2 neugc nsclc cancer science 2013 by P3DESIGN

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 regulate cell proliferation and differentiation. N-glycolylneuraminic acid (NeuGc)-containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal 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 cancer (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 inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc-containing gangliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc-containing ganglioside expression had a low overall survival rate and a significantly low progression-free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure 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-containing 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)

ung cancer is the most frequently diagnosed major cancer worldwide.(1) The World Health Organization classification of lung cancer identifies squamous cell carcinoma, adenocarcinoma, 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 treatment. The low rate of cure for NSCLC can be attributed to the high metastasis rate at diagnosis and the lack of effective treatments to cure such a metastatic disease. Gangliosides are ubiquitous membrane-associated glycosphingolipids 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 inhibition of epidermal growth factor receptor (EGFR) tyrosine

kinase.(4) GM3 is a ganglioside that binds to the extracellular domain of EGFR and inhibits its dimerization without inhibiting ligand binding.(5) Kawashima et al. described the significance 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 alphaGb5Cer 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 NeuGccontaining 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 primary 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 University approved the study protocol (approval no. 219-3). Immunohistochemistry. We determined N-glycolyated ganglioside expression in NSCLC tissues using mouse GMR8

7 To whom correspondence should be addressed. E-mail: hchiba@sapmed.ac.jp

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mAb.(13) Paraffin-embedded tumor tissues were cut into 5-lmthick sections and placed on glass slides. These sections were deparaffinized and pretreated by boiling the slides in citrate buffer (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–biotinylated horseradish peroxidase complex (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature. Finally, the peroxidase reaction was developed using diaminobenzidine as the chromogen. Slides were counterstained with hematoxylin. Next, we evaluated NeuGc-containing ganglioside expression. 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 evaluation 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 immunostained on a high-performance thin-layer chromatography plate (E, Merck, Darmstadt, Germany) to determine the ganglioside fraction. The solvent system used for developing chromatograms was chloroform-methanol-water (60:35:8, v/v/v). Gangliosides 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.

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-phenol-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′-GGTTGGAGGACCAG-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 denaturation 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 survivals. 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 analysis, only data before 31 December 2010 was considered.

In vitro GM3 (NeuGc)- and GM3 (NeuAc)-mediated inhibition of EGFR phosphorylation. Human ovarial epidermoid carcinoma

Patient characteristics. To investigate NeuGc-containing ganglioside 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 demographics 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.

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-AldrichJapan; number D5796, Tokyo, Japan) containing 10% heat-inactivated 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 performed as described by Kawashima et al.(6) and Zhou et al.(16) Aliquots of solution containing 250 lM GM3 (NeuAc) (Calbiochem; number 345733, Darmstadt, Germany) (average molecular weight = 1280) and 250 lM GM3 (NeuGc) (Alex number 302-019-MC01) (average molecular weight = 1264) in a chloroform-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, incubated 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 followed by western blotting using anti-EGFR and phosphotyrosine (PY20) antibodies. Sequencing of cDNA of CMP-NeuAc hydroxylase in NSCLC tissues. Lung cancer tissues were stored in RNAlater Tissue

Results

Table 1. Demographic patients

data

No. patients evaluable Mean age (range) (years) Male/female (n) Tumor histology (n) Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Other* Pathological stage I II III IV

the

non-small-cell

lung

cancer

93 66.3 (40–81) 58/35 60 22 6 5 59 10 20 4

*Pleomorphic carcinoma (n = 3); adenosquamous carcinoma (n = 2).

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 expression with GMR8 staining in NSCLC (not the same as the sample shown in Fig. 1A). (D) After removal of lipid components by incubating with chloroform–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 shown in Figure 1(A,B). No NeuGc-containing ganglioside expression was detected in normal lung tissues. If more than 5% of the tumor cells were GMR8 positive, then NeuGc-containing ganglioside expression was considered positive. NeuGc-containing gangliosides were expressed in 86 of 93 (93.5%) NSCLC samples. Details of the percentage of GMR8positive cells are shown in Table 2. In addition, Kaplan–Meier analysis was conducted to determine whether a difference in the percentage of GMR8-positive cells was associated with prognosis. Pathological stage IV patients whose prognosis was obviously poor were excluded from analysis. For survival analysis, the group with 80% GMR8-positive cells was separated from the group with <80% GMR8-positive cells (Fig. 2). We found that progression-free survival was significantly low in the group with a higher percentage of GMR8-positive cells (P < 0.05) and the overall survival was low, although this was not statistically significant (P = 0.156). The percentage of

Table 2. N-glycolyated ganglioside expression in tumor cells of non-small-cell lung cancer No. cases (%) High N-glycolyated ganglioside expression 80~100% positive cells in 15 (17.0) tumor cells Low N-glycolyated ganglioside expression 60~79% positive cells in 15 (17.0) tumor cells 40~59% positive cells in 12 (13.6) tumor cells 20~39% positive cells in 17 (19.3) tumor cells 6~19% positive cells in 16 (18.2) tumor cells 0~5% positive cells in 13 (14.8) tumor cells

OS (months)

52.4

47.5

55.9

56.4

55.4

58.6

57.8

56.9

58.8

OS, overall survival; PFS, progression-free survival.

Hayashi et al.

PFS (months)

GMR8-positive cells was not significantly correlated with the pathological stage of NSCLC. Immunoblotting of gangliosides on TLC plates. Using tumor tissues, we analyzed the composition of gangliosides recognized by GMR8 mAb. Using four NSCLC tissues that were positive for NeuGc-containing ganglioside expression in the immunohistochemistry study, we performed TLC immunostaining of gangliosides extracted from both normal and cancer tissues. N-glycolyated gangliosides (GD1a [NeuGc] and GM3 [NeuGc]) were detected only in cancer tissues using GMR8 mAb (Fig. 3A), whereas GM3 (NeuAc) was found in both normal and cancer tissues using mouse anti-GM3 (NeuAC) mAb (M2590 mAb) (Fig. 3B). In vitro GM3 (NeuGc) and GM3 (NeuAc)-mediated inhibition of EGFR phosphorylation. We investigated the difference in the

effects of GM3 (NeuAc) and GM3 (NeuGc) on inhibition of EGFR tyrosine kinase. The density ratio of anti-PY20 to antiEGFR 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 exposure to GM3 (NeuGC) was lower than that after exposure to GM3 (NeuAC). cDNA sequences of CMAH in NSCLC tissues. In three cancer tissues that were positive for NeuGc-containing ganglioside expression, we analyzed the cDNA sequence of the CMAH gene and found a 92-bp deletion in it and a frameshift mutation in the stop codon (TGA), as previously reported by Chou et al.(8) Discussion

In the present study, we demonstrated that N-glycolyated gangliosides were extensively expressed in NSCLC, although they have never been previously detected in normal human tissues. We used GMR8 mAb, which specifically reacts with gangliosides having NeuGc alpha 2,3Gal-terminal structures, generated by immunizing mice with purified GM3 (NeuGc). In contrast, van Cruijsen et al.(18) and Blanco et al.(19) reported that NSCLC tissues stained positive in a tissue microarray analysis using another anti-GM3 (NeuGc) mouse mAb, 14F7 mAb. Their studies demonstrated that GM3 (NeuGc) expression was Cancer Sci | January 2013 | vol. 104 | no. 1 | 45 © 2012 Japanese Cancer Association

100

Low N-glycolyated gangliosides expression Median progression free survival 57.4 months

(B)

100

Low N-glycolyated gangliosides expression Median overall survival 57.5 months

Overall survival (%)

Progression-free survival (%)

(A)

High N-glycolyated gangliosides expression Median progression-free survival 47.5 months

High N N-glycolyated glycolyated gangliosides expression Median overall survival 52.4 months 20

Log rank P = 0.156

Log rank P < 0.01 0

100

Time after primary tumor resection (months)

100

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-smallcell 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 NeuGccontaining 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 gangliosides on TLC demonstrated that at least two NeuGc-containing 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 chimpanzees 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 corresponding to the N-terminal domain of the hydroxylase, which is essential for enzyme activity.(9) We observed that cDNA 46

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, different 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 hypoxic culture markedly induced mRNA for the sialic acid transporter 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. 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. Phosporylation 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 transmembrane 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 tyrosine 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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after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). The difference in the inhibition of EGFR tyrosine kinase between the two types may be associated with the different manner of GM3 binding to EGFR via carbohydrate–carbohydrate interaction. Furthermore, our in vitro results were consistent with the results of survival analysis, indicating that prognosis is poor for NSCLC patients with high NeuGc-containing ganglioside expression. Thus, inhibition of EGFR tyrosine kinase with GM3 is weakened in NSCLC tissues with high NeuGc-containing ganglioside expression, possibly leading to an environment where a tumor can proliferate easily. Our immunohistochemical studies showed that NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC tissues. Their expression could be associated with patient survival due to the difference of GM3 (NeuGc) and GM3 (NeuAc) on the effects on inhibition of EGFR tyrosine kinase. Further study is required to determine the expression ratio of GM3 (NeuGc) and GM3 (NeuAc) in NSCLC. This work decribes that NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC and NeuGc-containing ganglioside expression is associated with patient survival. It might be useful for NSCLC therapy to 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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