HNK-1 glycan functions as a tumor suppressor for astrocytic tumor

Abstract

Astrocytic tumor is the most prevalent primary brain tumor. However, the role of cell surface carbohydrates in astrocytic tumor invasion is not known. In a previous study, we showed that polysialic acid facilitates astrocytic tumor invasion and thereby tumor progression. Here, we examined the role of HNK-1 glycan in astrocytic tumor invasion. A Kaplan-Meier analysis of 45 patients revealed that higher HNK-1 expression levels were positively associated with increased survival of patients. To determine the role of HNK-1 glycan, we transfected C6 glioma cells, which lack HNK-1 glycan expression, with β1,3-glucuronyltransferase-P cDNA, generating HNK-1-positive cells. When these cells were injected into the mouse brain, the resultant tumors were 60% smaller than tumors emerging from injection of the mock-transfected HNK-1-negative C6 cells. HNK-1-positive C6 cells also grew more slowly than mock-transfected C6 cells in anchorage-dependent and anchorage-independent assays. C6-HNK-1 cells migrated well after treatment of anti-β1 integrin antibody, whereas the same treatment inhibited cell migration of mock-transfected C6 cells. Similarly, α-dystroglycan containing HNK-1 glycan is different from those containing the laminin-binding glycans, supporting the above conclusion that C6-HNK-1 cells migrate independently from β1-integrin-mediated signaling. Moreover, HNK-1-positive cells exhibited attenuated activation of ERK 1/2 compared with mock-transfected C6 cells, whereas focal adhesion kinase activation was equivalent in both cell types. Overall, these results indicate that HNK-1 glycan functions as a tumor suppressor.

FIGURE 1.

Expression of HNK-1 glycan is inversely correlated with progression of human astrocytoma. A–F, representative images of immunohistochemistry for HNK-1 glycan in various types of human astrocytic tumor. Immunostaining was scored based on the ratio of immunoreactive cells to total cells evaluated. No staining (−) (A, glioblastoma), weak staining (+) (B, glioblastoma), moderate staining (++) (C, anaplastic astrocytoma), and strong staining (+++) (D, pilocytic astrocytoma) are shown. In non-tumorous tissue, staining is seen on the surface of neurons and neuropil (E, cerebral cortex; the inset shows an enlargement of the indicated area). A control experiment using second antibody only is shown (F). The arrow in A indicates multinucleated tumor cells. Scale bar = 50 μm. G, expression of HNK-1 glycan related to patients' prognosis. HNK-1 glycan is expressed at lower levels in aggressive tumors (anaplastic astrocytoma and glioblastoma) than in less aggressive tumors (pilocytic astrocytoma and diffuse astrocytoma). H, five-year survival of 45 patients was statistically analyzed using the Kaplan-Meier method. HNK-1 glycan expression was positively correlated with increased survival with statistical significance (p = 0.0058 by log-rank test).

FIGURE 2.

HNK-1 glycan expression following forced expression of GlcAT-P. A, C6 and COS-1 cells express HNK-1 glycan (green) only after transfection of GlcAT-P cDNA. Blue indicates nuclear staining by Hoechst 33342. G14 and H1 are C6 lines expressing HNK-1 glycan after stable transfection of GlcAT-P. B, RT-PCR analysis of transcripts for GlcAT-P and HNK-1ST. C, flow cytometry analysis of G14 and H1 clones of C6 cells transfected with GlcAT-P (left panel). Transient expression of GlcAT-P or GlcAT-p + HNK-1ST also results in HNK-1 glycan expression (right panel). D, immunoprecipitation (IP) with HNK-1 antibody followed by immunoblotting (IB) with anti-HNK-1, anti-NCAM, or anti-β1-integrin antibodies indicates that HNK-1 cells express HNK-1 glycan in several proteins (left panel), including NCAM (right top panel, arrow), myelin-associated glycoprotein (arrowhead), but not β1 integrin (right bottom panel). Equal amount of proteins from total cell lysate were loaded on IP (−) lanes. HNK-1 antibody immune complex was loaded on IP (+) lanes. *1, IgM band.

FIGURE 3.

C6 cells expressing HNK-1 glycan are less invasive than HNK-1-negative parental cells. Parental C6 and HNK-1 glycan-positive C6 cells (clones G14 and H1) were inoculated into the mouse brain using a stereotaxic frame. Mice were sacrificed 3 weeks later, coronal sections of the brain were prepared, and C6 cells were visualized with anti-vimentin antibody. A and B, in C57BL/6 mice, HNK-1 glycan-positive C6 cells, H1 and G14 formed smaller tumor than mock-transfected (Control) cells. The area of C6 cells is highlighted in the insets in B. The ratio of the area of vimentin-positive tumor cells per brain section is compared in bar graphs (right panels). n = 7 for both cell lines in A, n = 5 and 6 in B. C, almost identical results were obtained when nude mice were used as hosts. n = 10 and 9 for each cell type. Data are mean ± S.E. Statistical significance was evaluated by Student's t test.

FIGURE 4.

K_i-67 expression and TUNEL assay of HNK-1-positive or HNK-1-negative C6 cells tumor formed in mouse brain. The wild-type C57 BL/6 mice were injected with control C6, G14 (HNK-1-positive), or H1 (HNK-1-positive C6 cells) as described in the text. Three weeks later, mice were sacrificed, brains were fixed in 4% PFA, and paraffin sections were prepared. A, sections were stained with K_i-67 antibody to detect proliferating cells in the injected tumor. The larger amounts of K_i-67-positive cells were clearly observed in C6 tumor rather than in G14 or H1 tumor. The number of positive cells per field was counted (three fields per section, two mice) and shown as a graph. B, a TUNEL assay was performed on sections from the same mice as A. TUNEL-positive cells were not observed as much as K_i-67-positive cells. Because H1 tumor was small and no TUNEL positive cells were observed, the result of H1 was omitted in B.

FIGURE 5.

Migration of C6-HNK-1. A, migration of C6-HNK-1 and mock-transfected cells was measured using a transwell chamber. The migrated cells were stained with crystal violet, and cell numbers were counted under a light microscope. Migration of C6-HNK-1 and C6 cells is decreased when laminin (LN) or fibronectin (FN) is present in the upper chamber. B and C, E3 peptide (B), or anti-HNK-1 antibody (C) inhibits C6-HNK-1 cell migration. D, anti-β1 integrin antibody inhibits the migration of C6 cells but not HNK-1-positive C6 cells (G14). Anti-α6 integrin antibody or control IgG (10 μg/ml) did not have any effect. The bottom surface of membranes were coated with laminin (C), fibronectin (B and D), and fibronectin or laminin (A). The upper chamber was also coated with fibronectin or laminin in some experiments (in A). The assay was carried out three times for A, B, and D and five times for C. The error bars indicate S.D. The p value was calculated by Student's t test.

FIGURE 6.

Laminin-binding glycans and HNK-1 glycan are attached to different populations of α-dystroglycan. A–C, immunoblot analysis (IB) of α-dystroglycan-IgG·Fc released from C6 and C6-HNK-1 cells (G14). α-dystroglycan-expressing HNK-1 glycan is larger in the presence of LARGE (L). HNK-1 glycan was detected by anti-HNK-1 glycan antibody (A), whereas laminin-binding glycans were detected by IIH6 antibody (B) and laminin overlay (Lam O/L) (C). D, cell migration assay to laminin. Treatment of anti-laminin-binding glycan IIH6 antibody slightly facilitates migration of both C6-HNK-1- and mock-transfected C6 cells. The experiments were carried out three times. The arrows in B and C indicate IIH6-positive α-dystroglycan. The error bars indicate S.D.

FIGURE 7.

HNK-1 glycan suppresses cell proliferation. A, cell numbers of mock-transfected C6 cells and C6 cells expressing HNK-1 glycan (G14 and H1) grown on plates. The growth curves represent an average of four fields. B, C6 and C6-HNK1 clones G14 and H1 were cultured in soft agar for 4 weeks, and fixed cells were visualized by crystal violet staining. Colony size was determined by ImageJ scanning, and the average size is shown in the graph. The error bars represent S.E. The p value was calculated by Students' t test.

FIGURE 8.

Phosphorylation of ERK1/2 but not FAK is suppressed in C6 cells expressing HNK-1 glycan. A, phosphorylation of ERK1/2 was examined in control C6 cells and two lines of C6-HNK-1 cells, G14 and H1. Mono-dispersed cells were placed on laminin-coated plates, and cells were harvested at the indicated times. Cell lysates were prepared under phosphatase-free conditions and subjected to SDS-PAGE followed by immunoblotting with anti-phosphorylated ERK antibody. The blot was then stripped for blotting for total ERK1/2 protein. p and t correspond to phosphorylated and total ERK1/2, respectively. In HNK-1 positive G14 cells (upper panel), phosphorylation of ERK1/2 was suppressed. Comparable results were obtained in another C6-HNK-1 cell line, H1 (lower panel). B, a similar experiment was carried out for FAK. A decrease of the FAK phosphorylation signal was not observed. Bottom panel, anti-β1-integrin treatment caused a decrease in the phosphorylation of FAK for C6 cells but not C6-HNK-1 (G14) cells.