ORIGINAL ARTICLE

Gefitinib, but Not Erlotinib, is a Possible Inducer of
Fra-1-mediated Interstitial Lung Disease
Yasunari Takada and Koichi Matsuo

Laboratory of Cell and Tissue Biology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on December 9, 2011)(Revised for publication on May 31, 2012)(Accepted for publication on June 1, 2012)

Gefitinib is an anticancer drug developed to inhibit the tyrosine kinase activity of the epidermal growth
factor receptor (EGFR). Two structurally-related EGFR tyrosine kinase inhibitors, gefitinib (Iressa)
and erlotinib (Tarceva), are used as oral chemotherapy by patients with non-small-cell lung cancer.
Immediately after introduction of gefitinib to clinical practice, interstitial lung disease was identified
as a life-threatening adverse effect, although this condition can be well managed. It is still unclear
whether gefitinib and other EGFR inhibitors induce similar adverse effects in lung. We previously established mouse models of interstitial lung disease in which gefitinib induces expression of Fosl1 (which
encodes the AP-1 transcription factor Fra-1) in the presence of exogenous or endogenous Toll-likereceptor ligands, leading to abnormal cytokine and chemokine expression. Here, we compared andmonitored the effects of EGFR inhibitors gefitinib, erlotinib and AG1517 (PD153035) on the mRNA
expression levels of Fosl1, Tnf and Ccl2. Unexpectedly, gefitinib, but not the other tyrosine kinase inhibitors, elicited the Fosl1 expression profile proposed to be predictive of interstitial lung disease, suggesting that gefitinib-induced interstitial lung disease is an off-target effect not elicited by erlotinib.
(doi: 10.2302/kjm.2011-0009-OA; Keio J Med 61 (4) : 120–127, December 2012)

Keywords: gefitinib, erlotinib, EGFR inhibitor, interstitial lung disease

Introduction
Gefitinib (Iressa, ZD1839) was developed as a selective
inhibitor of the tyrosine kinase activity of the epidermal
growth factor receptor (EGFR) and has been used to treat
patients with advanced non-small-cell lung cancer.1–3
Other EGFR inhibitors, such as erlotinib (Tarceva), arealso widely used.4,5 Like other chemotherapeutic agents,
gefitinib treatment can cause adverse effects, most frequently skin rash and aminotransferase elevation, but
occasionally life-threatening interstitial lung disease canoccur. The incidence of interstitial lung disease in patients treated with gefitinib is currently about 1% (0.64%
in regions outside Japan).6,7 Since the initial reports ofgefitinib-associated fatalities appeared, gefitinib-induced
interstitial lung disease has been reduced in Japan (from5.8% in 2003–2004 to 1.28% in 2010), highlighting theimportance of appropriate patient selection based on risk
factors such as preexisting pulmonary fibrosis.6,7 Little is
known about the molecular target underlying gefitinibmediated interstitial lung disease, although there are reports that gefitinib has other targets in addition to EGFR.8
Tumor tissue destruction mediated by gefitinib andother chemotherapeutic agents likely produces endogenous Toll-like receptor (TLR) ligands, including hyaluronan and heparan sulfate.9,10 We recently reported that
gefitinib administration to mice along with the exogenous
TLR ligand lipopolysaccharide (LPS) or various endogenous ligands induces interstitial lung disease.11 Micetreated with gefitinib and LPS showed elevated expression of the transcription factor Fos-related antigen-1 (Fra1, encoded by Fosl1) in lung tissues. Fra-1, a member of
the activator protein 1 (AP-1) (Fos/Jun) transcription factor family, activates and/or represses various target genes

Reprint requests to: Koichi Matsuo, MD, PhD, Laboratory of Cell and Tissue Biology, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku, Tokyo 160-8582, Japan, E-mail: matsuo@z7. keio.jp
Copyright © 2012 by The Keio Journal of Medicine
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Keio J Med 2012; 61 (4): 120–127

by heterodimerizing with a Jun family protein, such as cJun, JunB or JunD.12 In the lungs of mice treated with gefitinib and LPS, Fra-1 upregulation inhibits expression of
the genes of proinflammatory cytokines such as Tnfa [encoding tumor necrosis factor-α (TNF-α)] and induces thatof cytokines such as Ccl2 [encoding monocyte chemoattractant protein 1 (MCP-1)]. In Fra-1 transgenic mice,
which show preexisting pulmonary fibrosis, LPS treatment alone results in severe lung injury associated with
reduced inflammatory cytokine production and increased
chemokine production.11 The observation of increased
chemokine production in Fra-1 transgenic mice was not
anticipated, since these mice show reduced inflammatory responses in bone and the intestinal tract.13,14 That
Fra-1 differentially modulates Tnfa and Ccl2 expression
is further supported by the observation that mice lacking
Fra-1 do not show skewed expression of cytokines andchemokines and are resistant to gefitinib/LPS-induced
lung injury.11 In addition, inhibition of MCP-1 by neutralizing antibody or a small-molecule inhibitor against its
receptor partially prevents lung injury. These and otherobservations suggest that gefitinib, in the presence ofchemotherapy-induced tissue destruction, induces lunginjury through transcriptional activation of Fra-1 and its
downstream targets.11
In this study, we used both cultured macrophages andbleomycin-induced lung injury experiments to determine
whether EGFR inhibitors such as gefitinib and erlotinib
act similarly to induce interstitial lung disease. We reportthat gefitinib is distinct from other EGFR inhibitors inthat it activates the interstitial lung disease-causing signaling cascade by an off-target effect.
Materials and Methods
EGFR inhibitors
For mouse studies, gefitinib (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy) quinazolin-4-amine; AstraZeneca, London, UK) or erlotinib(N-(3-ethynylphenyl) [6,7-bis (2-methoxyethoxy) quinazolin-4-yl]amine; Roche, Basel, Switzerland) tablets
were ground into powder using a mortar and pestle andsuspended in water. For cell culture studies, gefitinib, erlotinib and AG1517 (4-(3-bromoanilino)-6,7-dimethoxyquinazoline; PD153035, Calbiochem, La Jolla, CA, USA)
were suspended in dimethyl sulfoxide (DMSO) to makestock solutions (Fig. 1).
Cell culture
Mouse primary bone marrow macrophages were prepared as previously described.15,16 Briefly, tibiae and femora from adult wild-type C57BL/6 J mice were harvested
by flushing with Dulbecco’s modified Eagle’s medium

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Fig. 1 Structures of EGFR inhibitors used in this study.
Gefitinib, erlotinib and AG1517 all exhibit quinazoline and aniline
moieties. Morpholino, chloro and fluoro moieties specific to gefitinib are enclosed by dashed lines.

containing 10% fetal bovine serum and penicillin–streptomycin. After overnight culture in 10-cm tissue culture
dishes (Falcon, Franklin Lakes, NJ, USA), floating cells
were collected and expanded in the presence of 10 ng/ml
macrophage-colony stimulating factor (R&D, Minneapolis, MN, USA). Cells were re-plated and treated with 10
μM of gefitinib, erlotinib or AG1517 in DMSO or vehicle
for 30 min, and then 0.1 µg/ml LPS (S. minnesota Re595;
Sigma-Aldrich, St. Louis, MO, USA) was added to themedium for 12 h. Where indicated, each inhibitor wasadded to the culture medium at 30 min before, 30 min
after or 180 min after the start of LPS stimulation.
Chemotherapy-induced lung disease models
Six-week-old female ICR mice (a strain initiated at theInstitute for Cancer Research and also known as CD-1)
purchased from Clea Japan (Tokyo, Japan) were anesthetized, intratracheally administered 5 mg/kg bleomycin(Nippon Kayaku, Tokyo, Japan) and treated with gefitinibas described.17 Briefly, 250 mg/kg gefitinib or 100 mg/kg
erlotinib was given orally 1 h before bleomycin admin-

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Takada Y and Matsuo K: Gefitinib and Erlotinib in Interstitial Lung Diseaseistration, and then was administered 6 days a week for 3
weeks. The lung was isolated and fixed with 4% paraformaldehyde and embedded in paraffin. Sections 7 μm thickwere prepared and stained with hematoxylin and eosin.
All animal experiments were conducted in accordancewith institutional review board-approved protocols.
Quantitative reverse transcription polymerase chain
reaction
Total RNA was isolated using ISOGEN (NIPPON
GENE, Tokyo, Japan). The reverse transcriptase reaction was performed with 1 µg of total RNA using PrimeScript (TAKARA BIO, Tokyo, Japan). cDNA was subjected to real-time polymerase chain reaction (PCR)
using ABI 7500 Fast (Life Technologies, Grand Island,
NY, USA). TaqMan PCR was performed using Premix
Ex Taq (TAKARA BIO). TaqMan probes specific for
GAPDH (Mm03302249_g1), Tnfa (Mm00443258_m1),
Ccl2 (Mm00441242_m1), Fosl1 (Mm00487429_m1), Fos(Mm00487425_m1) and Fosl2 (Mm00484442_m1) werepurchased from TaqMan Assays-on-Demand Gene Expression Products (Life Technologies). All PCR assays
were performed in triplicate. Gene expression levels werenormalized to that of GAPDH.
Statistical analysis
Statistical significance was determined using an unpaired two-tailed Student’s t test.
Results
Gefitinib-dependent modulation of gene expression
changes seen following LPS challenge
Our previous studies suggested a pathogenesis modelof gene expression changes associated with gefitinibinduced interstitial lung disease (Fig. 2). These findings
prompted us to ask whether the EGFR inhibitors erlotinib and AG1517 could alter LPS-induced changes ingene expression in a similar way to that of gefitinib. Bonemarrow macrophages from mice were exposed to gefitinib, erlotinib or AG1517 from 30 min before 12-h LPS
treatment. Unexpectedly, LPS-stimulated Tnfa upregulation was blocked only in gefitinib-pretreated cells (Fig.
3A). Likewise, Ccl2 expression following LPS treatment
was enhanced only in gefitinib-pretreated cells (Fig. 3B).
Most importantly, Fosl1 expression, which is mildly upregulated in untreated cells stimulated with LPS, wassignificantly elevated by gefitinib pretreatment but notby treatment with erlotinib or AG1517 (Fig. 3C). Ectopic transgenic expression of Fosl2 in various organs reportedly promotes generalized fibrosis, especially in thelung.18 Therefore we investigated whether other Fos fam-Fig. 2 The mouse pathogenesis model of gefitinib-induced interstitial lung disease following chemotherapy.
Fosl1 is activated by gefitinib in the presence of TLR signaling.
Note that the direct target protein of gefitinib responsible for
Fosl1 activation has not yet been identified and is indicated by“X.” Dashed arrows indicate the LPS and bleomycin treatments
used in this study.

ily genes, such as Fos or Fosl2, were induced by gefitinib,
erlotinib or AG1517 treatment. None of the three EGFR
inhibitors tested induced Fos or Fosl2 in macrophages in
the presence or absence of LPS stimulation (Fig. 3D, E).
These results suggest that a gefitinib-specific mechanism
differing from that of other EGFR inhibitors underlies altered Fosl1 and cytokine–chemokine expression in LPStreated cells.
Next, we determined whether and how changing thetime frame of EGFR inhibitor treatment altered Tnfa,
Ccl2 and Fosl1 expression. Each inhibitor was added
30 min before LPS addition or 30 or 180 min after LPS
addition. When EGFR inhibitors were added after theKeio J Med 2012; 61 (4): 120–127

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Fig. 3 The effect of gefitinib on Tnfa, Ccl2, and Fosl1 expression in vitro.
Mouse primary bone marrow macrophages were pretreated for 30 min with 10 μM of gefitinib (Gef), erlotinib (Erl), or AG1517 (AG),and then stimulated with 0.1 µg/ml LPS. Expression levels of Tnfa (A), Ccl2 (B), Fosl1 (C), Fos (D) and Fosl2 (E) were analyzed byquantitative reverse transcription polymerase chain reaction (qRT-PCR) after 12 h. Results are shown relative to the expression of
GAPDH as means ±SEM of triplicate cultures. *p < 0.05.

start of LPS treatment, gefitinib administration had no
significant effect on Tnfa, Ccl2 or Fosl1 expression following LPS administration (Fig. 4A, B, C). These data
show that gefitinib must be added to the culture before
LPS treatment to alter the expression of these genes beyond the effect of LPS alone.
The effect of gefitinib in bleomycin-induced lung injury
Ccl2 and Fosl1 expression levels are upregulated in thelungs of wild-type mice treated with gefitinib and bleo-

mycin.11 We therefore examined whether erlotinib combined with bleomycin could modulate Ccl2 and Fosl1
mRNA levels in the lungs of wild-type mice in a similar way to that of gefitinib. Three weeks after bleomycin
administration, neither erlotinib nor gefitinib treatments
significantly affected Tnf expression, and both increased
Ccl2 expression (Fig. 5A, B). However, importantly, gefitinib induced Fosl1 expression, whereas erlotinib did
not (Fig. 5C). Thus, gefitinib treatment, but not erlotinib
treatment, enhanced bleomycin-induced lung injury,
which was characterized by accumulation of infiltrating

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Takada Y and Matsuo K: Gefitinib and Erlotinib in Interstitial Lung Disease

cells and thickened alveolar septa (Fig. 5D). These data
confirm that gefitinib functions specifically in inducing
the pivotal transcription factor Fosl1 in a mouse model ofinterstitial lung disease.
Discussion

Fig. 4 The effect on gene expression of EGFR inhibitor treatments
started at different times relative to LPS addition.
Mouse primary bone marrow macrophages were stimulated with
0.1 µg/ml LPS (time 0). EGFR inhibitors, 10 μM of gefitinib (Gef), erlotinib (Erl) or AG1517 (AG), were added 30 min before (Pre 30 min),30 min after (Post 30 min) or 180 min after (Post 180 min) LPS wasadded. Expression levels of Tnfa (A), Ccl2 (B) and Fosl1 (C) were analyzed by qRT-PCR 12 h after LPS addition. Results are shown relative
to the expression of GAPDH as means ±SEM of triplicate cultures.
*p < 0.05.

We showed that gefitinib, but not erlotinib or AG1517,
induced Fosl1 expression in macrophages treated with
LPS. Furthermore, in a bleomycin-induced lung injury
model, gefitinib, but not erlotinib, induced Fosl1 expression in the lung. Since Fosl1 induction is crucial for interstitial lung disease in mouse models,11 our data suggestthat the mechanism underlying interstitial lung disease isa gefitinib-specific off-target effect.
We found that gefitinib altered LPS-induced gene expression only when cells were treated before LPS stimulation. Currently, it is unclear why pretreatment is necessary to observe the off-target effect of gefitinib reported
in this study. We previously reported that gefitinib treatment and endogenous TLR ligands, such as hyaluronanand heparan sulfate, together induce interstitial lungdisease.11 It has recently been proposed that intracellular
proteins such as heat shock proteins, extracellular matrix
components such as fibrinogen, and oxidized low-density
lipoprotein released from damaged tissues may act as
PAMP-binding molecules (PBMs) or PAMP-sensitizing
molecules (PSMs), rather than genuine TLR ligands.19
Further studies are needed to determine the role of endogenous TLR4 ligands or PBMs/PSMs in the gefitinibinduced lung fibrosis seen in humans.
In the bleomycin-induced lung injury model, we observed that gefitinib enhanced Ccl2 and Fosl1 expression.
In contrast, erlotinib did not induce Fosl1 expression. Although erlotinib induced Ccl2, which can contribute tolung injury,11 histological analysis revealed no enhancement of bleomycin-induced lung injury by erlotinib. Since
gefitinib induces Ccl2 and several other chemokines, suchas Ccl6, Ccl8 and Cxcl5, through Fosl1 upregulation inlung injury models,11 these chemokines likely modulate
gefitinib-induced lung injury.
The EGFR inhibitors used in this study—gefitinib, erlotinib and AG1517—share the 4-anilinoquinazoline structure (Fig. 1). Both gefitinib and erlotinib bind near the
EGFR ATP binding site, as revealed by crystallographic analysis.20 Gefitinib has chloro and fluoro moieties,
whereas erlotinib and AG1517 have space-filling ethynyl
and bromo groups, respectively. In addition, gefitinib has
a morpholino group (a saturated nitrogen-containing cycloalkyl group), which neither erlotinib nor AG1517 has(Fig. 1). It is currently unclear whether chloro/fluoro ormorpholino groups confer gefitinib’s potential to induce
Fosl1 gene expression.
In addition to EGFR, several protein kinases are boundby and inhibited by gefitinib.8 The gefitinib analogue
AX14596 has an amino group at the position of the ge-Keio J Med 2012; 61 (4): 120–127

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Fig. 5 Analysis of lung tissues isolated from mice treated with EGFR inhibitors before (once) and after (6 days a week for 3 weeks)
intratracheal administration of bleomycin.
Effects of gefitinib (Gef) or erlotinib (Erl) treatment on Tnfa (A), Ccl2 (B) and Fosl1 (C) gene expression relative to that of GAPDH in
bleomycin (Bleo)-induced lung disease (n=6 mice per group). Total RNA was isolated from lung tissue, and the gene expression levels
were analyzed by qRT-PCR. *p < 0.05, ‡p=0.069. Hematoxylin and Eosin staining of lung tissues (D). Unt, untreated control. Images
shown are representative of three mice per group. Scale bar 50 μm.

fitinib morpholino group.21 When the amino group wasused to covalently immobilize AX14596 to beads forbinding analysis, multiple kinases in addition to EGFR
were identified,8 among them BRK (breast tumor kinase,or protein-tyrosine kinase 6), GAK (cyclin-G-associatedkinase) and RICK/Rip2 (receptor-interacting caspaselike apoptosis-regulatory kinase). Gefitinib inhibited kinase activities of all three kinases with a 50% inhibitory
concentration of less than 90 nmol.8
Recently, the kinase domain of the potential gefitinib
target GAK, encoded by exons 2, 3, and 4, was genetically deleted in mice.22 Whereas disruption of exon 1 of

the GAK gene (homozygous null mutations) results inembryonic lethality,23 mice expressing a smaller GAK
protein lacking kinase activity (kinase-dead form) diedperinatally from pulmonary dysfunction marked by anincreased number of alveolar compartments and reduced
thickness of the alveolar septum.22 In these mice, the distribution of surfactant protein A, E-cadherin and EGFR
was abnormal.22 Although the lung histology observed inkinase-dead GAK mice is not identical to that of interstitial lung disease in humans, GAK inhibition by gefitinib
may contribute to the pathogenesis of interstitial lung disease in humans. It will be interesting to discover whether

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Fosl1 expression is elevated in the lung of mice expressing the kinase-dead form of GAK.
Translational inhibition of heat shock protein 70(HSP70) has been suggested to promote interstitial lungdisease.24,25 Furthermore, lung fibrosis is reportedly suppressed by administering geranylgeranylacetone, an inducer of HSP70 used clinically to improve gastric mucosal lesions associated with gastritis.25 Curiously, both
gefitinib and erlotinib suppress HSP70 expression, implying that EGFR inhibition mediates this effect.25
Compared with standard chemotherapy, both gefitinib
and erlotinib improve progression-free survival with acceptable toxicity as a first-line treatment for patients with
EGFR mutation-positive advanced non-small-cell lungcancer.3,26 It is currently unclear whether the incidence ofinterstitial lung disease differs following treatment withgefitinib and treatment with erlotinib. Interestingly, Hottaet al reported that the incidence of interstitial lung disease
was somewhat lower following erlotinib therapy than following gefitinib therapy in Japanese patients.6 Fosl1 expression is critical to induce interstitial lung disease inmouse models, and here we found that, of the EGFR tyrosine kinase inhibitors tested, only gefitinib induced Fosl1
expression in LPS-treated cultured cells or bleomycintreated mice (Fig. 2). We conclude that gefitinib-dependent activation of Fosl1 may be a major determinant of
the induction of interstitial lung disease. Further work isneeded to identify the direct target protein of gefitinib.
Acknowledgments
We thank A. Oide and the Collaborative Research Resources, Keio University School of Medicine, for technical assistance.
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