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)
L
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
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 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
Cancer Sci | January 2013 | vol. 104 | no. 1 | 43–47