B3GNT5 is a novel marker correlated with stem‐like phenotype and poor clinical outcome in human gliomas

Abstract Aims Glioblastoma multiforme (GBM) is the most lethal tumor with a median patient survival of 14 to 15 months. Glioma stem cells (GSCs) play a critical role in tumor initiation and therapeutic resistance in GBM. B3GNT5 has been suggested as the key glycosyltransferase in the biosynthesis of the (neo‐) lacto series of glycosphingolipid. In this study, we evaluated the B3GNT5 expression in GSCs as well as the correlation with clinical data in GBM. Methods The mRNA levels of B3GNT5 in normal astrocytes, four glioma cell lines, and four GSCs were evaluated using real‐time PCR. Small interference RNAs (siRNAs) were used to inhibit B3GNT5 expression and analyze its ability to form neurospheres. Statistical analyses were conducted to determine the association with B3GNT5 expression and tumor grade and GBM subtypes as well as patient survival using public datasets. Results B3GNT5 expression was significantly elevated in GSCs compared with normal astrocytes, glioma cell lines, and their matched differentiated tumor cells. Knockdown of B3GNT5 in GSCs decreased the neurosphere formation. Patients with high B3GNT5 expression had a short overall survival. B3GNT5 is correlated with classical and mesenchymal GBM subtypes. Conclusion The findings suggest the central role of B3GNT5 in regulating malignancy of GBM.

into different series defined by their respective core structures. The major GSL types include ganglio-, lacto-, globo-, isoglobo-, and muco-series. 3 GSL series play a crucial role in cell adhesion, cell migration, regulation of signaling proteins, and binding of pathogens and toxins. 4,5 B3GNT5 (UDP-GlcNAc:β-Gal β-1,3-N-acetylglucosaminyltransferase 5) is a glycosyltransferase that transfers an Nacetylglucosamine (GlcNAc) from uridine diphosphate (UDP)-GlcNAc to galactose at the nonreducing end of carbohydrate chain. B3GNT5 is a key glycosyltransferase that mediates the biosynthesis of lactoand neolactoseries of GSLs. B3GNT5 transfers N-acetylglucosamine to the C-3 position of galactose in lactosylceramide resulting in the synthesis of lactotriaosylceramide, and thus, B3GNT5 is also known as lactosylceramide synthase. Several studies reported the biological significance of B3GNT5-mediated synthesis of glycolipids in B-cell activation, 6 pre-implantation development, 7 and development of nerve system, 8,9 The increased levels of B3GNT5 appear to be strongly correlated with the progression of breast cancer, 10 lung cancer, 11 and ovarian cancer. 12 In addition, B3GNT5-mediated glycosphingolipids play a key role in the differentiation of acute myeloid leukemia (AML) cells. 13 However, the role of B3GNT5 in GSC differentiation remain is incompletely understood. Therefore, in this study, we investigated the association between the expression of B3GNT5 and the malignancy of GBM using the Repository for Molecular Brain Neoplasia Data (REMBRANDT), Gene Expression Omnibus (GEO) (GSE4536), and Ivy Glioblastoma Atlas Project. In addition, we further evaluated the effects of B3GNT5 on the maintenance of stemness in cancer stem cells (CSCs) using GSCs derived from primary human GBM tissues.

| Dataset preparation for B3GNT5 expression and survival analysis
Repository for Molecular Brain Neoplasia Data (REMBRANDT),

| Tumorsphere assay
GSCs transfected with siRNA as described above were seeded onto 24-well plates at a density of 250 cells/well, followed by incubation for 10 days in the presence of 5% CO 2 at 37°C without disturbing the plates and without replenishing the medium. Growth factors (bFGF and EGF) were added once every three days. At the end of 10-day incubation, tumorspheres were collected at the center of the well by slowly swirling the plates, and the images were obtained to determine the number and size of tumorspheres using a digital microscope (Logos Biosystems, Anyang-si). The number of tumorspheres measuring more than 50 μm in diameter each was counted. 1.1.6r4). Significant quantitative differences between and among groups were determined by a two-tailed t test and one-way ANOVA, respectively, followed by Tukey's multiple comparison test. Kaplan-Meier survival analysis was used to estimate the survival distribution, followed by the log-rank test to evaluate the differences between stratified groups, using the median value as the cutoff.

| Expression of B3GNT5 in glioma stem cells was decreased in differentiated tumor cells
Using the available dataset, we first analyzed the expression of 361 glycosylation-related genes between glioma stem cells and their matched differentiated tumor cells. Heatmap and clustering analysis revealed that ST8SIA5, MT3, B3GNT5, and MGAT4A were commonly upregulated in both GSC0308 and 1228 in GSCs compared with the differentiated cells ( Figure 1A,B). We selected B3GNT5 as one of the top-ranked genes according to its fold change ( Figure 1C,D) and confirmed its significant expression using three different microarray probes ( Figure 1E,F). The results suggest that B3GNT5 exhibited glioma stem cell properties.

| Validation of B3GNT5 expression in GSCs
We

| Silencing of B3GNT5 inhibits neurosphere formation of GSCs
In order to elucidate the function of B3GNT5 in the maintenance of stem cell properties of GSCs, we designed two small interfering RNAs (siRNA) to silence the B3GNT5. Real-time PCR analysis showed that siB3GNT5 #1 and siB3GNT5 #2 inhibit B3GNT5 mRNA levels by 38% and 53% in GSC11, respectively ( Figure 3A). Knockdown of B3GNT5 decreased GSC markers (CD133 and CD15) mRNA levels ( Figure 3B,C) as well as neurosphere formation ( Figure 3D) compared with the scramble control indicating that B3GNT5 was required for self-renewal of GSC11.

| B3GNT5 is highly expressed in GBM
In order to understand the clinical significance of B3GNT5, we

| B3GNT5 is overexpressed in classical and mesenchymal subtypes
There are four different GBM subtypes-proneural, classical, mesenchymal, and neural-which are defined on the basis of genomewide analysis of mRNA expression in more than 300 GBM patient tissues. 16 These subtypes also exhibit their characteristic tumor histology and molecular alterations, and are associated with unique clinical outcomes. Using the REMBRANDT database, we investigated whether B3GNT5 was enriched in specific patient cohorts.
As shown in Figure  in PAN, which is associated with hypoxia, cell migration, and immune response. 17 In conclusion, we demonstrated that elevated B3GNT5 expression was associated with GBM aggressiveness and maintenance of self-renewal in glioma stem cells. We propose that B3GNT5 is a marker of poor prognosis in GBM. In this study, we found that B3GNT5 expression was significantly downregulated during GSC differentiation after serum supplementation. Previously, it was reported that neurobasal media containing EGF and bFGF (NBE) used to culture undifferentiated GSCs exhibit more robust tumorigenic potential, heterogenous morphology, and indefinite potential for self-renewal compared with serum-cultured differentiated GSCs. 14  GSLs are found abundantly on cellular membranes and contain either a ceramide or a sphingoid as the hydrophobic lipid moiety. GSL series are functionally important in cell adhesion, cell migration, regulation of signaling proteins, and binding of pathogens and toxins. 4,5 GSLs are classified into different series according to their core structure. Based on the core structure, GSLs exist as ganglio-series (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer), globo-(GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), isoglobo-series (GalNAcβ1-3Galα1-3Galβ1-4Glcβ1-1′Cer), muco-series (Galβ1-4Galβ1-4Glcβ1-1′Cer), and lacto-series (Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer). Among the GSL series, β1-3-linked GlcNAc structure exists only in the core structure of lacto-series GSL. Since our study demonstrated the upregulation of B3GNT5 in GSCs, an abundance of lacto-or neolacto-based GSL structures exist on GSCs.

| D ISCUSS I ON
Sulfoglucuronyl glycolipids (SGGL) such as SGGL-1 and SGGL-2 (SGGL-1; SO 4 3-GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer, SGGL-2; SO 4 3-GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer) represent extended structures of B3GNT5mediated nLc GSL. SGGL are detected during brain development and reported to play a critical role in cell-cell interaction in central nervous system. 19 B3GNT5 is the key enzyme involved in initiating the formation of root structure of the Lewis X carbohydrate Galβ1-4 (Fucα1-3)GlcNAcβ1-R, which mediates cellular adhesion and differentiation of the neuronal system. 20 Since our study demonstrated the elevated expression of B3GNT5 in GSCs, a comparative structural analysis of B3GNT5-based glycolipids such as SGGL and Lewis X on GSCs will be performed in our next study to elucidate the structure of the major glycolipid controlling the stemness of glioma stem cells.
In conclusion, our results indicate the potential of B3GNT5 as a prognostic biomarker used to characterize the most aggressive cells in GBM, with a possible therapeutic role in the targeted therapy of GBM.

ACK N OWLED G M ENTS
We thank the members of the Kim laboratory for consultation and assistance.