Journal of Cancer 2019, Vol.

10 2063

Ivyspring
International Publisher
Journal of Cancer
2019; 10(9): 2063-2073. doi: 10.7150/jca.29327
Research Paper

Simultaneous Inhibition of EGFR and HER2 via Afatinib

Augments the Radiosensitivity of Nasopharyngeal
Carcinoma Cells
Fangling Huang1,2, Xujun Liang1, Xiaoli Min1, Ye Zhang1, Guoqiang Wang1, Zhengrong Peng2, Fang Peng1,
Maoyu Li1, Lin Chen1, Yongheng Chen1
1. Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China
2. Department of Hyperbaric Oxygen, Xiangya Hospital, Central South University, Changsha 410008, China

 Corresponding author: Prof. Yongheng Chen, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South
University, 110 Xiangya Road, Changsha, Hunan 410078, P.R. China. E-mail:[email protected].

© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2018.08.18; Accepted: 2019.04.13; Published: 2019.05.12

Abstract
Ionizing radiation (IR) is the central component of the therapeutic scheme for nasopharyngeal
carcinoma (NPC) at present. Previous studies show that inhibition of epidermal growth factor
receptor (EGFR) enhances the radiosensitivity of NPC; however the effects of EGFR-targeted
agents are limited. In this study, we observed that simultaneously inhibition of EGFR and HER2 by
afatinib could augment the radiosensitivity of NPC cells; this approach has an advantage over
erlotinib-mediated inhibition of EGFR alone. The afatinib-induced augmentation of NPC cell
radiosensitivity was associated with increases in apoptosis and accumulation of DNA damage that
were induced by radiation. In addition, the crosstalk between radiation-induced activities and
EGFR-, and HER2-related downstream pathways may contribute to the enhancement of
radiosensitivity. Our findings indicate the potential of repositioning afatinib or other
ERBB-family-targeted agents for improving radiation response in NPC cells.
Key words: nasopharyngeal carcinoma, EGFR, HER2, radiosensitivity

Introduction
Currently, ionizing radiation (IR) is the investigated in the past decade that indicate that
foundation and central component of curative EGFR inhibition treatment could modulate cellular
treatment regimens for nasopharyngeal carcinoma resistance to radiation and enhance tumour response
(NPC). Improving radiosensitivity is crucial for the to IR in vitro and in vivo [7-9].
treatment of locally advanced NPC. Epidermal Although investigators have confirmed the
growth factor receptor (EGFR), an important member efficacy of EGFR monoclonal antibodies (cetuximab
of the ERBB family of receptors, is expressed at and nimotuzumab) and EGFR tyrosine kinase
elevated levels in most malignancies of epithelial inhibitors (TKIs) in optimizing radiotherapy effect in
origin. EGFR has diverse roles in promoting cellular preclinical and clinical research [10-12], definitive
proliferation, mediating differentiation and protecting benefit in local control and survival remains
cells from apoptosis [1, 2]. Increasing evidence unsatisfied for a fraction of patients [11]. Therefore,
supports the concept that EGFR is also correlated with rational integration of molecular targeted agents with
tumour responses to ionizing radiation (IR) [3-5]. radiotherapy is still needed for improved treatment of
Overexpression and activation of EGFR frequently NPC.
result in tumour resistance to radiation and poor Recent studies have identified HER2 activation
prognosis [6]. Several therapies have been as a potential mechanism compromising the

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effectiveness of radiation [13, 14]. HER2, as another (GIBCO). All cell lines were maintained at 37°C in a
member of the ERBB family of receptors without a humidified atmosphere of 5% CO2 and 95% air, as we
known ligand, could heterodimerize with EGFR and have previously reported [20].
transduce the subsequent signalling pathway.
Meanwhile, interreceptor kinase activation and Chemical reagents and antibodies
transmodulation of EGFR and HER2 are observed as Afatinib and erlotinib were purchased from
the results of heterodimerization [15, 16]. Moreover, Selleck Chemicals. For in vitro experiments, afatinib
HER2 amplification, overexpression and and erlotinib stock solutions were prepared in DMSO
heterodimerization obviate the need for activated and both compounds were diluted in culture medium
EGFR to initiate downstream signal pathways [17-19]. before dosing. The following antibodies were
For the purpose of seeking more effective and obtained from Cell Signaling Technology:
rational approaches to drug radiotherapy phospho-Erk1/2 Thr202/Tyr204 (#4370, 1:2000
combinations, we hypothesized that excessive dilution), phospho-Akt Ser473 (#4060, 1:2000
activation of HER2 suppresses the ability of dilution), Erk1/2 (#4695, 1:1000 dilution), Akt (#4691,
anti-EGFR therapies to regulate the biological effect of 1:1000 dilution), cleaved PARP Asp214 (#5625, 1:1000
radiation in NPC cells. Afatinib is a novel TKI for both dilution). The following antibodies were purchased
EGFR and HER2. The traditional EGFR TKIs, like from Abcam: phosphor-EGFR Tyr1068 (#ab32430,
erlotinib, bind in a reversible fashion to ATP banding 1:5000 dilution), phospho-HER2 Tyr1139 (#ab53290,
site of EGFR. However, afatinib, a second generation 1:1000 dilution), EGFR (#ab52849, 1:5000 dilution),
of EGFR TKIs, combines with EGFR, HER2 and other HER2 (#ab134182, 1:10000 dilution), ϒH2AX
ERBB family members via covalent bond, which phospho-Ser139 (#ab26350, 1:1000 dilution). The
irreversibly and more potently inhibit ERBB Caspase 3 (#19677-1-AP, 1:1000 dilution) antibody
signalling pathways. In 2013, afatinib was approved and Beta actin (#60008-1-Ig, 1:10000 dilution)
by the U.S. Food and Drug Administration as a new antibody were purchased from Proteintech.
first-line treatment for patients with metastatic
Cell antiproliferation assay
non-small lung cancer. With the goal of further
identifying mechanisms of radiation resistance in The human nasopharyngeal carcinoma cells
NPC, we performed a detailed analysis of the were seeded at 2000 cells/well in 96-well plates. After
synergistic effect between radiation and overnight incubation, the cells were treated with
pharmacological inhibition of both EGFR and HER2 afatinib or erlotinib (0, 0.625, 1.25, 2.5, 5, 7.5, or 10
by afatinib. Our data showed that pharmacological μM). After 72 hours of treatment, growth inhibition
inhibition of both EGFR and HER2 by afatinib was assessed through the MTT assay, which was
exhibited more improved radiosensitizing activity performed according to previously established
than inhibition of EGFR alone by erlotinib. methods [21]. The absorbance of the converted dye
Mechanisms of radiosensitizing activity of afatinib was measured at 570 nm using a microplate reader
were associated with the potent suppression of (Bio-Tek ELX800, USA). All experiments were
radiation-induced activation of ERBB-family-related repeated at least three times. The curves were fitted
signalling pathways to regulate proliferation, using a non-linear regression model with a sigmoidal
apoptosis and radiation-induced DNA repair. Our dose response.
results support the use of afatinib as a reliable Clonogenic assays
radiation sensitizer and, provide the direction for
5-8F and HNE2 human nasopharyngeal
optimizing the radiation response of advanced NPC.
carcinoma cells were cultured in 6-well plates and
Materials and methods were exposed to treatment the following day. The
cells were treated with different doses (0Gy, 2Gy,
Cells culture 4Gy, 6Gy, and 8Gy) of radiation following a 1-hour
Human NPC cells (5-8F, 6-10B, HNE2 and pretreatment with afatinib, erlotinib or vehicle on day
HNE3) were cultured in RPMI-1640 (GIBCO) 1. After culturing for 10 days, the cells were fixed in
supplemented with 10% foetal bovine serum ice-cold methanol and were strained with crystal
(GIBCO), and penicillin-streptomycin (GIBCO) to violet. Colonies with more than 50 cells were counted.
final concentrations of 100 U/ml and 100 μg/ml, At each concentration, the surviving fraction was
respectively. An immortalized nasopharyngeal determined by dividing the total number of colonies
epithelial cell line, NP69, was cultured in after irradiation by the number of colonies without
Keratinocyte-SFM medium (GIBCO) with 0.2 μg/ml irradiation.
EGF (GIBCO) and 30 μg/ml Bovine Pituitary Extract

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Determination of apoptosis with flow Data presentation and statistical analysis

cytometry Numerical results were expressed as the means ±
5-8F cells and HNE2 cells were pretreated for standard deviation and were graphically displayed
half an hour with vehicle or 2 μM afatinib before using GraphPad Prism version 7.0 for Windows,
irradiation (4Gy) and were incubated for 48 hours. (GraphPad Software; www.graphpad.com). Each
Next, the apoptotic cells were analysed via flow figure represents results from three independent
cytometry using the Annexin V-FITC Detection Kit I experiments. Statistical significance was evaluated
(BD Pharmingen, San Diego, CA) according to the using one-way ANOVA for normally distributed and
manufacturer's instructions. Flow cytometry data homoscedasic data or Kruskal-Wallis H tests for data
were plotted and analysed via the fluorescence which is non-normally distributed or with different
activated cell-sorting (FACS-Vantage) system using variances. P values less than 0.05 compared with the
the Cell quest software (Becton-Dickinson, San Jose, control were considered statistically significant.
CA) within 1 hour of staining. The percentages of
differently labelled cells were calculated using the Result
FlowJo software (Tree Star).
Afatinib inhibits NPC cell proliferation by
Ionizing irradiation treatment of cells blocking EGFR and HER2 signalling
5-8F cells and HNE2 cells in flask or plates were Since phosphorylations activate the EGFR and
exposed to X-rays (2-8 Gy, 4Gy per minute), using a HER2 signal pathways, we first examined the
6-MV photon beam from a linear accelerator (Varian phosphorylation levels and the total expression of
Medical Systems, Inc) at the Department of Clinical EGFR and HER2 in four NPC cell lines (5-8F, 6-10B,
Oncology of Xiangya Hospital (Changsha, China). HNE2, and HNE3) and an immortalized
The plates were covered with a tissue-equivalent gel nasopharyngeal epithelial cell line (NP69). Western
to promote dose build-up. blotting showed that the expression and
phosphorylation levels of EGFR and HER2 vary in all
Immunofluorescence these cell lines (Fig. 1A). Compared to immortalized
Cells were seeded on a glass slide, incubated nasopharyngeal epithelial cell line NP69, HNE2 cells
overnight and pretreated with either vehicle or 2 μM exhibited equivalent expression of phospho-EGFR,
EGFR- tyrosine kinase inhibitors (TKIs), afatinib or but only weak expression (p=0.000) of phospho-HER2
erlotinib, half an hour before IR (4 Gy). The number of (Fig. 1B). However, the expression levels of
ϒ-H2AX foci was determined at half an hour after IR. phosphorylated EGFR and HER2 in 5-8F cells are
The cells were treated as described previously [22] contrast to those in HNE2 cells. We chose 5-8F cells
and incubated with a primary antibody against and HNE2 cells in the following experiments to
γ-H2AX (Abcam). Next, the primary antibody was investigate the effect of afatinib in NPC cells.
washed off, and a secondary antibody conjugated to To address whether afatinib is a potent inhibitor,
FITC was applied to the slides. DNA damage was we evaluated its ability to reduce
visualized using laser scanning confocal microscopy autophosphorylation of EGFR, HER2 and their
(Leica, SP8). For each group, the ϒ-H2AX foci were downstream signalling properties (Erk1/2 and Akt) in
counted in at least 50 cells. The captured images were 5-8F cells and HNE2 cells without ligand stimulation
processed using Image-Pro Plus software (Media (Fig. 1C). The expression levels of phospho-EGFR,
Cybernetics, Rockville, Maryland, USA) and were phospho-HER2, phospho-Erk1/2 and phospho-Akt
displayed using Adobe Photoshop CS6. were generally reduced by afatinib in a
dose-dependent manner (Supplemental Fig. 1).
Western blot analysis Furthermore, to determine whether the inhibition of
Cells were lysed in lysis buffer (50 mM Tris pH = both EGFR and HER2 phosphorylation could more
6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and effectively inhibit cell proliferation, we treated 5-8F
1% protease inhibitor cocktail, with or without 1% cells and HNE2 cells with different doses of afatinib or
phosphatase inhibitor cocktail). An equal amount of with a standard EGRF-TKI (erlotinib). Consistent with
protein from each cell extract was separated via our signalling data, proliferation of NPC cells
SDS-PAGE gel (8–12% polyacrylamide), and the declined sharply with increasing dose of afatinib
separated proteins were transferred to PVDF (P=0.034 and p=0.022 in 5-8F and HNE2 cell lines with
membranes and immunoblotted with various 2.5μM afatinib, respectively. Fig. 1D). Compared to
antibodies, as we have previously reported [23]. The erlotinib, afatinib had a much better capacity to
proteins of interest were visualized via enhanced decrease the viability of NPC cells, with an IC50 of
chemiluminescence. approximately 5 μM in both NPC cell lines. These

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results demonstrate that afatinib effectively inhibit clonogenic survival assay was performed to estimate
proliferation of NPC cells by blocking the EGFR and the colony formation ability of 5-8F and HNE2 NPC
HER2 signalling pathways. cells after treatment with 0-8 Gy irradiation with or
without afatinib compared with that with erlotinib.
Simultaneous blockade of EGFR and HER2 As described in Fig. 2A, the colony formation assay
tyrosine kinase activities significantly demonstrated that afatinib and erlotinib
radiosensitizes nasopharyngeal carcinoma dose-dependently reduced the survival fraction of
cells 5-8F cells and HNE2 cells following 8 Gy irradiation.
To substantiate the statement that combined Moreover, afatinib was more successful in reducing
inhibition of EGFR and HER2 tyrosine kinase could the NPC colony numbers than erlotinib at the same
increase the radiosensitivity of NPC cells; the drug dose; the difference became obvious and

Figure 1. Afatinib block EGFR and HER2 signalling to inhibit NPC cell proliferation. (A) Lysates of the immortalized nasopharyngeal epithelial cell line
NP69 and 4 different cell lines (5-8F, 6-10B, HNE3, HNE2) were analysed via immunoblotting to detect phosphorylated EGFR (p- EGFR), total EGFR, phosphorylated
HER2 (p-HER2), total HER2 and ACTB. (B) Phosphorylated protein levels were quantified by densitometry and normalized by total protein levels. Compared with
the control group. *P< 0.05, ** P< 0.01, *** P< 0.001. (C) Lysates of 5-8F and HNE2 were treated for 30minutes with increasing concentrations of the EGFR TKI
afatinib (0, 0.125, 0.25, 0.5, 1 and 2μM) and were analysed via immunoblotting to detect p-EGFR, total EGFR, p-HER2, total HER2, p-Erk1/2, total Erk1/2, p-Akt,
total Akt and ACTB. D. Viability curve describing the viability of 5-8F and HNE2 NPC cells that were treated with vehicle or the indicated concentrations of afatinib
or erlotinib for 72 hours. Each data point represents the average value from 3 samples, and is expressed as a percentage of surviving cells relative to untreated
controls. Compared with the control group. *P< 0.05.

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significant with the increasing dose (P=0.006 for reduced the clonogenic survival rate with increase in
1.2μM drugs in 5-8F cells and p=0.013, p= 0.008 and drug dose (Fig. 2D and 2E). Our data strongly
p=0.000 for 0.4 μM, 0.8 μM and 1.2μM drugs in HNE2 suggests that afatinib is a satisfying radiosensitizing
cells, respectively.) of afatinib and erlotinib (Fig. 2B agent for NPC cells. Moreover, the ability of
and 2C). We further described the potent inhibition increasing radiosensitivity of afatinib is equal in 5-8F
ability of afatinib against colony formation with cells and HNE2 cells, indicating that afatinib could
different doses of radiation (Fig. 2D-2G). As shown in radiosensitize NPC cells regardless of the expression
Fig. 2F and 2G, the clonogenic survival rate of 5-8F levels of phosphorylated EGFR and HER2 in NPC
cells and HNE2 cells were mildly reduced with the cells before irradiation.
addition of erlotinib. However, afatinib sharply

Figure 2. Afatinib significantly radiosensitizes nasopharyngeal carcinoma cells. (A) 5-8F and HNE2 cells were seeded at 2000 cells/plate, and were treated with
either IR at 8Gy alone or the combination of 0.4μM, 0.8μM, or 1.2μM of afatinib or erlotinib with IR at 8Gy. Following treatment, the plates were fixed and stained
with crystal violet. All experiments were performed in triplicate. The representative plates are shown. (B and C) Colony numbers of 5-8F (B) and HNE2 (C) cells
were counted following the treatment described in Fig.2A. Data from three independent experiments was averaged. (bar graph). *p<0.05, **p<0.01, ***p<0.001. D-G.
5-8F (D, F) and HNE2 (E,G) cells either treated with IR (2Gy, 4 Gy, 6 Gy, or 8 Gy) alone or the combination of 0.4μM, 0.8μM, and 1.2μM of afatinib (D, E) or erlotinib
(F, G) with IR. Colony- forming efficiency was determined, and survival curves were generated approximated 10 days later. Data from three independent experiments
was averaged. *p<0.05, **p<0.01, ***p<0.001.

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Figure 3. Afatinib promotes the pro-apoptosis ability of IR. (A and B) Flow cytometry was performed to analysis cell apoptosis of 5-8F cells (A) and HNE2 cells
(B). Cells were labelled with Annexin V conjugated to FITC and propidium iodide (PI) 48 hours after IR at 4Gy or control treatment, with or without addition of 2μM
afatinib as indicated. (C and D) Percent of apoptosis cells in 5-8F cells (C) and HNE2 cells (D) that were treated as described in Figure 3A-B were quantified
following flow cytometry. (E) 5-8F cells and HNE2 cells were treated with IR at 4Gy and 2μM afatinib for half an hour before IR as indicated. Lysates from 5-8F and
HNE2 cells were analysed via immunoblotting to detect caspase-3, cleaved caspase-3 and ACTB 72 hours later. (F) Lysates from 5-8F and HNE2 cells that were treated
as described in Figure 3E were analysed via immunoblotting to detect cleaved PARP and ACTB. (G) Expression levels of caspase-3 (upper panel), Cleaved Caspase-3
(middle panel) and Cleaved PARP (bottom panel) were quantified by densitometry and normalized by ACTB levels.

Combined EGFR and HER2 tyrosine kinase (P=0.047 and p=0.000 in 5-8F and HNE2 cell lines,
inhibition promotes radiation-induced respectively.) after pretreatment with afatinib half an
hour before IR in both NPC cell lines, especially in the
apoptosis
HNE2 cell line (Fig. 3C and 3D). Furthermore, the
To further characterize the availability of afatinib increase in Annexin-V-positive cells treated with IR
as a radiation sensitizer in NPC cells, we examined its alone mainly counted on the contribution of early
pro-apoptosis effect in 5-8F cells and HNE2 cells via apoptotic cells (Annexin-V-FITC-positive and
flow cytometry using Annexin V/propidium iodide PI-negative), whereas afatinib equally promoted
(PI) staining (Fig. 3A and 3B). The population of IR-induced early apoptosis and advanced apoptosis
Annexin-V-positive cells was significantly increased (Annexin-V-FITC-positive and PI-positive). These

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results show that afatinib is capable of promoting be due to the inhibition of DNA damage repair
radiation-induced apoptosis. instead of enhancement in cytotoxicity caused by
We further assayed the expression levels of EGFR and HER2 inhibitors.
apoptosis-related proteins at 72 hours after treatment
with IR and afatinib. In line with the flow cytometry Concomitant EGFR and HER2 tyrosine kinase
results, pretreatment with afatinib significantly inhibition significantly suppresses radiation-
increased radiation-induced cleaved caspase-3 activated signalling pathways
(P=0.000 in both 5-8F and HNE2 cell lines. Fig. 3E and We sought to determine whether afatinib is an
3G) and poly ADP-ribose polymerase (PARP) effective therapeutic strategy for the inhibition of
(P=0.001 and p=0.002 in 5-8F and HNE2 cell lines, IR-activated EGFR and HER2 signalling pathways.
respectively. Fig. 3F and 3G). Caspase-3 preforms We treated 5-8F cells and HNE2 cells with the vehicle
different degrees of reduction owing to increasing of or afatinib 30 minutes before IR and assessed the
cleaved caspase-3 in 5-8F cells and HNE2 cells. effects of these treatments on the auto-
Caspase-3 plays a crucial role in the execution-phase phosphorylation of EGFR and HER2 and their
of cell apoptosis and is activated through both downstream signalling properties (such as
extrinsic and intrinsic pathways [24]. PARP, the most phospho-Erk 1/2, phospho-Akt). Hyperp-
important substrate of caspase-3, is the symbol of hosphorylation of HER2 (P=0.000 and p=0.017 in 5-8F
caspase-3 activation. These data indicate that the and HNE2 cells, respectively.) was observed in both
promotion of radiation-induced caspase-dependent NPC cell lines following IR, which suggested that
apoptosis is one of reasons that afatinib radiation induced hyperactivation of the HER2
radiosensitizes NPC cells. pathway and that it may led to the failure of
EGFR-targeted therapy (Fig 5A and 5C). Meanwhile,
Combined EGFR and HER2 tyrosine kinase IR damage conspicuously increased the activation of
inhibition enhances radiation-induced DNA EGFR, HER2, and all of their downstream signals in
damage 5-8F and HNE2 cells as previously reported (Fig 5A
Effective DNA damage, especially unrepaired and 5C) [25]. Afatinib is a potent inhibitor of
DNA double strand break (DSB) is the main IR-induced EGFR and HER2 autophosphorylation. As
mechanism that underlies the elimination of expected, activation of downstream Erk 1/2 and Akt
carcinoma cells via irradiation. Next, we investigated signalling were both inhibited after drug treatment.
whether afatinib modulated the level of We conclude that afatinib can significantly suppress
phosphorylated histone H2AX (γ-H2AX), which EGFR, HER2 and their downstream signalling
formed foci at DNA double-strand break areas. We properties that were activated by IR and improve the
first assessed the quantity of γ-H2AX using the radiosensitivity of NPC cells.
immunofluorescence technique. As shown in Fig.4A,
treatment with afatinib or erlotinib alone exhibited Discussion
little effect on the γ-H2AX foci. However irradiation Here, we provided compelling evidence that
led to a significant increase in the quantity of γ-H2AX demonstrated that blockage of HER2 augments the
foci. Half an hour of pretreatment with afatinib before radiotherapy sensitization ability of anti-EGFR
irradiation made further effort (P=0.005 and p=0.000 therapies through suppressing tumour proliferation,
in 5-8F and HNE2 cell lines, respectively.) on DNA improving apoptosis and increasing DNA damage
damage on the basis of irradiation in both 5-8F and induced by irradiation.
HNE2 cells (Fig. 4C) whereas erlotinib had less effect The role of EGFR in mediating the effect of
on it. Our data certify that, compared with EGFR radiotherapy is partly investigated. PI3K-Akt-mTOR
alone, combined EGFR and HER2 tyrosine kinase signalling is the most important downstream
inhibition enhances radiation-induced DNA damage pathway for EGFR to sensitize NPC cells to
to improve radiation response. Next, we examined the radiotherapy. PI3K-Akt-mTOR signalling is involved
protein expression level of γ-H2AX in both 5-8F and in elevating DNA-damage repair capability through
HNE2 cells. In accordance with immunofluorescence the activation DNA-dependent protein kinase
results, afatinib augmented the DNA damage (DNA-PK) [26]. DNA-PK is a crucial protein kinase in
triggered by IR (P=0.071 and p=0.033 in 5-8F and DNA non-homologous end joining (NHEJ) repair and
HNE2 cell lines, respectively. Fig. 4B and 4D) recombination for IR-induced DNA double strand
especially in HNE2 cells. Because DNA damage in breaks, and it mediates radiosensitivity [27, 28]. In
cells treated with afatinib or erlotinib alone was addition, PI3K-Akt-mTOR signalling mediates
considerably less than that in cells treated with IR, our inhibition of Nuclear factor-κB( NF-κB) kinase α
results suggest that the increase in DNA damage may phosphorylation and activation of NF-κB, in an

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Akt-dependent or Akt-independent manner [29, 30]. cells from apoptosis [32-34]. In our study,
In addition to regulating the induction of PI3K-Akt-mTOR signalling is the most dramatically
inflammatory cytokines and molecules via radiation inhibited downstream pathway by afatinib following
exposure [31], activated NF-κB upregulates the IR. The results demonstrate that afatinib is a potent
expression and activation of some oncogenes, such as potential therapeutic agent to inhibit IR-induced
MET, which promote IR-induced cell invasion, EGFR signalling activation and enhance the toxic
increase proliferation and angiogenesis and protect effect of IR.

Figure 4. Afatinib enhances radiation-induced DNA damage. (A) Confocal microscopy was used to obtain images of 5-8F cells and HNE2 cells that were
treated with IR at 4 Gy and /or 2 μM afatinib as indicated. Cells were fixed 30 minutes following the treatments, and the cell nuclei were stained with γ-H2AX
(green) and DAPI (blue). The scale bar represents 5μm. (B) Lysates of 5-8F and HNE2 cells that were treated as described in Figure 4A were analysed via
immunoblotting to detect γ-H2AX and ACTB. (C) γ-H2AX foci in the nuclei of 5-8F cells and HNE2 cells that were treated as described in Figure 4A were quantified.
For each group, at least 50 cells were counted, and the data are expressed as the means ± standard deviation. (D) Expression levels of γ-H2AX were quantified by
densitometry and normalized by ACTB levels.

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Figure 5. Afatinib inhibits radiation-activated EGFR and HER2 signalling. 5-8F (A) and HNE2 (C) were treated with IR at 4 Gy and /or 2 μM afatinib as
indicated. Lysates of 5- 8F cells and HNE2 cells were prepared 2 hours following treatments and were analysed via immunoblotting to detect p-EGFR, p-HER2,
and their downstream signalling phosphorylated proteins p-Erk1/2, p-Akt and ACTB. Phosphorylated protein levels of 5-8F (B) and HNE2 (D) were quantified by
densitometry and normalized by total protein levels.

The mechanisms for tumour resistance to In addition to drug efflux, populations of “cancer
anti-EGFR therapy are not completely understood; stem cells”, cellular metabolism [37] and reversible
however, one reason behind this may be the “drug-tolerant persisters” [38], epithelial-
upregulation, coexpression and heterodimerization of mesenchymal transition (EMT) may be an underlying
other EGFR family members, such as HER2. This is mechanism of EGFR-TKI drug resistance [39-41].
similar to the contribution of increased expression of Compared with the bulk tumour compartment of
EGFR to treatment resistance in HER-2-positive breast epithelial cells, mesenchymal cells tend to be more
cancer [35]. In cells co-expressing EGFR and HER2, resistant to EGFR inhibitors and antibodies. Previous
ligands of EGFR prefer to stimulate the formation of studies have revealed that activation of Src/focal
EGFR/HER2 heterodimers via cross-phosphorylation adhesion kinase (FAK) signalling was crucial in
[15, 36]. More importantly, reciprocal activation maintaining the mesenchymal status, and it partly
between ligand-independent tyrosine protein kinase generated EMT-associated drug resistance [39]. HER2
in c-Src and ERBB families contributes to promotes the expression levels of proteins associated
ligand-independent transmodulation of EGFR and with EMT via the activation of FAK [42].
HER2 [16]. Transactivation of EGFR and HER2 Consequently, combination inhibition of HER2 in
suggests that single-target inhibitors or antibodies mesenchymal cells may be a useful strategy to
may be insufficient to block EGFR signal overcome certain types of anti-EGFR drug resistance.
transduction. In our study, EGFR, HER2 and their vital

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