MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer
MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer
Danielle Queiroz Calcagno 0 1 3 4
Vanessa Morais Freitas 2
Mariana Ferreira Leal 0 3
Carolina Rosal Teixeira de Souza 1 4
Samia Demachki 7
Raquel Montenegro 1 4
Paulo Pimentel Assumpção 6
André Salim Khayat 1 4
Marília de Arruda Cardoso Smith 0 3
Andrea Kely Campos Ribeiro dos Santos 5
Rommel Rodriguez Burbano 1 4
0 Disciplina de Genética, Departamento de Morfologia e Genética, Escola Paulista de Medicina, Universidade Federal de São Paulo , Rua Botucatu 740, CEP 04023-900, São Paulo, SP , Brazil
1 Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém, PA , Brasil
2 Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo , São Paulo, SP , Brasil
3 Disciplina de Genética, Departamento de Morfologia e Genética, Escola Paulista de Medicina, Universidade Federal de São Paulo , Rua Botucatu 740, CEP 04023-900, São Paulo, SP , Brazil
4 Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém, PA , Brasil
5 Laboratório de Genética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém, PA , Brasil
6 Serviço de Cirurgia Hospital Universitário João de Barros Barreto, Universidade Federal do Pará , Belém, PA , Brasil
7 Laboratorio de Imunoistoquímica, Serviço de Anatomia Patológica, Faculdade de Medicina, Hospital Universitário João de Barros Barreto, Universidade Federal do Pará , Belém, PA , Brasil
Background: MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. Methods: We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. Results: MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. Conclusion: In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer.
Gastric cancer; MYC; FBXW7; TP53
Gastric cancer (GC) is the fourth most common
cancer and the second leading cause of cancer death
worldwide . GC is considered a major public health
concern, especially in developing countries, including
A fundamental aspect of carcinogenesis is
uncontrolled cell proliferation resulting from the accumulation
of changes that promote the expression or repression of
cell cycle-control genes . MYC is a transcriptional
factor involved in cell cycle regulation and cell growth
arrest that is commonly deregulated in cancers and has
been described as a key element of gastric carcinogenesis
[4,5]. Several different types of posttranslational
modifications of MYC have been described, including
phosphorylation, acetylation, and ubiquitination . The
ubiquitin-proteasome system is the major protein
degradation regulatory pathway involved in cell differentiation
and growth control . FBXW7 encodes an F-box protein
subunit of the Skp1/Cul1/F-box complex (SCF) ubiquitin
ligase complex. SCFFBXW7 induces degradation of the
products of positive cell cycle regulator genes, such as cyclin
E, MYC, NOTCH, and JUN, through
phosphorylationdependent ubiquitination . Among SCFFBXW7 substrates,
MYC is of particular importance in cell cycle exit because
it is thought to play a role in determining whether
mammalian cells divide or not .
Deregulated FBXW7 expression is a major cause of
carcinogenesis [10-12]. Loss of FBXW7 expression can
lead to MYC overexpression and has been associated
with poor prognosis in GC patients . However, MYC
activation by FBXW7 loss triggers activation of p53,
which plays a key role in the regulation of cellular
responses to DNA damage and abnormal expression of
oncogenes. Induction of cell cycle arrest by p53 allows
for DNA repair or apoptosis induction . Thus,
concomitant loss of FBXW7 and TP53 is necessary to
induce genetic instability and tumorigenesis .
In the present study, we investigated MYC, FBXW7,
and TP53 gene copy number variation and mRNA and
protein expression in GC samples and gastric
adenocarcinoma cell lines. Possible associations between our
findings and the clinicopathological features and/or
invasion and migration capability of the cell lines were
Samples were obtained from 33 GC patients who
underwent surgical treatment at the João de Barros Barreto
University Hospital in Pará State, Brazil. Dissected
tumor and paired non-neoplastic tissue specimens were
immediately cut from the stomach and frozen in liquid
nitrogen until RNA extraction.
The clinicopathological features of the patient samples
are shown in Table 1. GC samples were classified
according to Lauren . All GC samples showed the
presence of Helicobacter pylori, and the cagA virulence
factor was determined by PCR analysis of ureA and cagA
as described by Clayton et al.  and Covacci et al.
, respectively. All patients had negative histories of
exposure to either chemotherapy or radiotherapy before
surgery, and there were no other co-occurrences of
diagnosed cancers. Informed consent with approval of the
ethics committee of the Federal University of Pará was
Gastric adenocarcinoma cell lines ACP02 and ACP03
 were cultured in complete RPMI medium (Invitrogen
Corp., Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (FBS), 1% penicillin/streptomycin, and 1%
Copy number variation (CNV)
DNA was extracted using a DNAQiamp mini kit (Qiagen,
Hilden, Germany) according to the manufacturer’s
instructions. Duplex quantitative real-time PCR (real-time
qPCR) was performed using the FAM/MGB-labeled
TaqMan probes for MYC (Hs01764918_cn), FBXW7
(Hs01362464_cn), or TP53 (Hs06423639_cn), and VIC/
TAMRA-labeled TaqMan CNV RNAse P (#4403326) was
used for the internal control. All real-time qPCR reactions
were performed in quadruplicate with gDNA according to
the manufacturer’s protocol using a 7500 Fast Real-Time
PCR system (Life Technologies, Foster City, CA, USA).
The copy number of each sample was estimated by CNV
analysis using Copy Caller Software V1.0 (Life Technologies,
Foster City, CA, USA). Known Human Genomic DNA
(Promega, Madison, USA) was used for calibration.
Quantitative real-time reverse transcriptase PCR
Total RNA was extracted with TRI Reagent® Solution
(Life Technologies, Carlbad, CA, USA) following the
manufacturer’s instructions. RNA concentration and
quality were determined using a NanoDrop
spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and
1% agarose gels. Complementary DNA (cDNA) was
synthesized using a High-Capacity cDNA Archive kit
according to the manufacturer’s recommendations
(Life Technologies, Foster City, CA, USA). Real-time
qPCR primers and TaqMan probes targeting MYC
(Hs00153408_m1), FBXW7 (Hs00217794_m1), and TP53
(Hs01034249_m1) were purchased as Assays-on-Demand
Products for Gene Expression ((Life Technologies, Foster
City, CA, USA). Real time qPCR was performed using an
ABI Prism 7500 system (Life Technologies, Foster City,
CA, USA) according to the manufacturer’s instructions.
GAPDH (NM_002046.3; Life Technology, USA) was
selected as an internal control for monitoring RNA input
and reverse transcription efficiency. All real-time qPCR
reactions for target genes and internal controls were
performed in triplicate on the same plate. The relative
quantification (RQ) of gene expression was calculated
using the ΔΔCt method , in which the non-neoplastic
sample was designated as a calibrator for each paired
Immunohistochemical analyses for MYC and p53 were
performed on formalin-fixed, paraffin-embedded surgical
sections. Serial 3-μm sections were used. Heat-induced
antigen retrieval was employed (microprocessor-controlled
pressure Pascal® DakoCytomation, Carpinteria, CA, USA).
A universal peroxidase-conjugated secondary antibody kit
(LSAB System, DakoCytomation, Carpinteria, CA, USA)
was used for detection with diaminobenzidine (DAB) as
the chromogen. The following primary antibodies were
used: mouse monoclonal antibodies directed against MYC
(dilution 1:150; sc-40, Santa Cruz Biotechnology, Santa
Cruz, CA, USA and clone 9E10, Zymed®, San Francisco,
CA, USA), FBXW7 (dilution 1:50, Abnova Corp., Taipei
City, Taiwan), and p53 (dilution 1:50; DakoCytomation,
Carpinteria, CA, USA). Positive protein expression was
defined as clear nuclear staining in more than 10% of the
Migration and invasion assay
Migration and invasion assays were carried out in a
modified Boyden chamber with filter inserts (8-μm
pores) for 12-well plates (BD Biosciences, San Jose, CA,
USA). To assess invasion, filters were coated with 10 μl
of Matrigel (10–13 mg/ml) (BD Biosciences, San Jose,
CA, USA) while on ice. Cells (2 × 105) were plated into
the upper chamber in 1 ml of RPMI without FBS. The
lower chamber was filled with 1.5 ml of RPMI with FBS.
After 48 h in culture, cells were fixed with 4%
paraformaldehyde and post-fixed with 0.2% crystal violet in 20%
methanol. Cells on the upper side of the filter, including
those in the Matrigel, were removed with a cotton swab.
Invading cells (on the lower side of the filter) were
photographed and counted. Experiments were performed
Cells grown on glass coverslips were fixed with 1%
paraformaldehyde in phosphate-buffered saline (PBS) for
10 min, then permeabilized with 0.5% Triton X-100
(Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min
and blocked with 1% bovine serum albumin (BSA) in
PBS. The cells were stained with mouse antibodies
against MYC (diluted 1:50; Zymed®, USA), p53 (diluted
1:50; DakoCytomation, Carpinteria, CA, USA), and
FBXW7 (diluted 1:50; Abnova Corp., Taipei City,
Taiwan). Primary antibodies were revealed using an
antimouse Alexa-568-conjugated secondary antibody
(Invitrogen). All incubations were carried out for 60 min
at room temperature. Nuclei were stained with DAPI in
Prolong anti-fade mounting medium (Invitrogen).
Negative control samples were processed as described
above except that primary antibodies were omitted and
replaced with PBS alone.
Protein extraction from cells was performed according
to standard procedures. Briefly, total protein was extracted
from ACP02 and ACP03 cells using 50 mM Tris–HCl
buffer containing 100 mmol/L NaCl, 50 mM NaF, 1 mM
NaVO4, 0.5% NP-40, and complete protease inhibitor
cocktail (Roche, Germany). Protein concentration was
estimated using a Bradford assay (Sigma-Aldrich). About
30 μg of total protein extract was loaded onto a 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gel and electrophoresed. Resolved proteins
were then transferred from the gel onto a nitrocellulose
membrane. The membrane was blocked with 5% nonfat
milk in Tris-buffered saline containing 5% Tween
(SigmaAldrich, Sant Louis, MO, USA) and then incubated with
mouse monoclonal anti-MYC (Santa Cruz Biotechnology),
anti-FBXW7 (Abnova, Taipei City, Taiwan), anti-p53
(DakoCytomation, Carpinteria, CA, USA), and
anti-βactin (Sigma-Aldrich, Sant Louis, MO, USA) antibodies
diluted 1:200, 1:100, 1:100, and 1:2,000, respectively.
Subsequently, membranes were incubated with a 1:5,000
dilution of horseradish peroxidase (HRP)-conjugated
sheep anti-mouse antibody (Amersham Biosciences,
Piscataway, NJ, USA) for 1 h at room temperature.
Proteins were visualized by enhanced chemiluminescence.
ACP02 and ACP03 cells (5 × 104 of each) were plated
and allowed to adhere and spread for at least 8 h.
Adherent cells were washed three times with PBS, and the
culture medium was replaced with serum-free medium
for 24 h. The activity of MMP2 and MMP9 in the
conditioned medium was assessed by zymography.
Conditioned medium was collected, concentrated (Microcon
30 K, Merck Millipore, Darmstadt, Germany) and
resuspended in SDS-PAGE sample buffer (without
βmercaptoethanol). The remaining cells were lysed and
the protein concentration was estimated using a BCA
assay (Thermo Scientific Pierce, Rockford, IL, USA). A
total of 1 μg of protein from each conditioned medium
was separated on 10% polyacrylamide gels containing
0.2% gelatin. After electrophoresis, the gels were washed
in 2.5% Triton X-100 for 30 min, then equilibrated in
10 mM Tris (pH 8.0) and incubated at 37°C for 16–24 h
in a development buffer containing 50 mM Tris
(pH 8.0), 5 mM CaCl2, and 0.02% NaN3. The gels were
stained with 0.2% Coomassie blue R250 (GE Amersham,
Piscataway, NJ, USA) and destained with 1:1 acetic acid/
methanol solution. Experiments were performed in
triplicate. Zymographic bands, which are indicative of MMP
activity, were quantified by scanning densitometry.
The normality of variable distributions was determined
using the Shapiro-Wilk test. Associations between MYC,
FBXW7, and TP53 copy number variation, mRNA levels,
protein expression, clinicopathological features, and cell
invasion and migration capability were analyzed using
the chi-square (χ2) and Mann–Whitney tests.
Correlation between expression of the different target mRNAs
was determined using Spearman’s test, in which a value
below 0.3 indicated a weak correlation, 0.3-0.7 indicated
a medium correlation, and values above 0.7 indicated a
strong correlation. Data are shown as the median and
interquartile range; p values less than 0.05 were
Gastric tumor specimens showed amplification of MYC
and deletion of FBXW7 and TP53
Three or more copies of MYC were found in 51.5% (17/
33) of gastric tumor cells. In contrast, 45.5% (15/33) and
21.2% (7/33) of gastric tumor cells contained only one
copy of FBXW7 and TP53, respectively.
The association between clinicopathological features
and MYC, FBXW7, and TP53 copy number is
summarized in Table 1. One gastric tumor that contained three
copies of TP53 was excluded from the chi-square
analysis. No association was found between copy
number variation of the genes studied and
MYC mRNA expression was higher in tumors than in
non-neoplastic specimens, whereas FBXW7 and TP53
mRNA expression was lower in tumor specimens
The expression level of MYC mRNA (2.01 ± 1.72 fold
change) in tumor tissue samples was significantly higher
than in non-neoplastic tissue (p = 0.0002), whereas the
expression level of FBXW7 mRNA (0.53 ± 0.40 fold
change) and TP53 mRNA (0.84 ± 0.55 fold change) in
tumor tissue specimens was significantly lower than in
non-neoplastic tissue (p < 0.0001 and p = 0.0011,
respectively). We did not find a significant correlation between
MYC, FBXW7, and TP53 mRNA expression (MYC/
FBXW7 mRNA r = −0.3464, p = 0.0562; MYC/TP53
mRNA r = 0.0950, p = 0.6113; FBXW7/TP53 mRNA
r = −0.0745, p = 0.4747). Thus, only a tendency toward
correlation between an increase in MYC mRNA
expression and a decrease in FBXW7 mRNA expression
Table 2 summarizes the associations between various
clinicopathological features and the RQ of MYC, FBXW7,
and TP53 mRNA expression in tumor and paired
nonneoplastic specimens. An increase in MYC mRNA level
was associated with the presence of lymph node
metastasis (p = 0.016) and GC tumor stage III-IV (p = 0.036). A
significant reduction in FBXW7 mRNA level was also
associated with the presence lymph node metastasis
(p = 0.015) and tumor stage III-IV (p = 0.008).
Nuclear MYC protein staining is associated with
Positive staining for nuclear MYC and p53 was found in
64.5% (20/31) and 19.4% (6/31) of GC samples,
respectively (Figure 1). No positivity was found for
FBXW7. Table 1 summarizes the clinicopathological
features and MYC and p53 immunostaining results.
Expression of MYC was more frequent in intestinal-type
than diffuse-type GC (p = 0.007). Furthermore, MYC
immunostaining was associated with increased MYC
mRNA level (p = 0.0022). No association was found
between p53 immunostaining and clinicopathological
characteristics, TP53 copy number, or TP53 mRNA
Comparison of ACP02 and ACP03 cell lines
Both ACP02 and ACP03 cells contained three MYC
copies and only one FBXW7 copy. The number of TP53
copies was undetermined in both cell lines. Compared
with mRNA expression in ACP03 cells, ACP02 cells
expressed a higher level of MYC (1.34-fold) and lower
levels of FBXW7 and TP53 mRNA (0.62- and 0.73-fold,
Western blot analyses revealed that MYC expression
was significantly higher in ACP02 cells than ACP03 cells
(p = 0.048). Moreover, FBXW7 expression was significantly
lower in ACP02 cells than ACP03 cells (p = 0.049).
However, there was no significant difference in p53 expression
between the cell lines (p = 0.077) (Figure 2A-B).
Immunofluorescence analysis of both proteins showed
a punctiform pattern of labeling, supporting the Western
blot results showing an increase in MYC and reduction
in FBXW7 expression in ACP02 cells compared with
ACP03 (Figure 2). Matrigel invasion assay results
showed that ACP02 cells were more invasive than
ACP03 cells (p = 0.001). Migration assay results showed
that fewer ACP02 cells migrated compared with ACP03
cells (p = 0.0028) (Figure 2C-D).
Both ACP02 and ACP03 cells presented four gelatinase
activity bands: MMP-9 latent (92 kDa), MMP-9 active
(88 kDa), MMP-2 latent (72 kDa), and MMP-2 active
(66 kDa) (Figure 3). We found no significant differences in
MMP-9 latent (p = 0.9788), MMP-2 active (p = 0.7848),
and MMP-2 latent (p = 0.1678) between ACP02 and
ACP03 cells. However, significant differences were found
between ACP02 and ACP03 cells with respect to MMP-9
active (p = 0.0182).
In the current study, we observed that MYC mRNA
expression was increased in GC samples compared with
corresponding non-neoplastic samples. In addition, to
our knowledge, this is the first study to report an
association between increased MYC mRNA expression and
the presence of lymph node metastasis and CG stage
III-IV, reinforcing the idea that MYC deregulation is a
strong factor for malignancy in GC.
Age (y) (mean ± SD)
>50 (65.3 ± 9.1)
≤50 (42.1 ± 8.2)
Depth of tumor invasion
Lymph node metastasis
Table 2 MYC, FBXW7 and TP53 mRNA expression levels and clinicopathological factors of 33 gastric cancer patients
*p < 0.05; IQR: interquartile range.
Adams et al.  and Leder et al.  demonstrated
that MYC mRNA expression deregulation can promote
the development of cancer in transgenic mouse models.
The increase in MYC mRNA level in human cancers
may result from both direct and indirect mechanisms,
which could have several explanations. First, MYC
amplification is the most common mechanism of MYC
deregulation in GC . This mechanism leads to
increased production of oncogenic products in
quantities that exceed the transcriptional capacity of a normal
double copy gene. Here, we observed three or more
MYC gene copies in 51.5% of gastric tumors specimens.
Previous studies from our group also showed that MYC
amplification or trisomy of chromosome 8, on which
MYC is located, was present in all GC samples examined
from individuals in Northern Brazil, as well as in GC cell
lines established by our group from tumors of Brazilian
patients [18,22-27]. The presence of MYC amplification
has also been reported in plasma samples from
individuals with GC . However, no direct association
between MYC copy number variation and mRNA
expression was detected in the present study.
Second, the increase in MYC mRNA expression may
result from consistent recombination between the
immunoglobulin (Ig) locus and the MYC oncogene. This
phenomenon is frequently described in Burkitt’s
lymphoma and is associated with a longer half-life of MYC
mRNA in affected cells . Previously, our research
group observed MYC insertions in diffuse-type GC
mainly into chromosomes that are mapped to genes of
immunoglobulins (chromosomes 2, 14, and 22) .
Thus, chromosomal translocations involving the MYC
locus (8q24) in diffuse-type CG in individuals from
Northern Brazil might also reflect an increase in MYC
Immunohistochemistry (IHC) analysis revealed that
MYC expression is more frequently found in
intestinaltype GC than diffuse-type GC specimens. These
alterations could lead to an abnormal MYC protein that is
not recognized by either of the antibodies used in the
Figure 1 Immunohistochemical analysis of MYC and p53 protein expression in GC. (A) Negative MYC immunostaining in diffuse-type GC;
(B) MYC immune positivity in intestinal-type GC; (C) Positive p53 immunostaining in diffuse-type GC; (D) p53 immune positivity in intestinal-type
GC (magnification × 40).
Figure 2 MYC, FBXW7 and p53 expression, migration and invasion ability in ACP02 and ACP03. (A) Graph show mean ± SD of MYC,
FBXW7 and p53 protein expression in ACP02 and ACP03. These proteins were normalized to the level of beta actin; (B) Representative data of
MYC, FBXW7 and p53 protein expression; (C-D) Graphs show mean ± SD of migration and invasive cells triplicates assay; (E) Representative
results of MYC, FBXW7 and p53 immunofluorescence.
Figure 3 Representative gelatin zymography analysis of MMP-2 and MMP-9 activity in ACP02 and ACP03. (A) Bands corresponding to
both latent and active forms of MMP-2 and MMP-9 were observed in ACP02 and ACP03. Densitometric analyses are of the bands corresponding
to latent and active forms of MMP-2 (B) and MMP-9 (C).
present study. Moreover, we observed an association
between MYC mRNA expression level and MYC staining.
Furthermore, posttranscriptional mechanisms control
MYC stability [6,30]. MYC deregulation has been
associated with loss of FBXW7, a haploinsufficient tumor
suppressor gene. In general, FBXW7 loss may be caused
by loss of heterozygosity (LOH) and mutation . The
loss at 4q, the FBXW7 locus, is a recurring chromosomal
alterations in GC [31,32], and FBXW7 mutations have
been found in 3.7-6% of gastric tumors .
In the present study, we observed only one copy of the
FBXW7 gene in 45.16% of the gastric tumors studied.
Interestingly, FBXW7 mRNA expression in GC samples
is markedly decreased in comparison with corresponding
non-neoplastic tissue. In addition, FBXW7 mRNA
expression deregulation was associated with the
presence of lymph node metastasis and GC stage III-IV, as
was also observed with MYC mRNA. These findings
corroborate the work of Yokobori el al. , which also
showed an association between reduced FBXW7 mRNA
expression and lymph node metastasis that contributes
to the malignant potential of GC cells and results in
poor prognosis. Moreover, we observed that the
expression of MYC and FBXW7 mRNA tended to be inversely
correlated in the present study.
Several studies showed that MYC inactivation
suppresses tumors in animals, suggesting that MYC may be
a molecular target in cancer treatment [33-35].
Alternatively, Soucek et al.  proposed that FBXW7 might
facilitate “tumor dormancy therapy”. Thus, MYC
degradation by FBXW7 may not only induce a state of tumor
dormancy but could also have an anti-tumor effect.
Normally, MYC accumulation resulting from FBXW7
loss or another mechanism of MYC deregulation induces
p53-dependent apoptosis via MDM2 degradation. The
inactivation of both FBXW7 and p53 promotes MYC
accumulation and inhibits p53-dependent apoptosis via
MDM2 activation, which may in turn induce cell
In this study, we found that 21.2% of the gastric
tumors examined had one copy of the TP53 gene and
also found a substantial decrease in TP53 mRNA level
in GC tissues compared with paired non-neoplastic
gastric tissue samples. Loss of p53 function could be caused
primarily by LOH and mutations. TP53 mutations in
somatic cells are observed in about 50% of human
cancers, but the frequency and type of mutation varies
from one tumor to another and can be exchange of
sense, nonsense, deletion, insertion, or splicing
mutations [39,40]. In CG, the rate of mutations in this gene is
18-58% [41-43]. Some studies have shown that most
missense mutations in TP53 cause changes in the
conformation of the protein, thereby prolonging its
halflife and leading to accumulation in the nucleus of
neoplastic cells. This accumulation can be detected by
IHC in about 19-29% of GC tumors . Here, we
observed p53 immunostaining in 19.4% (6/31) of GC
samples. This finding was consistent with earlier studies
by our group that described LOH of TP53 and deletion
of 17p as frequent alterations in GC cell lines and
primary gastric tumors from individuals in Northern
Brazil [27,45]. The LOH may be related to the reduction
of TP53 mRNA expression observed in some of our GC
samples. However, no association was found between
this protein, TP53 mRNA level, copy number, or
clinicopathological features. The lack of association between
MYC, FBXW7, and TP53 copy number variation and
mRNA and protein expression observed in this study
highlights the complex relationship between gene copy
number, mRNA expression, and protein stability.
In our previous cytogenetic study using fluorescence
in situ hybridization (FISH), we described gains in MYC
copies and deletions in TP53 in ACP02 and ACP03
gastric adenocarcinoma cell lines, thus corroborating the
present results obtained using real-time qPCR . Both
alterations were observed in the primary tumors from
which these cell lines were established. Since ACP02 and
ACP03 cells present alterations similar to those of
gastric tumors, these cell lines may be useful as tools for
experimental modeling of gastric carcinogenesis and
may enhance understanding of the genetic basis
underlying GC behavior and treatment and perhaps may
change the landscape of GC.
In the present study, we also observed increased MYC
and reduced FBXW7 mRNA and protein expression in
ACP02 cells compared with ACP03 cells. Furthermore,
ACP02 cells were more invasive than ACP03 cells. On
the other hand, ACP03 cells had a higher migration
capability than ACP02 cells. Thus, despite the ability to
migrate, ACP03 cells probably do not have efficient
invasive machinery such as active proteases necessary to
degrade the substrate. These findings are in agreement
with observations in gastric tumors and reinforce the
hypothesis that deregulation of MYC and FBXW7 is
crucial for the invasive ability of GC cells. This result
encouraged us to investigate the MMP-2 and MMP-9
activities of cells using zymography. The MMPs are
synthesized as latent enzymes and later activated via
proteolytic cleavage by themselves or other proteins in
the intracellular space. Both proteases are synthesized
predominantly by stromal cells rather than cancer cells
and both contribute to cancer progression . Our
zymography analysis revealed no significant differences
in the activity of MMP2 between ACP02 and ACP03
cells. Additionally, MMP-9 was more active in ACP02
than ACP03 cells. Studies have shown that high levels of
MMP-2 and/or MMP-9 are significantly correlated with
GC invasion and are associated with poor prognosis
[47,48]. Sampieri et al.  showed that MMP-9
expression is enhanced in GC mucosa compared to
nonneoplastic mucosa and that gelatinase activity differs
significantly between cancerous and normal tissue.
In conclusion, our findings show that FBXW7 and MYC
mRNA levels reflect the potential for aggressive biologic
behavior of gastric tumors and may be used as indicators
of poor prognosis in GC patients. Furthermore, MYC
can be a potential biomarker for use in development of
new targets for GC therapy.
The authors declare that they have no competing interests.
DQC, VMF, ASK, MACS, AKRS and RRB participated in conception and design
of study. DQC, VMF, SD participated in acquisition and performed the
analysis of data. DQC, VMF, MFL, SD, CRTS performed interpretation of data.
DQC, VMF, RM, PPA and RRB involved in drafting the manuscript. All authors
read and approved the final manuscript.
This study was supported by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) as grants and fellowship awards. RRB, MACS,
RM, ASK and AKRS have research grants from CNPq. DQC, MFL and CRTS has
a fellowship granted by CNPq and Fundação de Amparo a Pesquisa do
Estado de São Paulo (FAPESP).
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