A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway
Liu et al. Journal of Hematology & Oncology
A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway
Jing Liu 0 1
Changqie Pan 0 1
Lihong Guo 0 1
Mengwan Wu 0 1
Jing Guo 0 1
Sheng Peng 1 2
Qianying Wu 0 1
Qiang Zuo 0 1
0 Department of Oncology, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong Province , China
1 Abbreviations: MACC1, Metastasis associated with the colon cancer 1; GC, Gastric cancer; PI3K/AKT, Phosphatidy linositol 3-kinase/Protein kinase B; LDHA , Lactate dehydrogenase A; HK2, Hexokinase 2; Ttzm, Trastuzumab; OX, Oxamate; 2-DG, 2-Deoxy-
2 Department of ICU, Zhujiang Hospital, Southern Medical University , Guangzhou, Guangdong Province , China
Background: Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive gastric cancer. However, recurrent therapeutic resistance presents revolutionary claims. Warburg effect and AKT signaling pathway was involved in the resistance to trastuzumab. Our previous studies have demonstrated that overexpression of metastasis associated with the colon cancer 1 (MACC1) predicted poor prognosis of GC and promoted tumor cells proliferation and invasion. In this study, we found that MACC1 was significantly upregulated in trastuzumab-resistant cell lines. Besides, downregulation of MACC1 reversed this resistance. Methods: The effect of trastuzumab and glycolysis inhibitor combination on cell viability, apoptosis, and cell metabolism was investigated in vitro using established trastuzumab-resistant GC cell lines. We assessed the impact of trastuzumab combined with oxamate on tumor growth and metabolism in an established xenograft model of HER2-positive GC cell lines. Results: Here, we found that MACC1 was significantly upregulated in trastuzumab-resistant cell lines. Besides, downregulation of MACC1 in trastuzumab-resistant cells reversed this resistance. Overexpression of MACC1-induced trastuzumab resistance, enhanced the Warburg effect, and activated the PI3K/AKT signaling pathway, while downregulation of MACC1 presented the opposite effects. Moreover, when the PI3K/AKT signaling pathway was inhibited, the effects of MACC1 on resistance and glycolysis were diminished. Our findings indicated that MACC1 promoted the Warburg effect mainly through the PI3K/AKT signaling pathway, which further enhanced GC cells trastuzumab resistance. Conclusions: Our results indicate that co-targeting of HER2 and the Warburg effect reversed trastuzumab resistance in vitro and in vivo, suggesting that the combination might overcome trastuzumab resistance in MACC1-over expressed, HER2-positive GC patients.
Metastasis-associated in colon cancer-1; Trastuzumab; Resistance; Warburg effect; PI3K/AKT signaling pathway; Gastric cancer
Gastric cancer is the fifth common cancer and the third
causes of tumor deaths worldwide [
]. Human epidermal
growth factor receptor 2 (HER2) is a member of the
receptor family associated with tumor cell proliferation,
adhesion, migration, and differentiation. Trastuzumab, a
humanized monoclonal antibody that targets HER2,
inhibits the HER2-mediated signaling pathway and
induces antibody-dependent cellular cytotoxicity [
HER2-positive advanced gastric cancer (GC) patients,
combining chemotherapy with trastuzumab is
significantly superior to chemotherapy alone with regard to
efficacy and safety [
]. Although the response rates to
this combination are far higher than those of
chemotherapy alone, the effects are usually transitory, suggesting a
high incidence of resistance [
]. Thus, more effective
predictors of trastuzumab response in HER2-positive
cancer, except for HER2, are required for personalized
Cancer cells prefer anaerobic breakdown of glucose for
energy rather than mitochondrial oxidative
phosphorylation, this phenomenon termed “Warburg effect” [
Warburg effect, which is the most common metabolic
phenotype in cancer cells, has been closely correlated with
drug resistance in cancer cells [
]. Since now, many
glycolysis inhibitors have been evaluated to overcome the
anticancer therapy resistance, such as MTCI inhibitors
], PDK inhibitors [
], lactate dehydrogenase A
(LDHA) inhibitors, and HK inhibitors [
glycolysis inhibitors are currently under pre-clinical and
clinical researches. 2-Deoxy-D-glucose (2-DG), is a glucose
analog that inhibits glycolysis via its actions on
hexokinase, presented a tolerable adverse effects in combination
usage with docetaxel [
]. Oxamate, a specific inhibitor of
the lactate dehydrogenase, is the most promising target to
develop glycolysis inhibitors with selective activity on
cancer cells because of its unnecessary for normal tissue
]. Therefore, appropriate combining glycolysis
inhibitor with anticancer reagents might be a key for
overcoming the drug resistance.
One of the major mechanisms underlying trastuzumab
resistance in breast cancer is the dysregulation of HER2
downstream signaling substrate, including the
phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT)
]. We recently found that activation of the
PI3K/AKT signaling pathway can leading to resistance of
HER2-positive GC cells to trastuzumab . As AKT
activation stimulates aerobic glycolysis in both solid tumors
and cancers of hematopoietic directly [
], it is reasonable
to hypothesize that activation of AKT might induce
enhancement of Warburg effect and resulted in
trastuzumab resistance in GC cells.
Metastasis associated in the colon cancer 1 (MACC1)
gene which was identified by Stein et al. is demonstrated
to be upregulated in several types of cancer and served
as a biomarker for cancer invasion and metastasis [
Previously, we found that MACC1 contributed to poor
prognosis of GC by promoting tumor cells proliferation
and invasion as well as epithelial-to-mesenchymal
]. Moreover, we discovered that MACC1 was
upregulated by metabolic stress in GC via adenosine
monophosphate-activated protein kinase signaling, which
increased the resistance to metabolic stress by promoting
the Warburg effect and consequently facilitated tumor
]. MACC1 is a regulator of MET/AKT
signaling pathway which has also been approved by our
previous work [
Here, we first found that MACC1 was significantly
upregulated in trastuzumab-resistant NCI-N87/TR and
MKN45/TR cell lines. MACC1 promoted the Warburg
effect mainly via the PI3K/AKT signaling pathway, which
further enhanced the resistance to trastuzumab. To
clarify this mechanism, we herein investigated the
relationship between MACC1 expression, the Warburg
effect, and trastuzumab resistance in HER2-positive GC
cells. Taken together, these findings provide evidence for
unraveling the mechanism of trastuzumab resistance
and improving the efficacy of treatment.
Cell lines and culture conditions
Human gastric cancer cells including SGC7901, MKN45,
and NCI-N87 were obtained from the American Type
Culture Collection (ATCC). BGC-823 and MKN-28 were
obtained from Foleibao Biotechnology Development
(Shanghai, China). Cells were cultured in complete
medium (Roswell Park Memorial Institute 1640 medium
(Invitrogen, Life Technologies, Carlsbad, CA) with 10 %
fetal bovine serum (Thermo Scientific HyClone, South
Logan, UT)) and incubated under 5 % CO2 at 37 °C.
Cells were collected in logarithmic growth phase for all
experiments as described in the following sections.
Antibodies and chemicals
MACC1-expressing and HER2-positive cells were selected
using Western blotting analyses. Antibodies against the
following proteins were used in this study: MACC1 (Abnova,
Taipei, China), GLUT1 (Epitomics, Burlingame, CA, USA),
hexokinase 2 (HK2), GAPDH and phosphorylation-AKT
(Ser473) (Cell Signaling Technology, Danvers, MA, USA)
and HER2, LDHA, and AKT (Abcam, USA). IHC staining
was done with the Dako Envision System (Dako, Glostrup,
Denmark). MitoTracker Red CMXRos (Invitrogen,
Carlsbad, CA, USA) or 4,6-diamidino-2-phenylindole was
stained when needed to label themitochondria or nucleus.
Trastuzumab was supported by Roche company. AKT
inhibitor MK2206 and PI3K inhibitor LY294002 were
obtained from Calbiochem (Selleck Chemicals, USA), and
the Warburg effect inhibitor sodium oxamate was obtained
from Sigma-Aldrich (Shanghai Trading Co., Ltd., China).
2-Deoxy-D-glucose (2DG) was obtained from
Biotechnology (Santa Cruz Biotechnology, Inc., California).
The cDNA encoding myristoylated-human AKT lacking
the PH domain (Myr-AKT) was cloned into the
pCAGGS-IRESEGFPpA vector to produce the active AKT
Induction of trastuzumab-resistant NCI-N87/TR and
The establishment of NCI-N87/TR has been previously
described in our last study [
]. Aliquots of MKN45
cells in the exponential growth phase were seeded into
25 cm2 culture bottles. Trastuzumab was added for 48 h
during the mitotic phase, and then, the cells were
transferred into drug-free culture medium until the next
mitotic phase, after which trastuzumab was added for the
next 48 h at twice the previous concentration. We
continued this process while observing cell death every day,
changing to fresh complete culture medium, and
performing the MTT assay regularly. This process was continued
until the concentration of trastuzumab in the medium
reached 2560 μg/ml after 150 days. Thus, MKN45 cells
were obtained, which were grown stably in trastuzumab
(2560 μg/ml)-containing medium, and these
trastuzumabresistant cells were named MKN45/TR cells.
Establishment of stably transfected cell lines
For MACC1 overexpression, the ectopic MACC1 coding
sequence was amplified by polymerase chain reaction
(PCR) (primer sequences in Additional file 1: Figure S1b)
and cloned into the pLVX-MCMV-ZsGreen-PGK-Puro
plasmid. For MACC1 silencing, sequences of short hairpin
RNA targeting MACC1 (shMACC1) and scramble were
cloned into the pLVX plasmid sequences (Additional file 1:
Figure S1b). Cell lines were transfected with these
constructed plasmids combined with the blank vector.
Stably transfected cell lines were selected with 0.5 mg/mL
(a minimum lethal dose) puromycin at 48 h after infection.
By this selection criterion, MACC1 expression was
markedly increased in the MACC1 overexpression group
and strongly inhibited in the MACC1 silencing group in
the transfected GC cells.
Cells were transfected with various plasmids using
Lipofectamine (Invitrogen) according to the
manufacturer’s instructions, seeded onto 6-well plates (3 × 105
cells per well) and incubated in 10 % FBS-containing
medium for 24 h prior to drug treatment.
Cell-based assay for glucose uptake and lactate assay
The levels of glucose uptake were measured using an
Amplex® Red Glucose/Glucose Oxidase Assay Kit
(Invitrogen). Cells were seeded in 96-well plates at a density of 5 ×
103 cells/well. After 24 h, glucose uptake assays were
performed according to the manufacturer’s protocol. Relative
fluorescence units were determined at 485–535 nm using a
VARIOSKAN FLASH Multimode Reader (Thermo). The
levels of lactate production were examined using a Lactate
Colorimetric/Fluorometric Assay Kit (Biovision, Milpitas,
CA, USA). Cells were plated in 96-well plates at a density
of 5 × 103 cells/well. After incubation for 24 h, the culture
medium was replaced with FBS-free DMEM. After an
additional 8 h, lactate assays were performed with culture
media collected from each sample according to the
manufacturer’s protocol, and the optical density was measured at
570 nm using Multiskan EX (Thermo).
Western blotting analysis
Harvested cells were lysed with lysis buffer containing
50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 % NP-40,
0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM NaF,
1 mM Na3VO4, and protease inhibitor (Roche,
Indianapolis, IN, USA). Protein concentrations in the cell
lysates were quantified using the BCA protein assay kit
(Thermo Scientific). Proteins were separated on
SDSPAGE gels and transferred onto nitrocellulose
membrane (Whatman, Maidstone, UK). After being blocked
with 5 % skim milk in TBS containing 0.05 % Tween-20,
the membranes were incubated in 5 % skim milk
containing the appropriate primary antibodies overnight,
followed by incubation with horseradish
peroxidaseconjugated secondary antibodies for 2 h. The protein
bands were visualized using a commercial ECL kit
Cell viability assay
Cells were seeded onto 96-well plates at a density of 3 × 103
cells/well. Twenty-four hours later, the cells were treated
with drugs at the indicated concentrations and incubated
for specific time periods. Cell viability was determined
3 days after treatment using the cell proliferation kit II
(Roche Molecular Biochemicals) with the
3′-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
(Sigma, St. Louis, MO, USA) according to the
manufacturer’s protocol. Experiments were performed in triplicate.
Flow cytometric analysis of apoptosis
Cells were treated with trastuzumab for 48 h, collected,
washed with phosphate-buffered saline (PBS) containing
0.1 % bovine serum albumin, and resuspended in 500 μl
binding buffer. Next, the cells were incubated with 5 μl
of Annexin V-FITC and 10 μl of PI solution for 15 min
at room temperature in the dark. Subsequently, the
samples were evaluated for apoptosis using a flow
cytometer (FACS Calibur; BD Biosciences, Franklin Lakes,
NJ, USA), Annexin V-FITC Apoptosis Detection Kit was
bought from Biovision USA.
Nude mice cancer xenograft model
All experimental procedures involving animals were
performed according to the Guide for the Care and Use of
Laboratory Animals (NIH publication no. 80-23, revised
in 1996) and were performed in compliance with the
institutional ethical guidelines for animal experimentation.
GC cells were pre-treated with different plasmids or NC.
The cells were suspended in 100 μl of PBS at a
concentration of 5 × 107 cells/ml and injected into either flank
of the same BALB/C female athymic nude mouse at 5–
6 weeks of age (six mice for each group, n = 6). The
tumor size was monitored by measuring the length (L)
and width (W) with calipers, and the volumes were
calculated using the formula: (L × W2) × 0.5.
Histology and immunohistochemistry
Mice were sacrificed with CO2, and half-dissected
tumors were snap-frozen in liquid nitrogen to prepare
protein lysates or were fixed in 10 % neutral-buffered
formalin overnight at room temperature, transferred to
70 % ethanol, embedded in paraffin, and sectioned at
5 μm for IHC staining. Hematoxylin and eosin staining
was performed in the Department of Pathology at
Nanfang Hospital. IHC staining for MACC1 was
performed using previously described methods [
After the animals were sacrificed, the formed tumors
were harvested, fixed with formalin, and embedded in
paraffin wax. Tissue was cut into 4-μm sections on
clean, charged microscope slides, and then heated in a
tissue-drying oven for 45 min at 60 °C. After
deparaffinization, antigen retrieval was performed. The slides were
incubated in 0.01 M sodium citrate buffer, pH 6.0 at
100 °C for 20 min, removed from the heat, cooled in
buffer at room temperature for 20 min, and rinsed in 1×
TBS with Tween at room temperature for 1 min. After
being blocked in 5 % BSA, the section was incubated
with diluted primary antibody at room temperature for
45 min, washed with 1× TBS with Tween three times,
incubated with specific biotinylated secondary antibody
at room temperature for 30 min, and finally color
developed by the addition of substrates.
The xenograft-bearing mice were fasted overnight and
anesthetized with inhaled isoflurane. 18F-FDG of about
200 μCi per mouse was injected into the tail vein. After
60 min of nonspecific clearance, the mouse was scanned
in microPET/CT Inveon scanner (Siemens, Knoxville,
TN, USA) and images were then reconstructed using a
two-dimensional ordered subsets expectation maximization
algorithm. PET and CT image fusion and image analysis
were performed using software ASIPro 22.214.171.124 (Siemens).
Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assays were performed on sections
using an DeadEndTM Colorimetric TUNEL assay kit
(Promega, Madison, USA) principally according to the
Using the CalcuSyn Version 2.11 (Copyright Biosoft, USA)
software, the combination index (CI) was calculated for
cells receiving combination therapy according to the Chou
and Talalay mathematical model for drug interactions. The
resulting CI theorem of Chou-Talalay offers quantitative
definition for an additive effect (CI = 1), synergism (CI < 1),
and antagonism (CI > 1) in drug combinations [
All data were represented as the mean of at least
triplicate samples ± standard deviation. Statistical analysis
included one-way ANOVA or Student’s t test using SPSS
20.0. P values less than 0.05 were considered statistically
MACC1 contributed to the resistance of HER2-positive GC cells in response to trastuzumab
In a previous study, we employed human gastric
carcinoma cell line NCI-N87 with high HER2 expressions to
generate trastuzumab-resistant NCI-N87/TR cell lines
via stepwise exposure to increasing doses of trastuzumab
]. Unexpectedly, compared with parental cells, the
expression of MACC1 was significantly increased in
trastuzumab-resistant cells (Fig. 1a).
To evaluated the effects of MACC1 on resistance to
trastuzumab in GC cells, first, we tested the expression of
HER2 and MACC1 protein levels (Additional file 1: Figure
S1a) and the sensitivity to trastuzumab of MKN28,
BGC823, SGC7901, MKN45, and NCI-N87 cell lines. Here,
MKN45 cell line was chosen as the relatively sensitive cells
to trastuzumab (Additional file 2: Table S1). We also
established MKN45 trastuzumab-resistant cell line by stepwise
exposure to increasing concentrations of trastuzumab
(Additional file 2: Table S2) and found that MACC1 was
also upregulated in MKN45/TR cells (Fig. 1a).
Next, to determine whether MACC1 was a regulatory
factor in resistance to trastuzumab in HER2-positive GC
cells, MACC1 was downregulated by small interfering
RNA (siRNA) in NCI-N87/TR and MKN45/TR cell lines
(Fig. 1b). Cell viability of the MACC1-downregulated
cell lines was much more inhibited by trastuzumab than
the resistant cells. Thus, targeting MACC1 reversed the
trastuzumab resistance observed in HER2-positive GC
cells (Fig. 1c).
To further identify the role of MACC1 in resistance to
trastuzumab in HER2-positive GC cells, colonies of
ectopicMACC1 and shMACC1 and their respective controls were
used to transfect NCI-N87 and MKN45 cells. Next, MACC1
overexpressing and downregulation NCI-N87 and MKN45
cells were treated with trastuzumab. Overexpression of
MACC1 significantly increased the cell viability. Conversely,
downregulation of MACC1 significantly inhibited the
sensitivity of cells to trastuzumab (Fig. 1d). Collectively, these
data indicated that MACC1 contributed to the resistance of
HER2-positive GC cells to trastuzumab.
MACC1 enhanced the Warburg effect in GC cells
As our previously reported, MACC1 upregulation
increased the resistance to metabolic stress by promoting the
Warburg effect [
]. Since Warburg effect was closely
correlated with trastuzumab resistance [
hypothesized that MACC1 may regulate resistance via Warburg
effect. The levels of glucose uptake and lactate production
were measured between the MACC1 upregulated and
downregulated cells. The glucose uptake (Fig. 2a) and
lactate production (Fig. 2b), which are hallmarks of
glycolysis, obviously increased in MACC1-upregulated cells,
while, decreased notably in MACC1-downregulated cells.
In addition, the expression of HK2 and LDHA, which are
rate-limited enzymes in the Warburg effect [
in MACC1-downregulated cells, whereas, increased in
MACC1-upregulated cells (Fig. 2c, d). When MACC1 was
silenced in NCI-N87/TR and MKN45/TR cells, the
expression of HK2 and LDHA proteins were also downregulated
(Fig. 2e). On the basis of our published paper [
] and these
results, MACC1 enhances Warburg effect in GC cells.
Synergistic cell growth and glycometabolism inhibition via the combination of trastuzumab and glycolysis inhibitor
Given that trastuzumab has been known to inhibited cell
glycolysis in breast cancer cells [
], we test if it has the
same effect in HER2-positive GC cells. NCI-N87 and
MKN45 cells were treated with trastuzumab, HK2 and
LDHA expression were downregulated after treatment
with trastuzumab (Fig. 3a). Meanwhile, the glucose
uptake and lactate production were also declined
(Fig. 3c), indicating that trastuzumab effectively inhibits
Warburg effect in GC cells.
Moreover, we found that when the GC cells became
resistant to trastuzumab, the expression of HK2 and LDHA
were highlighted (Fig. 3b). These results showed that the
Warburg effect play an important role in trastuzumab
resistance in GC cells, and it is logical to hypothesize that
the combination of trastuzumab with glycolysis
inhibitors may have more powerful antitumor effect
than either agent alone [
To testify this hypothesis, we use the inhibitor of
glycometabolism, 2-DG, or oxamate, combined with
trastuzumab to treat both parental and trastuzumab-resistant GC
cells. The combination of trastuzumab and 2-DG/oxamate
showed a strongly increased inhibitory efficacy in both cell
viabilities and glycometabolism of MKN45 and NCI-N87
parental cells and MKN45/TR and NCI-N87/TR cells
compared to use the agents individually (Fig. 3d, Additional
file 1: Figure S2). To evaluate whether the combination
effect is synergistic or not, the date generated from
NCIN87, MKN45, NCI-N87/TR, and MKN45/TR cells treated
with multiple concentrations of trastuzumab and 2-DG/
oxamate were analyzed by the CalcuSyn software [
combined index (CI) < 1.0 obtained in almost all combined
treatment groups. Moreover, the synergistic effect in
trastuzumab-resistant cells was more robust than in
parental cells (Fig. 3e, Additional file 1: Figure S3, Additional file
2: Tables S3 and S4). With these data, we confirmed that
the combination of trastuzumab with glycolysis inhibitors
better suppressed cell viability and glycometabolism in
HER2-positive GC cells, and this specific combination
could overcome trastuzumab resistance.
MACC1 promoted the Warburg effect via the PI3K/AKT signaling pathway and induced trastuzumab resistance in vitro
Trastuzumab could inhibit metabolism-regulating
molecules such as PI3K and mTOR. Here, we found that it
can also inhibit the AKT phosphorylation in NCI-N87
and MKN45 cell lines (Fig. 4a). Together with the results
from prior studies (Figs. 1e and 2c), the AKT signaling
pathway was activated and the Warburg effect was
enhanced upon MACC1 upregulation in GC cells,
meanwhile trastuzumab resistance was enhanced. Whereas,
resistance to trastuzumab was attenuated in
MACC1downregulated groups, simultaneously, the
phosphorylation of AKT decreased and the Warburg effect was
suppressed (Figs. 1e and 2d). Thus, there are
multirelationships between MACC1, the PI3K/AKT signaling
pathway, the Warburg effect, and trastuzumab resistance
in HER2-positive GC cells. Based on what we found, it
suggests that there is a regulatory axis associated with
trastuzumab resistance in HER2-positive GC cells:
MACC1 enhanced the Warburg effect via the PI3K/
AKT signaling pathway. To investigate this axis, we
treated MACC1-upregulated NCI-N87 and MKN45 cell
lines with MK2206, an AKT1/2/3 inhibitor. The results
showed that MK2206 suppressed the expression of HK2
and LDHA (Fig. 4c) and decreased glucose uptake and
lactate production (Fig. 4d), which were increased due to
MACC1 upregulation. Moreover, after inhibiting the
activity of PI3K/AKT signaling pathway by MK2206,
the enhanced drug resistance was reversed (Fig. 4e).
To further confirm the regulatory pathway
“MACC1PI3K/AKT-Warburg-trastuzumab resistance,” the AKT
constitutively activator Myr-AKT [
] and MACC1
siRNA were applied to co-transfect NCI-N87 and MKN45
cell lines. The inhibited PI3K/AKT pathway was rescued
as phosphorylation of AKT was enhanced (Fig. 4f). The
Warburg effect was suppressed when MACC1 expression
was disrupted by siRNA, but this effect was reversed when
Myr-AKT was added, as evidenced by the increased
expression of HK2 and LDHA (Fig. 4f ). The decreased
glucose uptake and lactate production caused by MACC1
downregulation were also reversed when the PI3K/AKT
signaling pathway was constitutively activated (Fig. 4g).
The enhanced cell inhibitory effect in response to
trastuzumab caused by the downregulation of MACC1 was
diminished when the PI3K/AKT signaling pathway was
activated again (Fig. 4h). These consistent data
demonstrate that inhibition of PI3K/AKT reverses
MACC1induced Warburg effect enhancement and trastuzumab
resistance in GC cells. Accordingly, MACC1 enhanced
trastuzumab resistance via regulation of the Warburg
effect, in which the PI3K/AKT signaling pathway was
mainly responsible for it.
MACC1 induced trastuzumab resistance and enhanced the Warburg effect in vivo
To confirm the trastuzumab resistance promoter role of
MACC1 in vivo, we established a BALB/c nude mouse
xenograft model using NCI-N87 MACC1-overexpressing/
silenced and their control cells. The tumor volume
was measured twice a week until they reached the
average volume (120 mm3). Subsequently, MACC1
expression was confirmed in xenografts using IHC
staining (Fig. 5a).
Our previous results showed that MACC1 enhanced
trastuzumab resistance in vitro. Here, to testify these
results in vivo, we treated tumor xenografts with PBS as
control or trastuzumab after the tumor had formed.
Xenografts with MACC1 overexpression were more
resistant to trastuzumab treatment than the vector group,
whereas, were more sensitive in MACC1-downregulated
xenografts than the scramble group (Fig. 5b). Compared
to PBS, trastuzumab could inhibit the tumor growth more
effectively when MACC1 was downregulated (Fig. 5c).
Animal PET scanning demonstrated that 18F-FDG
accumulation was markedly enhanced by MACC1
overexpression. Compared with PBS groups, 18F-FDG accumulation
was inhibited in trastuzumab-treated groups more
obviously in MACC1-silenced group rather than in
scramble groups, but the effect of 18F-FDG accumulation
mediated by trastuzumab did not show up when MACC1
overexpressing (Fig. 5d, Additional file 1: Figure S4).
Taken together, these results suggested that MACC1
could induce trastuzumab resistance and enhance the
Warburg effect in vivo.
We also investigated the apoptosis of tumor cells in
xenografts, which showed that trastuzumab induced cell
apoptosis when MACC1 was downregulated (Fig. 5e).
Combination of trastuzumab and oxamate effectively inhibited cell growth and the Warburg effect in MACC1overexpressing xenografts
Based on our findings that trastuzumab combined with a
glycolysis inhibitor synergistically inhibited the growth of
both trastuzumab-sensitive and trastuzumab-resistant
cells in vitro (Fig. 3d, e). To further confirm these
combined effect in vivo, we treated tumor xenografts with
trastuzumab (10 mg/kg, i.p., 2 times/wk × 3 weeks),
oxamate (750 mg/kg, i.p., daily for 21 days), or a combination
of the agents after tumor formation. The combination of
trastuzumab with oxamate more efficiently inhibited the
growth of tumors than the activity of any individual agent.
The synergistic inhibitory effect was stronger in the
MACC1-overexpressing group than the MACC1
downregulated and their control groups (Fig. 6a), which
indicated that MACC1 might be a prognosis factor
for the combined use of trastuzumab and glycolysis
inhibitors in HER2-positive GC. Tumor cell apoptosis
was also confirmed, which also followed the same
trend (Fig. 6b).
Furthermore, animal PET scanning demonstrated that
18F-FDG accumulation was markedly inhibited by the
combination of trastuzumab and oxamate compared to
the outcomes of single drug groups in
MACC1overexpressing groups rather than the other ones (Fig. 6c,
Additional file 1: Figure S5). The data of xenografts
suggesting that the combination of trastuzumab with
oxamate effectively inhibited tumor growth and the
Warburg effect in vivo and MACC1 may demonstrate
the synergistic inhibitory effect in HER2-positive GC.
These results further support the finding that
cotargeting of HER2 and the Warburg effect in
MACC1overexpressing HER2-positive GC cells contributed to
overcoming trastuzumab resistance.
Trastuzumab in combination with chemotherapy has
become the first-line treatment for advanced GC with
HER2 overexpression [
]. Although the response
rates to this combination are much higher than those of
chemotherapy alone, the effects are usually transitory,
suggesting a high incidence of acquired resistance [
The supposed mechanisms of trastuzumab resistance
presented so far include abrogation of productive
drug-target contact through overexpression of MUC4
glycoprotein ; upregulation of target-like tyrosine
kinase receptors or their ligands [
of target-downstream components in the PI3K/AKT
signaling pathway such as PI3KCA  and PTEN
]; cell reprogramming by deregulation of Bcl-2
], cycE [
], Mcl-1, and survivin [
]; and EMT
]. Owing to the heterogeneous nature of
tumors, different resistance mechanisms may coexist
in the same patient; thus, targeting single mechanism
is usually ineffective. To develop more powerful
regimens to overcome drug resistance, signaling nodes
which involved in multiple resistance mechanisms
need to be identified.
As a pivotal oncogene for tumor progression
influencing the HGF/c-MET pathway, MACC1, has been
shown to participate in many biological mechanisms that
produce poor clinical outcomes [
]. However, few
studies have so far been explored the upstream
mechanisms of MACC1, despite miR-338-3p, ORAI calcium
release-activated calcium modulator 1 (Orai1), and
stromal interacting molecule 1 (STIM1) being revealed as
able to modulate MACC1 in GC [
]. Previously, we
found that MACC1 contributed to the poor prognosis of
GC  and increased the resistance to metabolic stress
by promoting the Warburg effect that consequently
facilitated tumor progression [
], which elucidated its
key role in signaling networks associated with GC.
Besides, the Warburg effect was closely correlated to
trastuzumab resistance and facilitated tumor progression
]. In preliminary experiment, we unexpectedly
found that the expression of MACC1 protein was
significantly increased in trastuzumab-resistant cells. On the
basis of these findings, we hypothesized that MACC1
participated in trastuzumab resistance through regulating the
Warburg effect. As the gene encoding the hepatocyte
growth factor (HGF) receptor, MET, is a transcriptional
target of MACC1 , the downstream signaling pathway
of HGF-c-MET maybe involved in the mechanism of
the“MACC1 regulating the resistant to trastuzumab” in gastric
cancer cells. Since now, the PI3K/AKT [
], and STAT3 [
] signaling pathway
were identified as a regulatory axis in the resistance to
trastuzumab in HER2-positive cancers. Our previous
researches found that the PI3K/AKT pathway was involved
in the resistant mechanisms in HER2-positive GC cells
]. Together with MACC1-regulating cancer growth via
the activation of the HGF/c-MET/PI3K/AKT signaling
23, 46, 47
], we purposed that MACC1 might
induce trastuzumab resistance by regulating the Warburg
effect via the PI3K/AKT signaling pathway. This research
aimed to investigate the relationship between MACC1,
the Warburg effect, and the PI3K/AKT signaling pathway
in trastuzumab resistance in HER2-positive GC cells.
These three targets may provide new routes for
circumventing trastuzumab resistance and improving the
From our results, MACC1, PI3K/AKT signaling
pathway, and the Warburg effect exerted critical function in
trastuzumab-resistant GC cells. Here exits an effective
pathway in trastuzumab resistance of HER2-positive GC
cells: MACC1-PI3K/AKT-Warburg effect. Besides the
cell viability and glycometabolism, we also detected cell
apoptosis induced by trastuzumab in cells including
MACC1-overexpressed plus or not plus PI3K-inhibited
(by LY294002) cells, MACC1-silenced plus or not plus
AKT-activated (by Myr-AKT) cells. When MACC1 was
upregulated, the cell apoptosis caused by trastuzumab
was inhibited, while the effect was reversed when
LY294002 was added. When MACC1 was
downregulated, the cell apoptosis caused by trastuzumab was
enhanced, meanwhile, the effect was reversed when
Myr-AKT was added (Additional file 1: Figure S6).
The coupling of the MACC1, PI3K/AKT signaling,
and Warburg effect is an important event that
promotes survival of the resistant GC cells in the
presence of trastuzumab.
Activation of the PI3K/AKT signaling pathway is
known to mediate resistance to both molecularly
targeted therapy and chemotherapy in various cancers [
Targeting MACC1 could induce inactivate the
downstream signaling pathway such as PI3K/AKT.
Interesting, we found that blockade of AKT activation inhibited
the expression of MACC1 conversely; what is more,
activated AKT also upregulated MACC1 expression (Fig. 4c,
f ). MACC1 has been reported as the upstream regulator
of c-MET/AKT in hepatocellular cancer [
], ovarian cancer [
], and gastric cancer [
On the contrary, whether MACC1 could be regulated by
AKT-positive feedback loop has not been reported.
Based on many AKT and its regulators and positive
feedback phenomenon in tumor cells [
demonstrates a probable positive regulatory feedback loop
between MACC1 and the PI3K/AKT signaling pathway,
which needs to be testified and investigated deeply.
Warburg, in 1956, observed that many cancer cells
used glycolysis more than mitochondrial oxidative
phosphorylation for their energy requirements; this
phenomenon is called “the Warburg Effect”[
enzymes directly regulating glycolysis have also been
implicated in promoting a drug-resistant phenotype.
Targeting metabolic key enzymes can enhance
therapeutic efficacy or combat drug resistance by promoting
drug-induced apoptosis of cancer cells [
on our results, the Warburg effect may contribute to
chemoresistance or therapy failure in patients with GC,
making the disease difficult to cure using a single agent.
Targeting the Warburg effect improves the response to
cancer therapeutics, and combining targeted drugs with
cellular metabolism inhibitors may represent a
promising strategy to overcome drug resistance in cancer
]. A lot of tumor suppressors and
oncoproteins, including the PI3K/AKT/mTOR signaling
pathway, Myc, p53, and hypoxia-inducible factor-1 (HIF-1),
have been reported to be involved in the regulation of the
Warburg effect that favors tumor cell growth,
proliferation, and stress resistance [
]. In this study, we
demonstrated that trastuzumab and glycolysis inhibitors
synergistically suppressed the growth and
glycometabolism of HER2-positive GC cells in vitro and in vivo. More
importantly, this combined use effectively inhibits the
growth and Warburg effect of trastuzumab-resistant
cancer cells, suggesting a potential benefit of this regimen
in reversing trastuzumab resistance in HER2-positive GC.
LDHA is not necessary for normal tissue survival, as
witnessed by the observation that humans with a hereditary
deficiency of this LDH isoform do not show any symptom
under normal circumstances [
]. Therefore, we chose
oxamate, which specifically hinders enzymatic activity by
competing with pyruvate, combined with trastuzumab to treat
the xenografts. For the first time, we identified that
MACC1 as the predictor for synergistically inhibitory effect
of the combination of trastuzumab with oxamate in GC
cells’ growth and glycolysis. These novel findings indicated
that MACC1 promotes the Warburg effect via activation of
the PI3K/AKT signaling pathway and contributes to the
resistance of GC cells to trastuzumab. These in vitro and in
vivo results indicated that MACC1 might be a selective
factor in the use of co-targeting HER2 and glycolysis therapy
in trastuzumab-resistant HER2-positive GC. We have
provided new clues as to the mechanism by which the effect of
the Warburg effect on trastuzumab-resistant GC cells is
mediated. We also highlighted the involvement of MACCI
in the regulation of the Warburg effect.
In this study, we demonstrated that MACC1 participated
in trastuzumab resistance by promoting the Warburg
effect, which was mainly through PI3K/AKT signaling
pathway (Fig. 7). Importantly, we provide preclinical
evidence indicating that co-targeting HER2 and
glycolysis drastically benefits therapeutic outcomes for current
anticancer therapy, suggesting that this combination
may reverse trastuzumab resistance in patients with
MACC1 overexpression and HER2-positive GC.
Additional file 1: Figures S1 to S6. Figure S1: The expression of proteins
in GC cells and the sequences of ectopic MACC1 and shRNA. Figure S2:
The combination of trastuzumab and glycolysis inhibitors synergisticly
inhibit glycolysis in HER2 positive GC cells. Figure S3: The combination of
trastuzumab and glycolysis inhibitors synergisticly inhibit glycolysis in
HER2 positive GC cells. Figure S4: MACC1 enhanced the Warburg effect in
vivo. Figure S5: Combination of trastuzumab and oxamate effectively
inhibited the Warburg effect in vivo. Figure S6: The apoptosis of
indicated cells after treated with Ttzm. (ZIP 38363 kb)
Additional file 2: Tables S1 to S4. Table S1: IC50 of different GC cell
lines. Table S2: IC50 and RI (resistance index) of MKN45 cells after
treatment with trastuzumab at different inducing concentrations. Table
S3: Fa and CI for trastuzumab and glucolysis inhibitor combinations on
inhibition of cell viability. Table S4: Fa and CI for trastuzumab and glucolysis
inhibitor combinations on inhibition of glucose uptake. (DOCX 45 kb)
We thank the Pathology Department, Nanfang Hospital, Southern Medical
University for the help with immunohistochemistry data acquisition, Center
Laboratory of Southern Medical University for applying many experiment
instruments and equipment, and Dr. Huang shun from PET Center of
Nanfang Hospital for the assistance with microPET.
This work was supported by grants obtained from the Scientific Research
Foundation of Southern Medical University (PY2013N037) and new technology
and new profession of Nanfang Hospital, Southern Medical University (2013006).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
QZ and JL designed the study and drafted the manuscript. JL wrote and
reviewed the article. JG and SP participated in the manuscript preparation
and revisions. JL, CP, MW, LG, and QW carried out the experiments in vitro
and in vivo. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The experimental protocol for animal studies was reviewed and approved by
Ethics Committee of Nanfang Hospital. All experiments were conducted by
The Animal Experimental Center of Southern Medical University in
accordance with the recommendations of the Guide for Care and Use of
Laboratory Animals [
] with respect to restraint, husbandry, surgical
procedures, feed and fluid regulation, and veterinary care.
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