Vol$1,$Issue$1,$2019$(11-18)$
http://journal.unpad.ac.id/IdJP$
*Corresponding$author,$ $ $ $ $ $ $$$$https://doi.org/10.24198/idjp.v1i1.19582$
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Exploration of Proteins Involved in Acquisition of Resistance to Cetuximab
Hironori Nakamura
1,2
, Ayumu Nagamine
1,2
, Hideaki Yashima
1,2
, Takuya Araki
1,2,*
, Koujirou
Yamamoto
1,2
1. Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School of
Medicine, Maebashi 371-8511, Japan
2. Department of Pharmacy, Gunma University Hospital, Maebashi 371-8511, Japan
Received: 10 Dec 2018/Revised: 30 Dec 2018/Accepted :2 Jan 2019/Published
ABSTRACT
Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (Mabs) show high efficacy in about
50% of colorectal cancer (CRC) patients with wild-type KRAS. However, < 20% of patients with KRAS
wild-type CRC have continued therapeutic effects with these agents, and acquired resistance to treatment
has become a serious clinical problem. In this study, to clarify the factors related to acquisition of resistance
to cetuximab and establish countermeasures against such acquired resistance, we conducted a
comprehensive protein analysis via a proteomics approach using acquired resistance cell lines derived from
cetuximab -sensitive CRC cell lines and original cell lines. Cetuximab-acquired resistance cell lines were
generated by continuous exposure of SW48 and C99 cell lines to cetuximab. Expression of deoxycytidine
kinase (dCK) and zinc finger and BTB domain-containing protein 41 (ZBTB41) increased more than 10-
fold, and dual specificity protein phosphatase 3 (DUS3) expression decreased by less than 1/10 with
acquisition of resistance to cetuximab in both C99 and SW48 cell lines. Because overexpression of dCK is
known as a positive indicator of efficacy of nucleoside analogs such as cytarabine or gemcitabine, it is
considered that nucleoside analogs activated by dCK may be useful agents in treatment of cancers with
acquired cetuximab-resistance. In the future, we need to clarify the usefulness of these drugs for the
treatment of cetuximab resistant CRC and to assess the possibility of restoration of cetuximab sensitivity
by regulation of ZBTB41 and DUS3 expression.
Keywords:cetuximab, colorectal cancer, acquired resistance, protein, dCK, ZBTB41
1. Introduction
Cetuximab was launched in the United States
and Europe in 2004 as an anti-epidermal growth
factor receptor (EGFR) monoclonal antibody
(Mab) for treatment of colorectal cancer (CRC) and
pharyngeal cancer. Even in Japan, cetuximab has
been widely used to treat CRC and pharyngeal
cancer, and is considered a drug of choice,
especially in the treatment of CRC. A large-scale
clinical trial showed that the presence of genetic
variations of KRAS is a resistance factor for anti-
EGFR Mabs including cetuximab and that the
response rate to anti-EGFR Mabs in CRC patients
with wild-type KRAS was about 50%(1-3).
However, it is reported that < 20% of patients with
KRAS wild-type CRC who experience continued
therapeutic effects of anti-EGFR Mabs, and
acquired resistance to treatment has become a
serious clinical problem(1-3). As a countermeasure
to acquired resistance to anti-cancer drugs, factors
affecting development of acquired resistance were
investigated in several studies, and several factors
including the RAS-RAF-mitogen activated protein
kinase (MAPK)/Erk kinase (MEK)-MAPK
signaling pathway, which is significantly involved
in the proliferation of cancer cells, were reported to
affect tolerance to cetuximab(1-3). Troiani and
colleagues reported that MEK inhibitors showed
high growth inhibition efficacy against cells with
acquired resistance established by administering
cetuximab to mice inoculated with cancer cells or
exposing cultured cells to cetuximab, and that
concomitant use of cetuximab and a MEK inhibitor
showed stronger growth inhibition efficacy(4). In
addition, Zhang et al. reported that down-regulation
2019 Nakamura et al
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of phosphatase and tensin homolog (PTEN) and an
increase in phosphorylated protein kinase B, Akt
were found in acquired cetuximab-resistant cell
lines generated from cetuximab-sensitive cell lines
by exposing exosomes extracted from cetuximab-
resistant cell lines and acquired resistance to
cetuximab was canceled by use of Akt inhibitor (5).
However, those studies focused were on specific
pathways or proteins and could not sufficiently
account for factors affecting acquisition of
resistance to cetuximab, and thus countermeasures
against acquired resistance have not yet been
established. To establish appropriate
countermeasures against acquired resistance to
treatment, the influence of an enormous number of
proteins, including proteins other than targets of
analysis, have to be assessed and each mechanism
of resistance development caused by those factors
has to be clarified (6).
In recent years, the proteomics approach has
attracted attention as a comprehensive method of
protein analysis. The proteomics approach was
applied in many studies such as in the search for
predictive markers of the efficacy of erlotinib in
non-small cell lung cancer (7-11) and that of
platinum preparations for ovarian cancer (12), and
successfully established many predictive markers
of drug efficacy and factors affecting drug
susceptibility.
In this study, to clarify the factors related to
acquisition of resistance to cetuximab and establish
countermeasures against this acquired resistance,
we carried out a comprehensive protein analysis via
a proteomic approach using acquired resistance cell
lines generated from cetuximab-sensitive CRC cell
lines and original cell lines.
2. Method
2.1. Materials
Cetuximab sensitive CRC cell lines, C99 and
SW48 without genetic mutations affecting
sensitivity to anti-EGFR mAbs, such as KRAS,
NRAS, BRAF, and phosphatidylinositol-4,5-
bisphosphate 3-kinase catalytic subunit alpha
(PIK3CA) mutations, and PTEN overexpression,
were purchased from the European Collection of
Cell Cultures (Salisbury, UK) and the American
Type Culture Collection (Manassas, VA),
respectively. Reagents for culture and sample
preparation were purchased from Wako Pure
Chemical Industries (Osaka, Japan). All other
reagents were obtained from commercial sources,
and those used to analyze peptides were graded for
high-performance liquid chromatography, liquid
chromatography-tandem mass spectrometry (LC-
MS/MS), or analytical use.
2.2. Cell culture and sample preparation
The C99 cell line was cultured in a humidified
incubator at 37
o
C in the presence of 5% CO2, and
the SW48 cell line was cultured in a 37ºC incubator
with no supplemental CO2. Cetuximab-acquired
resistance cell lines (SW48-CR and C99-CR) were
generated upon continuous exposure of SW48 and
C99 cell lines to cetuximab according to the method
described by Troiani et al (13). Briefly, SW48 and
C99 cell lines were continuously exposed to
cetuximab, at a starting concentration of 0.1 μg/mL,
increased 2-fold every 30 days, to increase the
inhibition of 50% of cancer cell growth (IC50), up
to a final concentration of 12.8 μg/mL.
Cytoplasmic proteins were extracted from 80%
confluent cell lines using a Minute Plasma
Membrane Protein Isolation Kit (Invent
Biotechnologies, Inc., Plymouth, MN), and the
concentrations of the extracted proteins were
measured using a DC™ Protein Assay Kit (Bio-
Rad Laboratories, Inc., Hercules, CA).
Cytoplasmic protein extracts were diluted to 0.7
mg/mL, and 180 µL of the samples were mixed
with 20 μL of 5 mg/mL bovine serum albumin and
incubated at 37ºC for 90 min with 163 mg urea and
15.4 µL of 40 mg/mL dithiothreitol in 8 mol/L
urea/0.5 mol/L Tris HCl (pH 8.5) for reduction of
disulfide bonds. Reduced samples were alkylated
by reacting with 38.4 μL of 40 mg/mL
iodoacetamide in 8 mol/L urea/0.5 mol/L Tris-HCl
(pH 8.5) for 30 min at 37ºC. Subsequently, to digest
the proteins, 5.2 μL of 1 mg/mL trypsin in 20
mmol/L acetic acid was added to 984 μL of samples
diluted 4-fold with Milli-Q water, respectively, and
trypsinization was performed at 37ºC overnight.
Trypsinized samples were desalted using a
H.$Nakamura$et#al$/$Indo$J$Pharm$1$(2019)$11-18$
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MonoSpin C18 column (GL Sciences, Inc., Tokyo,
Japan).
2.3. MS analysis
Liquid chromatography/mass spectrometry
(LC/MS) analysis was performed with the
EksigentNanoLC 425 coupled to Triple TOF 6600
(AB Sciex, Tokyo, Japan) interfaced to a
NanoSpray III ion source. First, 10 μL of the
samples were loaded onto a trap column (Acclaim
PepMap 100 C18, 5 μm, 0.2 mm I.D. ×10 mm,
Thermo Fischer Scientific K.K., Tokyo, Japan),
and were then separated using an analytical column
(Acclaim PepMap 100 C18, 3 μm, 0.075 mm I.D.
×250 mm, Thermo Fischer Scientific) with a
gradient from 2 to 32% solvent B at a flow rate 300
nL/min for 120 min (solvent A: 0.1% formic acid
in water; solvent B: 0.1% formic acid in
acetonitrile). Ion source parameters were set as
follows: ion source voltage (ISVF) 2350 V, ion
source gas (GS1 and GS2) 5 and 0, interface heater
temperature150C, declustering potential (DP)
80V.
2.4. Sequential Window Acquisition of All
Theoretical Mass Spectra (SWATH) data analysis
SWATH runs were acquired using the 100
SWATH variable window method (AB Sciex Pte.
Ltd., Singapore) from m/z 100 to 1800 with each 25
ms accumulation time. Library samples were
prepared by mixing samples obtained from 4 cell
lines (C99, C99-CR, SW48, SW48-CR) equally,
and measured 6 times by data dependent
acquisition (DDA) selecting the top 25 highest
peaks found in the survey scan. Database searching
for the library was performed with ProteinPilot ver.
5.0 (AB Sciex) using the database downloaded
from Uniprot (uniprot_sprot.fasta, released 23 Nov.
2017). The 6 DDA runs were combined and
analyzed using the Paragon algorithm. The
resulting identified proteins with global false
discovery rate (FDR) 1% (1596 proteins) were
imported into SWATH Acquisition MicroApp ver.
2.0 (AB Sciex) on Peak View ver. 2.2 (AB Sciex)
as the library. The extracted ion chromatograms of
12 SWATH runs were mined for 5 transitions per
peptide and 5 peptides per protein, and then
processed with a peptide confidence threshold of
99% and a false discovery rate < 1%.
2.5. Data analysis
Data on expression levels of proteins analyzed
by SWATH was adjusted by peak intensity of
human serum albumin. Proteins for which
expression levels in acquired resistance cell lines
changed more than 2- or 0.5-fold compared to
original cell lines along with acquisition of
resistance were used as specific proteins in
cetuximab-resistant or cetuximab-sensitive cell
lines. Gene ontology (GO) analysis was performed
to analyze relevance of proteins specific to
cetuximab-resistant cell lines or -sensitive cell lines
was analyzed using The Database for Annotation,
Visualization and Integrated Discovery (DAVID)
v6. 8 (https://david.ncifcrf.gov).
3. Result
SWATH analysis detected 4,179 kinds of
peptides composed of 20,895 peaks; 1,294 kinds of
proteins were identified and quantified.
In the C99 cell line, the expression levels of 95
proteins increased more than two-fold with
acquisition of resistance, and 3 of 33 proteins
whose expression levels increased more than 10-
fold were glycolysis/gluconeogenesis-related
proteins (Figure 1). On the other hand, the
expression levels of 230 proteins decreased to half
or less, and 3 of 29 proteins whose expression
levels decreased to 1/10 or less were spliceosome-
related proteins (Figure 1). Likewise, in the SW48
cell line, the expression levels of 91 proteins
increased more than double with acquisition of
resistance, and the expression level increased more
than 10-fold in 30 kinds of proteins, but no
relationship was found among them. The
expression levels of 147 proteins decreased to half
or less and the expression levels of 20 kinds of
proteins decreased to 1/10 or less (Figure 2), but no
relationship was found for each protein.
H. Nakamura et al / Indo J Pharm 1 (2019)
14
Figure 1. Proteins whose expression level increased 2-fold or decreased to less than half with acquisition of
resistance to cetuximabin the C99 cell line (* and ** denote glycolysis/gluconeogenesis related proteins and
spliceosome related proteins, respectively)
Figure 2. Proteins whose expression level increased more than 2-fold or deceased to less than half with acquisition
of resistance to cetuximabin the SW48 cell line.
-100
-80
-60
-40
-20
0
20
40
60
80
100
CAH1
DOHH
ADH1G
PYRG1
CCDC6
IMA1
STAM1
CTNB1
HDDC2
G3BP1
AAGAB
DPP9
EFL1
SP16H
PFKAP
GABT
SYPL1
C99-CR / C99
C99 / C99-CR
340.2
257.5
104.2
111.6
Expression ratio
KCC2D
NPL4
NR2CA
MAP1S
AL3A1
ACOT2
JIP4
ARL3
ZBT41
LSM7
REG1B
IMA4
XPO5
2AAB
DCK
GFRP
H32
BLMH
H2B1M
GLNA
LAP2B
H12
DUS23
RAB10
PROF2
CPSM
PLPP
PARP1
SNW1
EI2BE
GNA1
RRBP1
DUS3
CSN1
GSTK1
EEA1
TBCB
H4
HNRPM
PRRC1
S10AG
DDX5
BCCIP
H2AJ
NFKB1
190.6
108.4
S10AG
ZPR1
TXD12
SNW1
YAP1
HAT1
GSTK1
FAHD1
FUBP3
ROA0
MEPCE
PTER
TYDP2
NLTP
PREP
AIP
GPAM1
CMBL
HNRL2
ARY1
PDCL3
ISG15
ZBT41
DCK
MIP18
GORS2
FABPL
ESRP1
PRKDC
GUAD
SW48-CR / SW48
SW48 / SW48-CR
232.5
Expression ratio
ACADM
DUS3
TRIR
PRC2C
PRKRA
LAGE3
ATE1
MP2K2
UBQL2
SSA27
DRG1
ISOC2
MYO1E
ATPA
SRSF3
UCHL5
SAP3
ZNF66
ECHB
FKB10
H.$Nakamura$et#al$/$Indo$J$Pharm$1$(2019)$11-18$
15
Figure 3. Proteins whose expression level increased more than 2-fold with acquisition of resistance to
cetuximabin both C99 and SW48 cell lines (* denotes proteins with “molecular function” of “protein binding”)
Figure 4. Proteins whose expression level decreased to less than half with acquisition of resistance to cetuximabin
both C99 and SW48 cell lines (*1, denotes proteins with “molecular functions” of “poly(A) RNA binding” and
“cellular component” of “nucleus”, *2 denotes ribosomal proteins, and *3 denotes spliceosome proteins,
respectively.
As proteins which increased more than doubled
with acquisition of resistance to cetuximabin both
of C99 and SW48 cell lines, 19 kinds of proteins
were found. GO analysis showed that 12 of 19
proteins have "protein binding as a molecular
function" as a common factor, but no relationship
0 20 40 60
SW48-CR/SW48
0204060
C99-CR/C99
CAH1*
CCDC6*
DOHH*
HDDC2*
KCC2D*
MIP18*
SBP1*
TPM1*
ZBT41
ADH1G
CATS
DCK
GNAT3
ISOC1*
LEG4
PYRG2*
SYPL1
VP26A*
ZPR1*
93.8
340.2
190.6
257.5
80.9
Expression ratio Expression ratio
CATZ
DRG1
EEA1
GNA1
H4*
1
KHDR1*
1
MVP
PUF60 *
3
RL10*
1,2
RL18*
2
RS27L*
1,2
RS9*
1,2
SFPQ*
1
SSBP*
1
THOC4*
1
VIGLN*
1
0 20 40 60
0204060
CAD17
CCD58
DUS3
FKB10
H2B1M
IVD
LAP2B
NEMF
RISC
RL10A*
2
RL7*
1,2
RS5*
2
SC16A
SRSF3*
3
TCOF*
1
UCHL5*
1
75.6
104.2
82.9
72.8
Expression ratio Expression ratio
C99 / C99-CR SW48 / SW48-CR
H. Nakamura et al / Indo J Pharm 1 (2019) 11-18
16
was found among these proteins (Figure 3).
Deoxycytidine kinase (dCK) and zinc finger and
BTB domain-containing protein 41 (ZBTB41)
increased more than 10-fold after acquiring
resistance in both of C99 and SW48. On the other
hand, the expression levels of 32 proteins decreased
to half or less in both of C99 and SW48 with
acquisition of resistance to cetuximab(Figure 4).
GO analysis showed that 12 of 32 proteins have
"nucleus as a cellular component" and "poly (A)
RNA binding as a common factor" as common
factors. In addition, 7 of 32 proteins were
constituent proteins of ribosomes and other 3
proteins were constituent proteins of spliceosomes.
Dual specificity protein phosphatase 3 (DUS3)
decreased to less than 1/10 after acquiring
resistance in both of C99 and SW48 (Figure 4).
4. Discussion
The proteins obtained from cetuximabresistance
cell lines generated by exposing cetuximab-
sensitive cell lines to cetuximaband that from
original cell lines were analyzed, and increase of
dCK and ZBTB41 and decrease of DUS3 were
found as a common factor related to acquisition of
resistance to cetuximabin both C99 and SW48 cell
lines.
dCK is an enzyme catalyzing the phosphate
esterification reaction of the 5'-OH of
deoxynucleosides, which is the rate-determining
step in the nucleoside salvage pathway. In cancer
chemotherapy, dCK is an enzyme needed for
phosphorylation of several deoxyribonucleosides
and their nucleoside analogs such as gemcitabine
and cytarabine and is known as a rate limiting
enzyme in activation of these drugs. Furthermore,
in addition to a report that the deficiency of dCK is
involved in resistance to gemcitabine and
cytarabine, it has also been reported that these
drugs show very high anti-tumor efficacy in cancer
cells that overexpress dCK(14-17). We found for
the first time that the expression levels of dCK
increase with the acquisition of resistance to
cetuximabin this study. Although the reasons and
mechanisms for activation of the metabolic
pathway of nucleic acids regarding acquisition of
resistance to cetuximabinhibiting EGFR pathway is
unknown, it is considered that the pathway with the
activation of dCK as the final reaction may be
activated as an alternative pathway to the EGFR
pathway. In addition, although more detailed study
using more cetuximab-resistant cell lines is needed,
nucleoside analogs activated by dCK, such as
gemcitabine or cytarabine, may be also be useful
agents for treating cancers that have acquired
tolerance to cetuximab.
On the other hand, the impact of increased
ZBTB41 expression and decreased DUS3
expression on the efficacy of chemotherapy has not
been reported. ZBTB41 is a protein involved in the
stabilization of various proteins such as ribosomal
proteins and is involved in the regulation of
expression of various proteins (18), and
overexpression of ZBTB41 is known to be a poor
prognostic factor in liver cancer (19). DUS3 is a
member of the dual-specificity protein phosphatase
subfamily. By dephosphorylation of
phosphorylated residues, DUS3 negatively
regulates certain pathways, such as the MAPK
pathway, which are associated with cellular
proliferation and differentiation (20). Although
both of ZTBT41 and DUS3 regulate several protein
expression levels, the impact of fluctuation of those
proteins on the efficacy of chemotherapy has not
been studied. In the future, it is necessary to clarify
the significance of variations in ZBTB41 and
DUS3 expression levels by analyzing the change in
expression levels of several proteins along with
changes in ZBTB41 and DUS3 expression levels
and assess the possibility of restoration of
cetuximab sensitivity by regulation of ZBTB41 and
DUS3 expression.
In addition, glycolysis/gluconeogenesis-related
proteins were detected as a cetuximab resistance-
related factor in C99 cells. The relationship
between drug resistance and changes in expression
of glycolysis/gluconeogenesis-related proteins has
been reported in several papers, and cancer cells are
considered to produce energy as required for
survival in the microenvironment by altering the
energy production pathway. However, because an
increase in glycolysis/gluconeogenesis-related
proteins was observed in only C99 but not in SW48
cells, the significance of this change varies greatly
depending on the cell line, and thus it would be
H. Nakamura et al / Indo J Pharm 1 (2019) 11-18
17
difficult to establish a therapeutic target for
cetuximab-resistant tumors.
In this study, because proteomic assay was
performed using whole cells, we could not evaluate
proteins with low expression such as EGFR
pathway-related proteins, which is the underlying
pathway in the mechanism of action of cetuximab.
To evaluate the factors affecting acquisition of
resistance to cetuximab, more detailed study
focusing on proteins with low expression is needed.
5. Conclusion
In conclusion, we have revealed for the first time
a change in dCK, ZBTB41, and DUS3 expression
levels, largely with acquisition of resistance to
cetuximab. Our data suggest that nucleoside
analogs such as cytarabine and gemcitabine may be
useful for treating CRC with acquired resistance to
cetuximab. In future, it would be necessary
elucidate the usefulness of these drugs for the
treatment of cetuximab-resistant CRC and to assess
the possibility of restoration of cetuximab
sensitivity by regulation of ZBTB41 and DUS3
expression.
Acknowledgements
We thank Dr. Touko Hirano (Laboratory for
Analytical Instruments, Education and Research
Support Center, Gunma University Graduate
School of Medicine) and Dr. Mitsue Miyazaki
(Division of Endocrinology, Metabolism and
Signal Research, Gunma University Initiative for
Advanced Research) for excellent technical
assistance. This work was supported by JSPS
KAKENHI Grant Number JP16H00504 and
JP18K06743.
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