Combination of cetuximab with met inhibitor in control of cetuximab-resistant oral squamous cell carcinoma

Abstract

Objective: To investigate the underlying molecular mechanisms contributing to oral squamous cell carcinoma (OSCC) cell resistance to the epidermal growth factor receptor (EGFR) inhibitor. Materials and methods: OSCC cell lines HSC-2 and HSC-3 were assessed in vitro for drug treatment, cell viability, and gene expression and the online gene expression in OSCC tissues was analyzed for association with OSCC prognosis. Results: HSC-2 and HSC-3 cells expressed high EGFR levels, but hepatocyte growth factor (HGF) treatment induced cetuximab resistance, whereas the Met inhibitor PHA-665752 as well as Met siRNA was able to restore OSCC cell sensitivity to cetuximab. HGF treatment induced tumor cells to express p-Akt and p-ERK1/2. In contrast, the activity of Akt and ERK1/2 was suppressed by treatment with PHA-665752, Met siRNA, or their combination. Furthermore, Met was highly expressed in OSCC tissues and associated with a poor patient survival, while Met/HGF-activated Akt also was associated with a poor patient survival. Conclusions: This study demonstrates that Met/HGF expression results in OSCC resistance to cetuximab and tumor recurrence after cetuximab therapy; thus, inhibition of Met/HGF activity could restore OSCC sensitivity to cetuximab.

Full text

Am J Transl Res 2019;11(4):2370-2381
www.ajtr.org /ISSN:1943-8141/AJTR0088717
Original Article
Combination of cetuximab with met inhibitor in control
of cetuximab-resistant oral squamous cell carcinoma
Hua Yang1, Chuzi Mo1, Yang Xun1, Leyna G Liu2, Wenxing Li3,4, Jieying Guan1, Jing Liu1, Jianquan Wu1, Anping
Yang1, Songguo Zheng5, Dahai Liu1, Fang Liu1
1Department of Basic Medicine and Biomedical Engineering, School of Stomatology and Medicine, Foshan
University, Foshan 528000, Guangdong, China; 2Portola High School, 1001 Cadence, Irvine, CA 92618, U.S.A;
3The Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming 650223, Yunnan, China; 4Kunming College of Life Science, University of Chinese
Academy of Sciences, Kunming 650204, Yunnan, China; 5Department of Medicine, Division of Rheumatology,
Milton S. Hershey Medical Center at Penn State University, Hershey, PA 17033, USA
Received November 20, 2018; Accepted January 29, 2019; Epub April 15, 2019; Published April 30, 2019
Abstract: Objective: To investigate the underlying molecular mechanisms contributing to oral squamous cell car-
cinoma (OSCC) cell resistance to the epidermal growth factor receptor (EGFR) inhibitor. Materials and methods:
OSCC cell lines HSC-2 and HSC-3 were assessed in vitro for drug treatment, cell viability, and gene expression and
the online gene expression in OSCC tissues was analyzed for association with OSCC prognosis. Results: HSC-2 and
HSC-3 cells expressed high EGFR levels, but hepatocyte growth factor (HGF) treatment induced cetuximab resis-
tance, whereas the Met inhibitor PHA-665752 as well as Met siRNA was able to restore OSCC cell sensitivity to
cetuximab. HGF treatment induced tumor cells to express p-Akt and p-ERK1/2. In contrast, the activity of Akt and
ERK1/2 was suppressed by treatment with PHA-665752, Met siRNA, or their combination. Furthermore, Met was
highly expressed in OSCC tissues and associated with a poor patient survival, while Met/HGF-activated Akt also was
associated with a poor patient survival. Conclusions: This study demonstrates that Met/HGF expression results in
OSCC resistance to cetuximab and tumor recurrence after cetuximab therapy; thus, inhibition of Met/HGF activity
could restore OSCC sensitivity to cetuximab.
Keywords: Oral squamous cell carcinoma, cetuximab resistance, HGF, Met, Akt, ERK1/2
Introduction
Head and neck cancer, which occurs in the oral
cavity, nose, throat, larynx, sinuses, salivary
glands, nasopharynx, or hypopharynx, signi-
cantly affects the quality of life of patients [1].
Oral squamous cell carcinoma (OSCC) is a com-
monly diagnosed head and neck squamous cell
carcinoma (HNSCC). The risk factors include
tobacco smoking and alcohol consumption [2]
as well as excessive consumption of processed
meats and red meat [3], human papillomavirus
infection [4], and Frequently chewing betel nuts
[5]. OSCC management is usually surgery,
chemoradiotherapy, targeted therapy, or photo-
dynamic therapy [6, 7]. Early-stage OSCC has a
favorable prognosis after treatment, but drug
resistance, disease recurrence, and metasta-
sis result in overall 5-year survival rates ≤ 50%
[8]. Targeted therapy is a hot topic for the treat-
ment of OSCC, and clinical drugs used frequent-
ly include epidermal growth factor receptor
(EGFR) inhibitors, such as cetuximab, bevaci-
zumab, and erlotinib, which have shown
improvement of OSCC patient survival [9, 10].
Indeed, EGFR is a transmembrane receptor
tyrosine kinase that is highly overexpressed in
head and neck, lung, and breast cancers [11,
12]. EGFR promotes cancer development by
increasing cell growth, migration, and survival
[13]. Cetuximab is a chimeric monoclonal anti-
body that can bind to EGFR and in turn inhibit
EGFR tyrosine kinase activity to suppress EGFR-
positive cancer progression [14]. In the treat-
ment of HNSCC, cetuximab has gained much
attention as a novel therapeutic strategy and
was approved by the European Medicines
Agency in 2004 and the US Food and Drug

Drug combination in treatment of oral cancer
2371 Am J Transl Res 2019;11(4):2370-2381
Administration (FDA) in 2006 [15]. Unfor-
tunately, long-term cetuximab treatment results
in HNSCC drug resistance, even in tumors with
high EGFR expression [16, 17]. Thus, a better
understanding of the underlying molecular
events of this drug resistance is critical to
restore the sensitivity of cancer cells to cetux-
imab [18-20]. To date, there is increasing evi-
dence implicating that receptor tyrosine kinas-
es play a pivotal role in regulating cetuximab
resistance in colon and lung cancers [21, 22],
and the combined treatment with receptor tyro-
sine kinase inhibitors may overcome cetuximab
resistance [22].
Hepatocyte growth factor (HGF) plays a crucial
role in cell motility, growth, and morphogenesis
through binding to hepatocyte growth factor
receptor (also known as Met) to activate the
receptor tyrosine kinase cascade, which is
related to cancer development [23-25]. HGF is
highly expressed in OSCC, and the HGF/Met
signaling pathway has been shown to induce
OSCC cell migration, invasion, and metastasis
through lamellipodia and lopodia formation
[26] and the destruction of E-cadherin [27]. The
cross-talk of Met with other signaling proteins,
like EGFR, vascular endothelial growth factor
receptor, and Wnt, also has been revealed in
various cell lines [28-30], indicating the role of
HGF/Met in cancer development. In addition, it
has been shown that Met activation during
cetuximab treatment of recurrent and meta-
static HNSCC is associated with poor outcomes
[31].
In this study, we investigated the underlying
molecular events contributing to OSCC cell
resistance to cetuximab using an in vitro OSCC
cell line model. Our data provide novel insight
into a therapeutic strategy to restore cetuximab
sensitivity of OSCC.
Materials and methods
Cell culture
The human OSCC cell lines HSC-2 and HSC-3
(Immuno-Biological Laboratories Co. Shanghai,
China) were maintained in Dulbecco’s modied
Eagle medium (DMEM; Gibco, Gaithersburg,
MD, USA) supplemented with 10% fetal bovine
serum (Gibco), 100 units/mL penicillin, and
100 µg/mL streptomycin (Gibco) in a humidi-
ed incubator with 5% CO2 at 37°C.
Cell viability assay
Cell viability was measured by using the 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) method. In brief, 2 × 103 tumor
cells/well were seeded into 96-well plates and
grown for 24 h and then treated with various
concentrations of cetuximab (Merck, Germany),
PHA-665752 (Selleck Chemicals, TX, USA),
HGF (R&D Systems, Minneapolis, MN, USA),
and a goat anti-human HGF neutralizing anti-
body, normal goat IgG (R&D Systems) for 72 h.
At the end of each experiment, 50 μL of MTT
solution (2 mg/mL; Sigma Chemicals, St. Louis,
MO, USA) was added to each well of the cell cul-
ture plate, and the cells were incubated for an
additional 2 h. The culture medium was
replaced with 100 μL of dimethyl sulfoxide. The
absorbance rate was measured with a micro-
plate reader (BioTek, Winooski, VT, USA) at a
wavelength of 490 nm. The percentage of cell
viability was calculated by comparison to
untreated controls.
Western blot
A western blot assay was performed, as
described previously [32]. Briey, cells after
treatment were lysed in cell lysis buffer, and the
protein concentration was measured using a
bicinchoninic acid protein assay kit (Beyotime
Biotechnology, Shanghai, China). Each total
protein sample (30 µg per lane) was separated
by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred onto a polyvi-
nylidene uoride membrane (Millipore, Billerica,
MA, USA). After that, the membranes were
blocked by 5% skimmed dry milk solution in
phosphate-buffered saline (PBS) at room tem-
perature for 1 h and then incubated with a pri-
mary antibody at 4°C overnight (Table 1). On
the following day, the membranes were washed
briey three times with PBS-Tween 20 and then
incubated with the corresponding horseradish
peroxidase-conjugated secondary antibody at
room temperature for 1 h. The target protein
bands were visualized by ECL plus western blot-
ting detection reagent and exposed to X-ray
lms.
RNA interference (RNAi) assay
For the RNAi assay, Duplexed Stealth RNAi tar-
geting ErbB3 and Met or Stealth RNAi Negative
Control Low GC Duplex #3 were purchased
from Invitrogen (Carlsbad, CA, USA). OSCC cells

Drug combination in treatment of oral cancer
2372 Am J Transl Res 2019;11(4):2370-2381
were seeded into 24-well plates at a density of
2 × 104 per well in 400 μL of antibiotic-free
DMEM and grown overnight. On the next day,
the cells were transfected with siRNA (50 μmol)
using Lipofectamine 2000 (Invitrogen) for 24 h,
according to the manufacturer’s instructions,
then washed with ice-cold PBS and reseeded
into 96-well plates for treatment with cetux-
imab (1 μg/mL) and/or recombinant human
HGF (20 ng/µL) for 72 h, and nally subjected
to the MTT assay or another assay. The siRNA
sequences targeting ErbB3 and Met were
5’-GGCCAUGAAUGAAUUCUCUACUCUA-3’ and
5’-UCCAGAAGAUCAGUUUCCUAAUUCA-3’, res-
pectively.
Online gene expression database and survival
analysis
Transcriptome data of OSCC tissues were
retrieved from the NCBI-GEO database with the
series ID of GSE42743 (https://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE42743). R
statistical software v3.3.3 (https://www.rpro-
ject.org/) was used for data analysis. In brief,
we utilized the Robust Multichip Average algo-
rithm in the “oligo” package [33] to normalize
the raw data on gene expression and generat-
ed the normalized expression matrix. The gene
annotation and integration of the expression
matrix was conducted by using a custom-
designed Python code, according to a previous
study [34]. We then removed probes without
any gene annotations or those that matched
multiple gene symbols. After that, we calculat-
ed the average expression value for each gene
when there were multiple probe IDs that
matched one ofcial gene symbol and made
this value to represent the expression intensity
“survival” package in R. Kaplan-Meier survival
curves were used to show the prognostic differ-
ences between two groups.
Statistical analysis
All data were expressed as the mean ± stan-
dard deviation of triplicate experiments. The
two-tailed unpaired Student’s t test or the one-
way analysis of variance test was used to deter-
mine the p values; P < 0.05 was considered as
a signicant difference.
Results
HGF induction of OSCC cell resistance to ce-
tuximab by an increase in akt and ERK1/2
phosphorylation
First, we detected the levels of EGFR and c-Met
expression in the OSCC cell lines HSC-2 and
HSC-3 using western blot. We found that both
cell lines signicantly expressed EGFR and MET
(Figure 1A). We then assessed the effect of
cetuximab on OSCC viability using the MTT
assay and found that HSC-2 and HSC-3 cell
growth was moderately inhibited by cetuximab
in a dose-dependent manner (Figure 2B). To
obtain cetuximab resistance, we cotreated
HSC-2 and HSC-3 cells with cetuximab (5 µg/
mL) and HGF (20 µg/mL); however, we did not
nd a signicant change in cell viability (Figure
1B), indicating that both OSCC cell lines became
resistant to cetuximab after HGF treatment. At
the protein level, HGF treatment induced Met
activity (Figure 1C), which could be partially
blocked by cetuximab treatment. Specically,
cetuximab treatment downregulated the phos-
phorylation of Akt and ERK1/2, but it recovered
back to the previous levels after the addition of
Table 1. Antibodies used for Western blot
Antibody Cat # Source Dilution
Anti-EGFR R&D Systems, AF231 Rabbit 1 µg/mL
Anti-phospho-EGFR Abcam, ab32578 Rabbit 1:1000
Anti-Met CST, 3127 Mouse 1:1000
Anti-phospho-Met Abcam, ab47402 Rabbit 1:1000
Anti-Akt CST, 9272 Rabbit 1:1000
Anti-phospho-Akt CST, 4060 Rabbit 1:1000
Anti-ErbB3 CST, 4754 Rabbit 1:1000
Anti-phospho-ErbB3 CST, 4791 Rabbit 1:1000
Anti-ERK1/ERK2 R&D Systems, AF1576 Rabbit 0.2 μg/mL
Anti-phospho-ERK1/ERK2 R&D Systems, AF1018 Rabbit 0.1 μg/mL
CST, Cell Signaling Technology (Danvers, MA, USA); Abcam, Cambridge, MA, USA.
of the corresponding gene
symbol. Differentially expres-
sed genes were then obtained
by using the empirical Bayes
method in the “limma” pack-
age [35]. The upregulated
genes were considered to be
those with logarithmic trans-
formed fold-change (log2FC)
≥ 1, and the downregulated
genes were considered to be
log2FC ≤ -1. A false discovery
rate-adjusted P value ≤ 0.05
indicated statistical signi-
cance. All survival analyses
were conducted using the

Drug combination in treatment of oral cancer
2373 Am J Transl Res 2019;11(4):2370-2381
HGF (Figure 1C). Under these conditions,
p-ErbB3 was dramatically inhibited with cetux-
imab, even after the addition of HGF, indicating
that p-ErbB3 is the target of cetuximab but not
involved in in the HGF-induced cetuximab resis-
tance of OSCC cells; whereas the Met, PI3K/
Akt, and MAPK pathways may be involved in the
HGF-induced cetuximab resistance of OSCC
cells. For further conrmation, we pretreated
these cells with the HGF neutralizing antibody
to block HGF signaling and found that HSC-2
cells regained their sensitivity to cetuximab
treatment (Figure 1D).
Met inhibitor restoration of cetuximab sensitiv-
ity of HGF-treated OSCC cells by decreasing
akt and ERK1/2 phosphorylation
The biological effects of HGF are through bind-
ing to its receptor, Met; thus, we treated the
cells with the Met inhibitor PHA-665752 in the
same setting. Our MTT assay data showed that
PHA-665752 signicantly reduced the tumor
cell viability in a dose-dependent manner
(Figure 2A). Without the stimulus of HGF, PHA-
665752 and cetuximab together strongly
induced inhibition of cell proliferation, even at a
low dose of PHA-665752 (< 1 µM). After stimu-
lus with HGF, the OSCC cells showed cetuximab
resistance in low-dose PHA-665752-treated
cells, but the cell viability was dramatically
reduced after the PHA-665752 dose reached 1
µM, which was similar to that without the HGF
stimulus.
To evaluate the mechanism by which HGF res-
cues OSCC cancer cells from cetuximab-
induced inhibition of proliferation, we deter-
mined activation of the downstream signaling
molecules and found that cetuximab not only
Figure 1. HGF induction of OSCC resistance to cetuximab. A. Western blot. HSC-2 and HSC-3 cells were subjected
to protein extraction and showed high protein expression levels of EGFR and MET by western blot. B. The MTT as-
say. HSC-2 and HSC-3 cells were treated with different doses of cetuximab (0-5 µg/mL) and HGF (20 ng/mL) for
72 h, and the cell proliferation levels were shown by the MTT assay. C. Western blot. HSC-2 and HSC-3 cells were
pretreated with control, HGF (20 ng/mL), cetuximab (1 µg/mL), or their combination. D. The MTT assay. HSC-2 cells
treated with HGF (20 ng/mL) and the HGF neutralizing antibody or cetuximab (1 µg/mL) for 72 h showed different
proliferation abilities by the MTT assay. *P < 0.05.

Drug combination in treatment of oral cancer
2374 Am J Transl Res 2019;11(4):2370-2381
inhibited EGFR phosphorylation, but also AKT
and ERK1/2 activation in HSC-2 and HSC-3
cells. Treating the cells with a combination of
cetuximab with HGF led to MET phosphoryla-
tion and reactivation of AKT and ERK1/2.
However, when treating the cells with a combi-
nation of cetuximab with PHA-665752, inhibi-
tion of EGFR and MET phosphorylation led to
sustained inhibition of the AKT and MAPK path-
ways in the presence of HGF (Figure 2B).
Met siRNA restoration of cetuximab sensitivity
of HGF-treated OSCC cells
Next, we assessed whether Met siRNA pos-
sesses the same effects as PHA-665752 on
HSC-2 cells; it is because cetuximab had even
better inhibitory effects on the tumor cells and
ErbB3 siRNA was used as a negative control
since ErbB3 is not involved in this effect. The
western blot data showed that Met siRNA was
able to knock down Met expression in OSCC
cells, compared to scrambled and ErbB3 siRNA
transfection (Figure 3A). Moreover, Met siRNA
reduced the OSCC cell viability and reversed
the cetuximab resistance induced by HGF treat-
ment, while ErbB3 siRNA did not show these
effects (Figure 3B). At the protein level, p-Akt
and p-ERK1/2 expression was blocked by
cetuximab treatment and was restored partially
by HGF; however, the expression of these pro-
Figure 2. Treatment with the Met inhibitor restored OSCC cell sensitivity to cetuximab in HGF-induced cetuximab-
resistant OSCC cells and decreased Akt and ERK1/2 phosphorylation. (A) The MTT assay. HSC-2 and HSC-3 cells
were grown and treated with different doses of PHA-665752 or pretreated with or without HGF (20 ng/mL) and
cetuximab (1 µg/mL). (B) Western blot. The cells described in (A) were subjected to western blotting.

Drug combination in treatment of oral cancer
2375 Am J Transl Res 2019;11(4):2370-2381
teins was almost completely blocked by Met
siRNA transfection (Figure 3C).
Association of met and akt expression with
poor OSCC outcomes
To relate our current data clinically, we assess-
ed and retrieved data on the differentially
expressed genes in OSCC tissue samples from
the NCBI-GEO database and then identied the
expression of HGF, Met, ERK1 (also called
MAPK3 in Figure 4D), ERK2 (also called MAPK1
in Figure 4E), and Akt in OSCC tissues. The
Kaplan-Meier curves were plotted against the
expression of these genes and then statistical-
ly analyzed by using the log rank test. We found
that the expression of Met and Akt was associ-
ated with a poor survival of OSCC patients (P =
0.021 and 0.011, respectively; Figure 4A and
4F), whereas HGF expression was associated
with a better patient survival (P = 0.026; Figure
4B). Moreover, interactive analysis of HGF and
Met expression did not yield any statistical sig-
nicance for patient survival (Figure 4C). In
addition, the expression of ERK1 and ERK2
also did not show any signicant association
with patient survival (P = 0.20 and 0.51; Figure
4D and 4E).
Discussion
Cetuximab is a monoclonal antibody used to
treat various human cancers by blockage of
EGFR activation to inhibit the downstream
pathways in tumor cell growth and invasion
[36]. However, primary and acquired cetuximab
resistance occurs in most cancer cases, even if
the tumor cells express a high level of EGFR
[37]. The underlying molecular mechanisms of
drug resistance are complicated. For example,
in colon cancer, resistance to anti-EGFR anti-
bodies is mainly through the irregular activation
of the EGFR downstream proteins by other pro-
teins, like RAS [38], STAT [39], PTEN [40], or
Figure 3. Met siRNA restoration of OSCC cell sensitivity to cetuximab in HGF-induced cetuximab-resistant OSCC
cells. A. Western blot. HSC-2 cells were grown and transiently transfected with Met, negative control, or ErbB3 siRNA
for 48 h and then subjected to western blotting. B. The MTT assay. HSC-2 cells were grown and transiently trans-
fected with Met, negative control, or ErbB3 siRNA for 48 h and then subjected to the MTT assay. C. Western blot.
HSC-2 cells were grown and transiently transfected with Met siRNA for 48 h and then treated with cetuximab (1 µg/
mL) for 72 h before being subjected to western blotting.

Drug combination in treatment of oral cancer
2376 Am J Transl Res 2019;11(4):2370-2381
Met [41]. A single nucleotide polymorphism of
the EGFR gene has been reported to occur in
more than 40% of HNSCC patients, a change
that could affect the afnity of cetuximab to
bind to EGFR protein [42]. In another previous
study, Smad4 expression was found to be criti-
Figure 4. Association of gene expression with the survival of OSCC patients. The online database data were retrieved
and statistically analyzed. A. Association of Met expression with the overall survival (OS) of OSCC patients. High Met
expression predicted a poor OS (P = 0.021). B. Association of HGF expression with the OS of OSCC patients. A high
HGF level was associated with a better OS (P = 0.026). C. Association of Met/HGF expression with the OS of OSCC
patients. The combined expression of Met and HGF failed to predict the OS of OSCC patients. D and E. Association
of ERK1 and ERK2 expression with the OS of OSCC patients. ERK1 and ERK2 expression failed to predict the OS
of OSCC patients. F. Association of Akt expression with the OS of OSCC patients. A high Akt expression predicted a
poor OS of OSCC patients (P = 0.011).

Drug combination in treatment of oral cancer
2377 Am J Transl Res 2019;11(4):2370-2381
cal in mediating cetuximab sensitivity and
resistance [43]. In addition, in cetuximab-sen-
sitive HNSCC A431 cells, cetuximab downregu-
lated p-ERK and p-Akt expression, whereas Akt
phosphorylation was unchanged in cetuximab-
resistant cells [44]. In our current study, we
found that HGF-induced cetuximab resistance
occurred in both HSC-2 and HSC-3 cells with
high levels of p-Akt and p-ERK1/2 when they
were cotreated with cetuximab, indicating that
Akt and ERK1/2 were responsible for mediat-
ing cetuximab resistance in OSCC.
Met protein is frequently highly expressed in
various human cancers. It is activated by its
ligand HGF to trigger activation of the down-
stream signaling pathways and multiple cellular
events [45], like cancer development and
metastasis. However, it has been shown in lung
cancer and hepatocellular carcinoma that aber-
rant HGF-independent Met activation occurs
and then in turn triggers the ErbB3/PI3K/Akt
signaling pathway, leading to cancer progres-
sion [46, 47]. In HNSCC, a previous study has
revealed that ErbB3 is required to activate
EGFR and drug resistance [48]. However, in our
current study, we did not provide any evidence
showing the importance of ErbB3 in HGF-
induced cetuximab resistance in OSCC cells.
Furthermore, in non-small cell lung cancer, it
has been demonstrated that there is a syner-
gistic effect of HGF and EGF on cancer cell
growth and that the cross-talk between Met
and HGF, at the molecular level, is crucial for
tumor cell growth [49]. In colorectal cancer,
HGF induction of Met activation resulted in
cetuximab resistance [50]. Furthermore, Met
siRNA was able to reduce HNSCC cell viability
and migration in vitro and tumor xenograft
growth in vivo [51]. In addition, in human OSCC
tissues, Met expression was signicantly upreg-
ulated compared with that of the adjacent nor-
mal tissues [35]. Met inhibitors exhibited a
strong effect on inhibition of OSCC cell growth
and induction of OSCC cell apoptosis by sup-
pression of Akt and ERK1/2 activities [52]. In
our current study, HGF induced Akt and ERK1/2
phosphorylation, leading to cetuximab-resis-
tant OSCC cells. With the combination of the
Met inhibitor and Met siRNA, OSCC cell viability
was signicantly reduced and Akt and ERK1/2
phosphorylation was dramatically decreased.
Taken together, we speculated that Met and
EGFR cross-talk could synergistically promote
cancer progression and that anti-Met therapy
could be effective even in OSCC cells resistant
to anti-EGFR therapy. In this context, the combi-
nation of cetuximab and Met inhibitor could be
a better option for the treatment of OSCC
patients with primary or acquired cetuximab
resistance.
Indeed, different Met inhibitors are under clini-
cal trials, and crizotinib and cabozantinib were
approved by the US FDA to treat NSCLC and
medullary thyroid cancer, respectively [53].
Another Met inhibitor, PHA-665752, is a com-
pound used to inhibit Met phosphorylation and
the downstream signaling cascades [54]. In
ovarian cancer, PHA-665752 and Met siRNA
have been shown to inhibit tumor cell growth
and overcome cisplatin resistance [55]. While
in colorectal cancer, the combination of PHA-
665752 and cetuximab signicantly decreased
tumor cell proliferation compared with that of
either agent alone [56]. In our current study,
PHA-665752 was shown to resensitize OSCC
cells to cetuximab resistance, and its combina-
tion with cetuximab had a better antitumor ef-
cacy. At the molecular level, our current data
showed that both HGF and cetuximab were
able to regulate Akt and ERK1/2 activities.
Indeed, Yu et al. have demonstrated that the
PI3K/Akt/mTOR pathway is an ideal target for
controlling OSCC because the combination of
PI3K/Akt inhibitor with radiation improved the
radiation efcacy for the treatment of OSCC
[57]. Targeting of the PI3K/Akt/mTOR pathway
also improved the effects of doxorubicin on its
antitumor activity in OSCC [58]. Furthermore,
sulfasalazine (SSZ), an anti-inammatory drug,
has been demonstrated to have a potential
therapeutic ability in the treatment of OSCC by
promoting autophagy-induced tumor cell death
and inhibiting the PI3K/Akt and MAPK path-
ways [59]. Another previous study has revealed
that activation of the ERK, Akt, and p38 path-
ways is involved in HGF- and EGF-induced OSCC
cell migration [60]. Taken together, the PI3K/
Akt and MAPK pathways could be involved in
OSCC development and progression, and these
signaling pathways are activated by different
upstream factors, e.g. EGFR and Met. When
OSCC cells became sensitive to cetuximab
treatment, the Akt and ERK activities were sig-
nicantly suppressed in OSCC cells, leading to
a decrease in OSCC progression; whereas in
cetuximab-resistant cells, the cetuximab-EGFR-

Drug combination in treatment of oral cancer
2378 Am J Transl Res 2019;11(4):2370-2381
Akt/ERK axis was aberrant, but there was an
increase in HGF-induced Met activity to induce
Akt/ERK phosphorylation, leading to OSCC cell
proliferation. However, blockage of these two
signaling pathways using the combination of
cetuximab and Met inhibitor or Met siRNA inhib-
ited Akt and ERK phosphorylation, resulting in
cell growth inhibition. Thus, our current data
provide insightful information regarding the
effects of the combined treatment of cetux-
imab and Met inhibitor as a novel therapeutic
strategy in the control of OSCC.
In addition, our ex vivo data analysis showed
that the expression of the cell growth signaling
proteins Akt and Met was associated with a
poor survival of OSCC patients. However, due to
the small sample size, the expression of Met
with HGF did not show statistically signicant
data that predict OSCC patient survival. Future
studies with a large sample size should be per-
formed to conrm our current ndings. However,
our current study does have some limitations;
for instance, all the experiments were per-
formed in vitro, and in vivo experiments are
needed to assess and validate the effects of
the combined treatment of cetuximab and Met
inhibitor on the control of OSCC in the future.
Furthermore, our ex vivo data lacked a suf-
cient sample size as well as cetuximab or Met
inhibitor treatment data for OSCC patients.
Conclusions
Our current study revealed that HGF activated
Met, subsequently increasing the phosphoryla-
tion of the downstream PI3K/Akt and MAPK
pathways, which were responsible for the
cetuximab resistance of OSCC, and that inhibi-
tion of Met expression or activity restored OSCC
sensitivity to cetuximab treatment. Future stud-
ies are needed to conrm the effects of the
combination treatment of cetuximab with Met
inhibitor in the control of OSCC clinically.
Acknowledgements
This study was supported in part by grants from
the Foshan Science and Technology Innovation
Project (#2017AB002001 and #2015AG1-
0010), the National Natural Science Founda-
tion of China (#81570202, #81570376 and
#81870307), the University Special Innovative
Research Program of Department of Education
of Guangdong Province (#2017KTSCX189) and
a Project of DEGP (#2015KTSCX154).
Disclosure of conict of interest
None.
Abbreviations
Akt, Protein kinase B; c-Met, hepatocyte growth
factor receptor; EGFR, epidermal growth factor
receptor; ERK1/2, Extracellular signal-regulat--regulat-
ed kinase; HGF, Hepatocyte growth factor;
OSCC, Oral squamous cell carcinoma.
Address correspondence to: Dahai Liu and Fang Liu,
Department of Basic Medicine and Biomedical
Engineering, The School of Stomatology and Me-
dicine, Foshan University, 5 Hebin Road, Chancheng
District, Foshan 528000, Guangdong, China. E-mail:
seansean2014@126.com (DHL); 542998816@qq.
com (FL)
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