the ratios for IC50WT/IC50 L858R for STAT5 and PLCg phosphorylation, with the largest

difference being STAT5 (Fig. 3B). This large difference implies thateffectiveness of erlotinib in patients with EGFR mutation may belargely driven by inactivation of the STAT5 pathway perhaps inconcert with PLCg1. Similar results were obtained with cancer celllines in which a 404-fold difference in erlotinib sensitivity wasnoted for STAT5 (Fig. 4B).

The differential erlotinib activity against Y845 suggests thatL858R EGFR might also be more dependent on Y845 activationrelative toWT EGFR. Therefore, inhibiting Y845 phosphorylationby erlotinib might provide a further explanation for the effective-ness in the context of EGFR L858Rmutation. Phosphorylation ofY845 has shown to be ligand-independent and associated withSTAT-dependent gene expression (36). Upon phosphorylation,the Y845 residue interactswith Src through an SH2domain that inturn activates STAT5 signaling. Y845 phosphorylation could beindirectly inhibited because of Src inhibition by drugs. However,as shown in Supplementary Tables S2 and S3, Src activity is notsubstantially changed by the 2 drugs. Therefore, it seems plausiblethat Y845 phosphorylation is directly inhibited by drugs ratherthan via Src inhibition. The lack of Src inhibition by gefitinibsuggests that L858R activation is Src-independent. Interestingly,studies done by Fu and colleagues showed that phosphorylationof Y845 is important for constitutive activation of L858R EGFR.They also found that L858R EGFR is more sensitive to PP2, a Srcinhibitor, compared withWT EGFR (36). This implies that L858REGFR uses Y845 phosphorylation as an activation mechanismand that Src inhibition by PP2 would preclude Y845 phosphor-ylation from exhibiting enhanced sensitivity toward PP2. Otherwork by Yang and colleagues suggests that mutation in the kinasedomain of EGFR alone is sufficient for phosphorylation of Y845and that Src kinase activity is dispensable (36). An additionalstudy by Chung and colleagues showed that NSCLC-associatedmutant forms of EGFR displayed enhanced association with Src(37). Activation of Y845 in L858R EGFR was also observed bySordella and colleagues (38).

Whether or not Y845phosphorylation is Src-dependent, L858REGFR signaling seems to involve Y845 activation. The resultsdescribed in the current study of gefitinib and erlotinib are inaccord with a similar mechanism in which L858R EGFR becomesconstitutively active due to the phosphorylation of the Y845residue. Therefore, the inhibitory drugs are more effective towardL858R EGFR by specifically targeting Y845.

Another important consequence of Src-mediated phosphory-lation of Y845 in EGFR signaling is the activation of STAT5 (39,40). Phosphorylation of Y699 within STAT5b is responsible formediating EGF-induced DNA synthesis. Interestingly, a connec-tion in EGFR signaling between EGFR Y845 and STAT5 is repre-sented in Figs. 3B and 4B and Supplementary Tables S2B and S3B.In the case of erlotinib, the level of phosphorylation for STAT5wasmuch lower in the presence of drug suggesting a possible linkbetween Y845 phosphorylation and STAT5 activation. Src phos-phorylation of EGFR on Y845 has a major role in regulation ofcellular functions, as described in reports by Sato and colleagues(41). Src is negatively regulated byphosphorylation and its signal-dependent transient activation contributes to cellular responsessuch as proliferation and differentiation. Although Y845 is dis-pensable for cellular functions regulated by EGFR, transpho-sphorylation of Y845 by Src plays an important role in severalaspects of cellular functions. Thus, it was reported by Gotoh andcolleagues (42) that the Y845F mutant of EGFR was able totransform NIH3T3 cells. STAT is a possible mediator of the






Y845 phosphorylation–dependent synergy between EGFR andSrc. In breast cancer cells, STAT5b was identified as a majortyrosine-phosphorylated protein and expression of the Y845Fmutant of EGFR had a negative effect on activation of STAT5b.Moreover, activated EGFR–STAT3 signaling promotes tumor sur-vival in vivo in NSCLC (43).

Our study suggests that the efficacy of erlotinib in L858REGFR-expressing 32D cells is a result of drug effects on Y845and STAT5 phosphorylation. Sordella and colleagues reportedthat activating mutations in EGFR selectively activate Akt andSTAT pathways that are important in lung cancer cell survival byactivating an anti-apoptotic pathway and gefitinib inhibits thissurvival pathway in EGFR-dependent cells (38). Other studiessupport a role for Src family kinases to link signals from growthfactors to downstream effector pathways (44, 45). An under-standing of these phosphorylation events in signal propagationenables prediction of drug sensitivity to small-molecule inhibi-tors such as gefitinib and erlotinib. Song and colleagues showedthat a Src kinase inhibitor, dasatinib, selectively induces apo-ptosis in lung cancer cells dependent on EGFR signaling forsurvival (46) suggesting that Src is in fact themain player in EGFRsignaling. Our studies further validate the importance of Src inlinking EGFR signaling to activation of the STAT pathway viaphosphorylation of tyrosine 845 in EGFR and suggest that drugssuch as gefitinib and erlotinib are much more effective in thecontext of EGFR-activating mutations due to their specificinhibition of phosphorylation of tyrosine residue 845, leadingto apoptosis. Activation of Src and STAT5 has also beenreported in squamous cell carcinoma of the head and neck(SCCHN; refs. 47, 48). Koppikar and colleagues showed thatcombined treatment with Src inhibitor AZD0530 and gefitinibled to greater inhibition of SCCHN compared with single-inhibitor treatment (49). In another study, Koppikar and col-leagues presented that STAT5 activation contributes to theresistance of SCCHN cells to the EGFR tyrosine kinase receptor,erlotinib (50). Taken together with our studies, combinationtherapy using inhibitors of both STAT5 and EGFR would bemore effective treatment in patients with lung cancer. Manyinhibitors targeting STAT pathway in various cancers have beendeveloped (51).

In summary, we have examined the effect of 2 FDA-approvedtyrsoine kinase inhibitors, gefitinib and erlotinib on inhibition ofEGFR and its downstream signaling proteins. The IC50 values forgefitinib and erlotinib inhibition with respect to key tyrosineresidues in EGFR and essential downstream signaling proteinswere determined for normal and oncogenic EGFR signaling. Themost significant difference in terms of EGFR activation was foundto be erlotinib activity toward tyrosine 845 in L858R EGFRsignaling, which exhibited about 30-fold lower IC50 value thannormal WT signaling. For downstream signaling proteins, a 288-fold difference in IC50 WT/IC50 L858R was observed for Cblinhibition following gefitinib in 32D cells. Erlotinib showed a267-fold difference in IC50 WT/IC50 L858R for STAT5 phosphor-ylation in 32D cells. Similarly, in comparing A431 cells andH3255, there was 100-fold difference in IC50 WT/IC50 L858R forCbl inhibition following gefitinib and 404-fold difference in IC50

WT/IC50 L858R for STAT5 inhibition following erlotinib. Figure 5provides a diagrammatic representation of gefitinib and erlotinibeffects on EGFR downstream signaling pathways. These studieshighlight the importance of Y1045 in EGFR and downstreamsignaling protein Cbl for the efficacy of gefitinib on L858R EGFR.In addition, they suggest that constitutive activation of L858REGFR is mediated through phosphorylation of Y845 followed byactivation of the STAT5 downstream pathway, and erlotinibseems to specifically target these pathways in the context ofoncogenic mutant forms of EGFR. This information provides amore detailed understanding of the effectiveness of gefitinib anderlotinib. Moreover, this in-depth analysis may point to newavenues to explore for designing better drugs and combinationtherapy targeting not only EGFRbut also its downstream signalingproteins when treating various forms of cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.S. AndersonDevelopment of methodology: M. Apetri, K.S. AndersonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Kim, M. Apetri, B. Luo

TK

C-terminus

Erlotinib

Gefitinib

L858R

Src

STAT5Y845

Y1045

Y1068Y1148

Y1173

Y992

STAT3

Grb2

Shc Raf

PI3K AKT

ERKMEK

Cbl

PLCg

Figure 5.Diagram of EGFR signaling pathway showingimpact of gefitinib and erlotinib on EGFRdownstream signaling proteins.

Kim et al.





Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Kim, B. Luo, J.E. Settleman, K.S. AndersonWriting, review, and/or revision of the manuscript: Y. Kim, J.E. Settleman,K.S. AndersonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Luo

AcknowledgmentsThe authors thank Dr. Carlos Arteaga at Vanderbilt University School of

Medicine for kindly providing cell lines and Dr. Christal Sohl for helpfulcomments on this article.

Grant SupportThis research was supported by National Institute of Health grants NCI

CA127580 and CA125284 to K.S. Anderson.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 9, 2014; revised December 11, 2014; accepted December 16,2014; published OnlineFirst January 8, 2015.

References1. Mauro MJ, Druker BJ. STI571: a gene product-targeted therapy for leuke-

mia. Curr Oncol Rep 2001;3:223–7.2. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan

BW, et al. Activating mutations in the epidermal growth factor receptorunderlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med 2004;350:2129–39.

3. YunCH, BoggonTJ, Li Y,WooMS,GreulichH,MeyersonM, et al. Structuresof lung cancer-derivedEGFRmutants and inhibitor complexes:mechanismof activation and insights into differential inhibitor sensitivity. Cancer Cell2007;11:217–27.

4. Shan Y, Eastwood MP, Zhang X, Kim ET, Arkhipov A, Dror RO, et al.Oncogenic mutations counteract intrinsic disorder in the EGFR kinase andpromote receptor dimerization. Cell 2012;149:860–70.

5. Kim Y, Li Z, Apetri M, Luo B, Settleman JE, Anderson KS. Temporalresolution of autophosphorylation for normal and oncogenic forms ofEGFR and differential effects of gefitinib. Biochemistry 2012;51:5212–22.

6. Gajiwala KS, Feng J, Ferre R, Ryan K, Brodsky O, Weinrich S, et al. Insightsinto the aberrant activity of mutant EGFR kinase domain and drugrecognition. Structure 2013;21:209–19.

7. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–25.

8. Wu CJ, Cai T, Rikova K, Merberg D, Kasif S, Steffen M. A predictivephosphorylation signature of lung cancer. PLoS One 2009;4:e7994.

9. KlammerM, Kaminski M, Zedler A, Oppermann F, Blencke S, Marx S, et al.Phosphosignature predicts dasatinib response in non-small cell lungcancer. Mol Cell Proteomics 2012;11:651–68.

10. Chen YR, Fu YN, Lin CH, Yang ST, Hu SF, Chen YT, et al. Distinctiveactivation patterns in constitutively active and gefitinib-sensitive EGFRmutants. Oncogene 2006;25:1205–15.

11. MulloyR, FerrandA,KimY, Sordella R, BellDW,HaberDA, et al. Epidermalgrowth factor receptormutants fromhuman lung cancers exhibit enhancedcatalytic activity and increased sensitivity to gefitinib. Cancer Res 2007;67:2325–30.

12. Perez-Torres M, Guix M, Gonzalez A, Arteaga CL. Epidermal growth factorreceptor (EGFR) antibody down-regulates mutant receptors and inhibitstumors expressing EGFR mutations. J Biol Chem 2006;281:40183–92.

13. Mukohara T, Engelman JA,HannaNH, YeapBY, Kobayashi S, LindemanN,et al. Differential effects of gefitinib and cetuximab on non-small-cell lungcancers bearing epidermal growth factor receptor mutations. J Natl CancerInst 2005;97:1185–94.

14. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFRmutations in lung cancer: correlation with clinical response to gefitinibtherapy. Science 2004;304:1497–500.

15. Tracy S, Mukohara T, Hansen M, Meyerson M, Johnson BE, Janne PA.Gefitinib induces apoptosis in the EGFRL858R non-small-cell lung cancercell line H3255. Cancer Res 2004;64:7241–4.

16. Fujishita T, Loda M, Turner RE, Gentler M, Kashii T, Breathnach OS, et al.Sensitivity of non-small-cell lung cancer cell lines established frompatientstreated with prolonged infusions of Paclitaxel. Oncology 2003;64:399–406.

17. Yang S, Qu S, Perez-Tores M, Sawai A, Rosen N, Solit DB, et al. Associationwith HSP90 inhibits Cbl-mediated down-regulation of mutant epidermalgrowth factor receptors. Cancer Res 2006;66:6990–7.

18. Yokouchi M, Kondo T, Houghton A, Bartkiewicz M, Horne WC, Zhang H,et al. Ligand-induced ubiquitination of the epidermal growth factor

receptor involves the interaction of the c-Cbl RING finger and UbcH7.J Biol Chem 1999;274:31707–12.

19. Levkowitz G, Waterman H, Ettenberg SA, Katz M, Tsygankov AY, Alroy I,et al. Ubiquitin ligase activity and tyrosine phosphorylation underliesuppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell 1999;4:1029–40.

20. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ. c-Src-mediatedphosphorylation of the epidermal growth factor receptor on Tyr845 andTyr1101 is associated with modulation of receptor function. J Biol Chem1999;274:8335–43.

21. Maa MC, Leu TH, McCarley DJ, Schatzman RC, Parsons SJ. Potentiation ofepidermal growth factor receptor-mediated oncogenesis by c-Src: implica-tions for the etiology of multiple human cancers. Proc Natl Acad Sci U S A1995;92:6981–5.

22. Tice DA, Biscardi JS, Nickles AL, Parsons SJ. Mechanism of biologicalsynergy between cellular Src and epidermal growth factor receptor. ProcNatl Acad Sci U S A 1999;96:1415–20.

23. Yarden Y, SliwkowskiMX.Untangling the ErbB signalling network. Nat RevMol Cell Biol 2001;2:127–37.

24. Reungwetwattana T, Dy GK. Targeted therapies in development for non-small cell lung cancer. J Carcinog 2013;12:22.

25. Harada D, Takigawa N, Kiura K. The role of STAT3 in non-small cell lungcancer. Cancers 2014;6:708–22.

26. Fumarola C, Bonelli MA, Petronini PG, Alfieri RR. Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer. Biochem Pharmacol 2014;90:197–207.

27. Thomas SM, Coppelli FM, Wells A, Gooding WE, Song J, Kassis J, et al.Epidermal growth factor receptor-stimulated activation of phospholipaseCgamma-1 promotes invasion of head and neck squamous cell carcinoma.Cancer Res 2003;63:5629–35.

28. Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell 2010;141:1117–34.

29. Furdui CM, Lew ED, Schlessinger J, Anderson KS. Autophosphorylation ofFGFR1 kinase is mediated by a sequential and precisely ordered reaction.Mol Cell 2006;21:711–7.

30. Lew ED, Furdui CM, Anderson KS, Schlessinger J. The precise sequence ofFGF receptor autophosphorylation is kinetically driven and is disrupted byoncogenic mutations. Sci Signal 2009;2:ra6.

31. Grovdal LM, Stang E, Sorkin A, Madshus IH. Direct interaction of Cbl withpTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR tolysosomes for degradation. Exp Cell Res 2004;300:388–95.

32. Ravid T, Heidinger JM, Gee P, Khan EM, Goldkorn T. c-Cbl-mediatedubiquitinylation is required for epidermal growth factor receptor exit fromthe early endosomes. J Biol Chem 2004;279:37153–62.

33. Gui A, Kobayashi A, Motoyama H, Kitazawa M, Takeoka M, Miyagawa S.Impaired degradation followed by enhanced recycling of epidermalgrowth factor receptor caused by hypo-phosphorylation of tyrosine1045 in RBE cells. BMC Cancer. 2012;12:179.

34. Jiang X, Huang F, Marusyk A, Sorkin A. Grb2 regulates internalization ofEGF receptors through clathrin-coated pits. Mol Biol Cell 2003;14:858–70.

35. Guedat P, Colland F. Patented small molecule inhibitors in the ubiquitinproteasome system. BMC Biochem 2007;8 Suppl 1:S14.

36. Fu YN, YehCL,ChengHH, YangCH, Tsai SF,Huang SF, et al. EGFRmutantsfound in non-small cell lung cancer show different levels of sensitivity tosuppression of Src: implications in targeting therapy. Oncogene 2008;27:957–65.






37. Chung BM, Raja SM, Clubb RJ, Tu C, George M, Band V, et al. Aberranttrafficking of NSCLC-associated EGFR mutants through the endocyticrecycling pathway promotes interaction with Src. BMC Cell Biol 2009;10:84.

38. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFRmutations in lung cancer activate anti-apoptotic pathways. Science2004;305:1163–7.

39. KlothMT, Catling AD, Silva CM.Novel activation of STAT5b in response toepidermal growth factor. J Biol Chem 2002;277:8693–701.

40. Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ, Silva CM.STAT5b, a mediator of synergism between c-Src and the epidermal growthfactor receptor. J Biol Chem 2003;278:1671–9.

41. Sato K. Cellular functions regulated by phosphorylation of EGFR onTyr845. Int J Mol Sci 2013;14:10761–90.

42. Gotoh N, Tojo A, Muroya K, Hashimoto Y, Hattori S, Nakamura S, et al.Epidermal growth factor-receptor mutant lacking the autophosphorylationsites induces phosphorylationof Shc protein and Shc-Grb2/ASHassociationand retains mitogenic activity. Proc Natl Acad Sci U S A 1994;91:167–71.

43. Haura EB, Zheng Z, Song L,Cantor A, Bepler G. Activated epidermal growthfactor receptor-Stat-3 signaling promotes tumor survival in vivo in non-small cell lung cancer. Clin Cancer Res 2005;11:8288–94.

44. Ishizawar R, Parsons SJ. c-Src and cooperating partners in human cancer.Cancer Cell 2004;6:209–14.

45. Yu H, Jove R. The STATs of cancer–newmolecular targets come of age. NatRev Cancer 2004;4:97–105.

46. Song L, Morris M, Bagui T, Lee FY, Jove R, Haura EB. Dasatinib (BMS-354825) selectively induces apoptosis in lung cancer cells dependent onepidermal growth factor receptor signaling for survival. Cancer Res 2006;66:5542–8.

47. Xi S, Zhang Q, Gooding WE, Smithgall TE, Grandis JR. Constitutiveactivation of Stat5b contributes to carcinogenesis in vivo. Cancer Res2003;63:6763–71.

48. Xi S, Zhang Q, Dyer KF, Lerner EC, Smithgall TE, Gooding WE, et al. Srckinases mediate STAT growth pathways in squamous cell carcinoma of thehead and neck. J Biol Chem 2003;278:31574–83.

49. Koppikar P, Choi SH, Egloff AM, Cai Q, Suzuki S, Freilino M, et al.Combined inhibition of c-Src and epidermal growth factor receptor abro-gates growth and invasion of head andneck squamous cell carcinoma. ClinCancer Res 2008;14:4284–91.

50. Koppikar P, Lui VW, Man D, Xi S, Chai RL, Nelson E, et al. Constitutiveactivation of signal transducer and activator of transcription 5 contributesto tumor growth, epithelial-mesenchymal transition, and resistance toepidermal growth factor receptor targeting. Clin Cancer Res 2008;14:7682–90.

51. Furqan M, Akinleye A, Mukhi N, Mittal V, Chen Y, Liu D. STAT inhibitorsfor cancer therapy. J Hematol Oncol 2013;6:90.


Kim et al.




2015;13:765-774. Published OnlineFirst January 8, 2015.Mol Cancer Res Youngjoo Kim, Mihaela Apetri, BeiBei Luo, et al. Oncogenic EGFR Signaling and Downstream EffectorsDifferential Effects of Tyrosine Kinase Inhibitors on Normal and

Updated version

10.1158/1541-7786.MCR-14-0326doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2015/01/09/1541-7786.MCR-14-0326.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/13/4/765.full#ref-list-1

This article cites 51 articles, 24 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/13/4/765To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-14-0326

http://mcr.aacrjournals.org/content/suppl/2015/01/09/1541-7786.MCR-14-0326.DC1

http://mcr.aacrjournals.org/content/13/4/765.full#ref-list-1

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/13/4/765


Documents

Differential Effects of Tyrosine Kinase Inhibitors on ... · Signal Transduction Differential Effects of Tyrosine Kinase Inhibitors on Normal and Oncogenic EGFR Signaling and Downstream