Detection of N-glycolyated gangliosides
in non-small-cell lung cancer using GMR8
monoclonal antibody
Nobuyoshi Hayashi,1 Hirofumi Chiba,1,7 Koji Kuronuma,1 Shinji Go,2 Yoshihiro Hasegawa,1 Motoko Takahashi,3
Shinsei Gasa,4 Atsushi Watanabe,5 Tadashi Hasegawa,6 Yoshio Kuroki,2 Jinichi Inokuchi2 and Hiroki Takahashi1
1
Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo; 2Division of Glycopathology, Institute of Molecular
Biomembranes and Glycobiology, Tohoku Pharmaceutical University, Miyagi, Sendai; Departments of 3Biochemistry, 4Chemistry, 5Second Department of
Surgery, 6Clinical Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan
(Received March 12, 2012 ⁄ Revised September 5, 2012 ⁄ Accepted September 13, 2012 ⁄ Accepted manuscript online September 24, 2012 ⁄ Article first published online October 30, 2012)
Gangliosides are glycosphingolipids found on the cell surface.
They act as recognition molecules or signal modulators and regu-
late cell proliferation and differentiation. N-glycolylneuraminic
acid (NeuGc)-containing gangliosides have been detected in some
neoplasms in humans, although they are usually absent in nor-
mal human tissues. Our aim was to evaluate the presence of
NeuGc-containing gangliosides including GM3 (NeuGc) and assess
their relationship with the prognosis of non-small-cell lung can-
cer (NSCLC). NeuGc-containing ganglioside expression in NSCLC
tissues was analyzed immunohistochemically using the mouse
monoclonal antibody GMR8, which is specific for gangliosides
with NeuGc alpha 2,3Gal-terminal structures. On the basis of
NeuGc-containing ganglioside expression, we performed survival
analysis. We also investigated the differences in the effects of
GM3 (N-acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhi-
bition of epidermal growth factor receptor (EGFR) tyrosine kinase
in A431 cells. As a result, the presence of NeuGc-containing gan-
gliosides was evident in 86 of 93 (93.5%) NSCLC samples. The
NSCLC patients with high NeuGc-containing ganglioside expres-
sion had a low overall survival rate and a significantly low pro-
gression-free survival rate. In the in vitro study, the inhibitory
effect of GM3 on EGFR tyrosine kinase in A431 cells after expo-
sure to GM3 (NeuGc) was lower than that after exposure to GM3
(NeuAc). In conclusion, NeuGc-containing gangliosides including
GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc-contain-
ing ganglioside expression is associated with patient survival.
The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc)
on the inhibition of EGFR tyrosine kinase might contribute to
improvement in the prognosis of NSCLC patients. (Cancer Sci
2013; 104: 43–47)
L ung cancer is the most frequently diagnosed major cancer
worldwide.(1) The World Health Organization classification
of lung cancer identifies squamous cell carcinoma, adenocarci-
noma, large cell carcinoma and small cell carcinoma as its
four major types. These tumors are commonly divided into
small-cell lung cancer and non-small-cell lung cancer
(NSCLC) depending on differences in their biology and treat-
ment. The low rate of cure for NSCLC can be attributed to the
high metastasis rate at diagnosis and the lack of effective treat-
ments to cure such a metastatic disease.
Gangliosides are ubiquitous membrane-associated glycos-
phingolipids containing at least one sialic acid residue. They
act as recognition molecules or signal modulators and regulate
cell proliferation and differentiation.(2,3) Delay in cell growth
of the human epidermoid carcinoma cell line A431 is caused
by GM3 (N-acetylneuraminic acid [NeuAc])-mediated inhi-
bition of epidermal growth factor receptor (EGFR) tyrosine
doi: 10.1111/cas.12027
© 2012 Japanese Cancer Association
kinase.(4) GM3 is a ganglioside that binds to the extracellular
domain of EGFR and inhibits its dimerization without inhibit-
ing ligand binding.(5) Kawashima et al. described the signifi-
cance of carbohydrate–carbohydrate interactions between
N-glycans with N-acetylglucosamine (GlcNAc) termini on
EGFR and oligosaccharides on GM3.(6)
NeuAc and N-glycolylneuraminic acid (NeuGc) are the most
common types of sialic acids found in vertebrates. The only
structural difference between them is a single oxygen atom at
the C-5 position of NeuGc, which is catalyzed by the cytidine
monophospho-N-acetylneuramic acid hydroxylase (CMAH).(7)
In general, NeuGc is abundant in mammals other than humans,
in whom CMAH is inactivated due to a mutation in the
CMAH gene.(8,9) However, the composition and production of
gangliosides is altered in many tumor types.(10,11) It is known
that anti-NeuGc-containing ganglioside antibodies recognize
tumor tissues in breast cancer, Wilms tumor, melanoma and
neuroblastoma.(11–14)
A mouse GMR8 monoclonal antibody (mAb) was generated
by immunizing mice with purified GM3 (NeuGc).(15) GMR8
mAb binds specifically to gangliosides with NeuGc alpha
2,3Gal-terminal structures, such as GM3 (NeuGc), IV3NeuGc
alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-
Gb5Cer and GD1a (NeuGc, NeuGc). Furthermore, it does not
recognize any other ganglioside with internal NeuGc alpha
2,3Gal-terminal structures, such as GM2 (NeuGc) and GM1
(NeuGc), corresponding gangliosides with NeuAc alpha
2,3Gal-terminal structures and neutral glycolipids. Thus, the
epitope structures recognized by mAb are exclusively NeuGc
alpha 2,3Gal-terminal structures.
In the present study, we evaluated the presence of NeuGc-
containing gangliosides in NSCLC by immunohistochemistry
using GMR8 mAb and performed survival analysis based on
NeuGc-containing ganglioside expression.
Materials and Methods
Patients. Ninety-three NSCLC patients who underwent pri-
mary tumor resection at Sapporo Medical University between
July 2003 and October 2006 were included in the present
study. Informed consent was obtained from all patients. The
Human Ethics Review Committees of Sapporo Medical Uni-
versity approved the study protocol (approval no. 219-3).
Immunohistochemistry. We determined N-glycolyated gangli-
oside expression in NSCLC tissues using mouse GMR8
7
To whom correspondence should be addressed.
E-mail: hchiba@sapmed.ac.jp
Cancer Sci | January 2013 | vol. 104 | no. 1 | 43–47
mAb.(13) Paraffin-embedded tumor tissues were cut into 5-lm-
thick sections and placed on glass slides. These sections were
deparaffinized and pretreated by boiling the slides in citrate buf-
fer (pH 6.8) for 10 min. The sections were then immersed in
0.3% hydrogen peroxide for 30 min to block endogenous tissue
peroxidase activity. After blocking non-specific proteins by
incubation in diluted normal serum for 30 min, sections were
incubated with mouse GMR8 mAb (1:200) at 4°C overnight.
Subsequently, sections were biotinylated with anti-mouse IgM
for 30 min, followed by incubation with avidin DH–biotinylat-
ed horseradish peroxidase complex (Vector Labs, Burlingame,
CA, USA) for 30 min at room temperature. Finally, the peroxi-
dase reaction was developed using diaminobenzidine as the
chromogen. Slides were counterstained with hematoxylin.
Next, we evaluated NeuGc-containing ganglioside expres-
sion. We defined GMR8-positive cells as cells with complete or
partly stained cytoplasmic membranes. Three 0.25 9 0.25 mm
squares were randomly marked in the tumor area. The mean
percentage of GMR8-positive cells among the total number of
tumor cells was determined for each NSCLC sample. The eval-
uation was performed by two independent observers.
TLC immunostaining of gangliosides. Gangliosides were
extracted from approximately 10 mm blocks of formalin-fixed
tumor and normal tissues using a chloroform-methanol (2:1)
solution. Gangliosides extracted from tissues were immuno-
stained on a high-performance thin-layer chromatography plate
(E, Merck, Darmstadt, Germany) to determine the ganglioside
fraction. The solvent system used for developing chromato-
grams was chloroform-methanol-water (60:35:8, v/v/v). Gan-
gliosides were visualized with orcinol stain. The plate was air
dried, blocked with phosphate-buffered saline (PBS) containing
3% bovine serum albumin for 1 h, and incubated with mouse
GMR8 mAb and mouse anti-GM3 (NeuAC) mAb M2590
(Cosmo Bio Co. Ltd, Tokyo, Japan) for 1 day at 4°C. Bound
primary antibodies were visualized using the Vectastain kit
(Vector Labs) according to the manufacturer’s instructions.
In vitro GM3 (NeuGc)- and GM3 (NeuAc)-mediated inhibition of
EGFR phosphorylation. Human ovarial epidermoid carcinoma
A431 cells known to overexpress EGFR in the membrane
were used in the present study. A431 cells were cultured in
Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich-
Japan; number D5796, Tokyo, Japan) containing 10% heat-inac-
tivated fetal bovine serum (Japan Bio Serum; number 82225,
Fukuyama, Japan) and 100 lg/mL sodium pyruvate at 37°C in
5% CO2. An in vitro EGFR phosphorylation assay was per-
formed as described by Kawashima et al.(6) and Zhou et al.(16)
Aliquots of solution containing 250 lM GM3 (NeuAc) (Calbio-
chem; number 345733, Darmstadt, Germany) (average molecu-
lar weight = 1280) and 250 lM GM3 (NeuGc) (Alex number
302-019-MC01) (average molecular weight = 1264) in a chlo-
roform-methanol solution were dried under vacuum. Ethanol
was added and the sample was dried again. Serum-free DMEM
(400 lL) was added to completely dried GM3 (NeuAc) or GM3
(NeuGc), mixed using a vortex mixer for 3 h and sonicated for
30 min at 4°C. A431 cells were cultured separately in 12-well
plates in DMEM containing 10% fetal bovine serum and starved
in serum-free DMEM for 24 h before confluence, then washed
with serum-free DMEM and incubated in serum-free DMEM
containing GM3 (NeuAc) or GM3 (NeuGc) for 3 h at 37°C.
Cells were washed with PBS to induce phosphorylation, incu-
bated for 1 h in 400 lL of EGF (10 ng/mL) or 400 lL of EGF
(100 ng/mL) at 4°C, washed and lysed in 100 lL of lysis buffer
(1% SDS, 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4) for
30 min at 4°C. The cell lysate was subjected to SDS-PAGE fol-
lowed by western blotting using anti-EGFR and phosphotyro-
sine (PY20) antibodies.
Sequencing of cDNA of CMP-NeuAc hydroxylase in NSCLC
tissues. Lung cancer tissues were stored in RNAlater Tissue
44
Protect tubes (Qiagen, Duesseldorf, Germany) immediately
after pulmonary resections of RNA stabilization, and total
RNA was extracted from specimens using the single-step
method of RNA isolation by acid guanidinum thiocyanate-phe-
nol-chloroform extraction.(17) Reverse transcription of total
RNA was performed to generate the first strand cDNA. For
detection of cDNA of human CMP-sialic acid hydroxylase, we
performed reverse–transcription polymerase chain reaction
(RT-PCR) assays using the primer set based on sequences of
CMP-sialic acid; 5′-CCAGTCAGGAAGTC-3′ (forward primer,
the lesion upper 92-bp deletion) and 5′-GGTTGGAGGAC-
CAG-3′ (reverse, the lesion lower 92-bp deletion primer).(8)
Amplification was initiated by preincubation for 15 min at 94°
C for the initial activation, followed by 30 cycles of denatur-
ation for 1 min at 94°C, and primer annealing for 1 min at 54°
C and elongation for 2 min at 72°C using Hotstar Taq Master
Mix Kit (Qiagen). A 20 lL aliquot of each reaction mixture
was electrophoresed through 1% agarose gel and stained using
ethidium bromide for visualization. DNA fragments were
extracted with agarose gel using the QIAquick Gel Extraction
Kit (Qiagen). DNA sequencing was performed with the use of
dye terminator chemistry and a Capillary ABI 3100 sequencer
(Applied Biosystems, Tokyo, Japan).
Statistical analysis. The statistical analysis with categorical
data was done using the Pearson’s Chi-squared test. Kaplan–
Meier plots and log-rank analysis were used to determine the
significance of differences in progression-free and overall surviv-
als. P < 0.05 (two-tailed) was considered statistically significant.
Progression-free survival was the time between diagnosis and
the date of recurrence or remaining alive, while overall survival
was the time between diagnosis and the date of death or the date
when patients were last known to be alive. In the survival analy-
sis, only data before 31 December 2010 was considered.
Results
Patient characteristics. To investigate NeuGc-containing gan-
glioside expression in NSCLC tissues, we examined expression
levels in surgically resected tumor tissues of 93 NSCLC
patients treated at Sapporo Medical University Hospital and
retrospectively reviewed their clinical records. Patient demo-
graphics are shown in Table 1.
N-glycolyated ganglioside expression in NSCLC tissues. To
confirm that lipid components were recognized by GMR8
mAb, immunostaining was performed after removal of lipid
components from lung tissues using a chloroform-methanol
(2:1) solution. As shown in Figure 1(C,D) staining was lost
after removal of the lipid components. Therefore, the
molecules immunohistochemically detected by GMR8 were
gangliosides.
Table 1. Demographic data of the non-small-cell lung cancer
patients
No. patients evaluable 93
Mean age (range) (years) 66.3 (40–81)
Male/female (n) 58/35
Tumor histology (n)
Adenocarcinoma 60
Squamous cell carcinoma 22
Large cell carcinoma 6
Other* 5
Pathological stage
I 59
II 10
III 20
IV 4
*Pleomorphic carcinoma (n = 3); adenosquamous carcinoma (n = 2).
doi: 10.1111/cas.12027
© 2012 Japanese Cancer Association
 A B

C D

Fig. 1. N-glycolyated ganglioside expression in non-small-cell lung cancer (NSCLC). (A) Positive N-glycolyated ganglioside expression with GMR8
staining in NSCLC. (B) Negative N-glycolyated ganglioside expression with GMR8 staining in NSCLC. (C) Positive N-glycolyated ganglioside expres-
sion with GMR8 staining in NSCLC (not the same as the sample shown in Fig. 1A). (D) After removal of lipid components by incubating with chlo-
roform–methanol solution (2:1), negative N-glycolyated ganglioside expression with GMR8 staining in NSCLC (same sample as shown in Fig. 1C).

Positive and negative GMR8 expression in NSCLC tissues is GMR8-positive cells was not significantly correlated with the
shown in Figure 1(A,B). No NeuGc-containing ganglioside pathological stage of NSCLC.
expression was detected in normal lung tissues. If more than Immunoblotting of gangliosides on TLC plates. Using tumor
5% of the tumor cells were GMR8 positive, then NeuGc-con- tissues, we analyzed the composition of gangliosides recog-
taining ganglioside expression was considered positive. Neu- nized by GMR8 mAb. Using four NSCLC tissues that were
Gc-containing gangliosides were expressed in 86 of 93 positive for NeuGc-containing ganglioside expression in the
(93.5%) NSCLC samples. Details of the percentage of GMR8- immunohistochemistry study, we performed TLC immuno-
positive cells are shown in Table 2. In addition, Kaplan–Meier staining of gangliosides extracted from both normal and cancer
analysis was conducted to determine whether a difference in tissues. N-glycolyated gangliosides (GD1a [NeuGc] and GM3
the percentage of GMR8-positive cells was associated with [NeuGc]) were detected only in cancer tissues using GMR8
prognosis. Pathological stage IV patients whose prognosis was mAb (Fig. 3A), whereas GM3 (NeuAc) was found in both nor-
obviously poor were excluded from analysis. For survival anal- mal and cancer tissues using mouse anti-GM3 (NeuAC) mAb
ysis, the group with  80% GMR8-positive cells was sepa- (M2590 mAb) (Fig. 3B).
rated from the group with <80% GMR8-positive cells (Fig. 2). In vitro GM3 (NeuGc) and GM3 (NeuAc)-mediated inhibition of
We found that progression-free survival was significantly low EGFR phosphorylation. We investigated the difference in the
in the group with a higher percentage of GMR8-positive cells effects of GM3 (NeuAc) and GM3 (NeuGc) on inhibition of
(P < 0.05) and the overall survival was low, although this was EGFR tyrosine kinase. The density ratio of anti-PY20 to anti-
not statistically significant (P = 0.156). The percentage of EGFR bands is shown in the bar graph (Fig. 4). As shown in
Figure 4, the inhibitory effect of GM3 (NeuAc) and GM3
(NeuGc) on EGFR tyrosine kinase induced by 10 or 100 ng
EGF in A431 cell membrane fractions was reduced after expo-
Table 2. N-glycolyated ganglioside expression in tumor cells of sure to GM3 (NeuGC) was lower than that after exposure to
non-small-cell lung cancer GM3 (NeuAC).
cDNA sequences of CMAH in NSCLC tissues. In three cancer
No. cases (%)
OS PFS tissues that were positive for NeuGc-containing ganglioside
(months) (months) expression, we analyzed the cDNA sequence of the CMAH
gene and found a 92-bp deletion in it and a frameshift muta-
High N-glycolyated ganglioside expression
tion in the stop codon (TGA), as previously reported by Chou
80~100% positive cells in 15 (17.0) 52.4 47.5
et al.(8)
tumor cells
Low N-glycolyated ganglioside expression
60~79% positive cells in 15 (17.0) 55.9 56.4 Discussion
tumor cells
In the present study, we demonstrated that N-glycolyated gan-
40~59% positive cells in 12 (13.6) 55.4 55.4
gliosides were extensively expressed in NSCLC, although they
tumor cells
have never been previously detected in normal human tissues.
20~39% positive cells in 17 (19.3) 58.6 57.8
We used GMR8 mAb, which specifically reacts with ganglio-
tumor cells
sides having NeuGc alpha 2,3Gal-terminal structures, generated
6~19% positive cells in 16 (18.2) 56.9 56.9
by immunizing mice with purified GM3 (NeuGc). In contrast,
tumor cells
van Cruijsen et al.(18) and Blanco et al.(19) reported that
0~5% positive cells in 13 (14.8) 58.8 58.8
NSCLC tissues stained positive in a tissue microarray analysis
tumor cells
using another anti-GM3 (NeuGc) mouse mAb, 14F7 mAb.
OS, overall survival; PFS, progression-free survival. Their studies demonstrated that GM3 (NeuGc) expression was

Hayashi et al. Cancer Sci | January 2013 | vol. 104 | no. 1 | 45

© 2012 Japanese Cancer Association
 (A) (B) 100 Low N-glycolyated gangliosides expression
100 Low N-glycolyated gangliosides expression
Median overall survival 57.5 months
Median progression free survival 57.4 months

80

Progression-free survival (%)

80

Overall survival (%)

60 60

40 40
High N
N-glycolyated
glycolyated gangliosides expression
High N-glycolyated gangliosides expression Median overall survival 52.4 months
Median progression-free survival 47.5 months
20 20
Log rank P = 0.156
Log rank P < 0.01

0 0

0 20 40 60 80 100 0 20 40 60 80 100
Time after primary tumor resection (months) Time after primary tumor resection (months)

Fig. 2. Kaplan–Meier plots for (A) progression-free survival and (B) overall survival based on N-glycolyated ganglioside expression in non-small-
cell lung cancer patients.

(A)

(B)

Fig. 3. (A) TLC immunostaining using GMR8 mAb. Normal tissues = 1, 2, 3 and 4; cancer tissues = 5, 6, 7 and 8. (B) TLC immunostaining using
anti-GM3 (NeuAc) mAb, M2590. Normal tissues = 1, 2, 3 and 4; cancer tissues = 5, 6, 7 and 8.

associated with a better prognosis in NSCLC patients. On the
contrary, our results indicated that patients with high NeuGc-
containing ganglioside expression had a low overall survival
rate and a significantly low progression-free survival rate. The
reason for this discrepancy might be that GMR8 mAb have a
different specificity for recognizing molecules compared with
the 14F7 mAb. In NSCLC tissues, our immunostaining of gan-
gliosides on TLC demonstrated that at least two NeuGc-con-
taining gangliosides, GM3 (NeuGc) and GD1a (NeuGc), were
recognized by GMR8 mAb. In addition, our investigation used
93 patients, which is a larger sample size compared with that in
their investigation, which used 26 patients. Thus, differences in
the specificity of the antibodies and the number of patients
might lead to differences in results.
Human tissues and body fluids contain little or undetectable
amounts of NeuGc, whereas corresponding samples from chim-
panzees and bonobos express high levels.(20) Human deficiency
of NeuGc is due to a mutation in the CMAH gene.(7–9) Human
CMAH is inactive because of the deletion of the region corre-
sponding to the N-terminal domain of the hydroxylase, which
is essential for enzyme activity.(9) We observed that cDNA
sequences of the CMAH gene in human NSCLC tissues were
the same as those in normal tissues. Thus, CMAH enzyme
activity in human NSCLC tissues is considered to be inactive,
similar to that in normal tissues. This suggests the existence of
an alternative pathway for NeuGc-containing gangliosides, dif-
ferent from the normal pathway that is mediated by CMAH
enzyme activity. Yin et al. reported a hypoxic culture induced
NeuGc-containing ganglioside expression on human cells as a
possible candidate for the alternative pathway because a hyp-
oxic culture markedly induced mRNA for the sialic acid trans-
porter sialin.(21) This might be the cause of NeuGc-containing
ganglioside expression in NSCLC tissues because tumor cells
in a hypoxic environment promote incorporation of NeuGc-type
sialic acid in extracellular fluid via activation of the sialic acid
transporter. Selection of hypoxia-resistant cancer cells by the
hypoxic environment in locally advanced tumor sites is known
to result in the culture expansion of cancer cells with higher
invasive and metastatic activities.(22,23) Overall, NSCLC with
high NeuGc-containing ganglioside expression might occur in a
severely hypoxic environment, which might be related to poor
survival as suggested earlier.

46 doi: 10.1111/cas.12027
© 2012 Japanese Cancer Association

Fig. 4. In vitro inhibitory effect of GM3 (N-glycolyl neuraminic acid
[NeuGc]) and GM3 (N-acetylneuraminic acid [NeuAc]) on epidermal
growth factor receptor (EGFR) tyrosine phosphorylation. Phosporyla-
tion was analyzed using western blotting with mAb PY20 in the upper
section. The density of band analyzed as a percentage of the control
value is shown in the lower section.
Epidermal growth factor receptor belongs to the erbB trans-
membrane receptor family and plays a role in cell proliferation
and differentiation at the epithelial surface.(24) Delay in cell
growth of the human epidermoid carcinoma cell line A431 is
caused by GM3 (NeuAc)-mediated inhibition of EGFR tyro-
sine kinase.(2) GM3 (NeuAc) inhibits EGFR tyrosine kinase
via carbohydrate–carbohydrate interactions between N-glycans
with GlcNAc termini on EGFR and oligosaccharides on
GM3.(6) In the present study, we found that the inhibitory
effect of GM3 on EGFR tyrosine kinase reduced in A431 cells
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decrease the NeuGc-containing ganglioside ratio in NSCLC tissues.
Acknowledgments
The authors are grateful to Dr I. Kawashima at Tokyo Metropolitan
Institute of Medical Science for his valuable comments on the present
study.
Disclosure Statement
The authors declare no competing financial interests.
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