Jacob Francis, Hitchins Megan P, Fedier André, Brennan Kevin, Nixdorf Sheri, Hacker Neville F, Ward Robyn, Heinzelmann-Schwarz Viola A
Gynecological Research Group, Department of Biomedicine, University Hospital Basel and University of Basel, Hebelstrasse 20, CH-4013 Basel, Switzerland.
BMC Mol Biol. 2014 Oct 7;15:24. doi: 10.1186/1471-2199-15-24.
The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated.
RT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2'-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells.
GBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.
GBGT1基因编码球苷α-1,3-N-乙酰半乳糖胺基转移酶1。该酶催化福斯曼（Fs）抗原多步生物合成的最后一步，Fs抗原是球系列糖鞘脂中的一种五糖基神经酰胺。虽然在多种人类组织中观察到GBGT1 mRNA表达存在差异，在胎盘和卵巢中表达最高，但尚未研究GBGT1的表达以及参与Fs生物合成的糖基转移酶和糖苷酶基因的表达，也未研究DNA甲基化在GBGT1表达转录调控中的可能作用。
RT-qPCR分析显示，在正常卵巢表面上皮（HOSE）细胞系中GBGT1表达较高，而在除一个（OVCAR3）外的所有研究的卵巢癌细胞系（如A2780、SKOV3）中GBGT1表达较低，这一发现通过蛋白质印迹分析得到证实。层次聚类分析表明，GBGT1甚至是Fs生物合成相关糖基因中表达变化最大的基因，在所研究的细胞系中，而编码水解Fs的α-N-乙酰半乳糖胺酶的NAGA没有差异表达。对GBGT1启动子区域CpG岛甲基化状态的亚硫酸氢盐和COBRA分析表明，所有卵巢癌细胞系（除OVCAR3外）中GBGT1 DNA甲基化水平较高或中等，但在两个HOSE细胞系中DNA甲基化水平较低。DNA甲基化程度与GBGT1 mRNA和蛋白质表达呈负相关。对癌症基因组图谱卵巢癌数据集中GBGT1的生物信息学分析表明，在原发性卵巢癌组织样本中也发现了这种负相关，证实了我们基于细胞系的研究结果。通过5-氮杂-2'-脱氧胞苷处理，低表达GBGT1的A2780细胞中GBGT1 mRNA和蛋白质表达得以恢复，这些处理后的细胞螺旋蜗牛凝集素染色增加，反映了这些细胞上Fs二糖的存在增加。
在卵巢癌中，GBGT1表达通过启动子高甲基化在表观遗传上被沉默。我们的研究结果不仅提示DNA甲基化参与了Fs抗原的合成，还可能解释了早期研究中显示GBGT1在各种人类组织样本和疾病阶段表达存在差异的原因。
