Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer

Abstract

Epithelial ovarian cancer is the deadliest female reproductive tract malignancy in Western countries. Less than 25% of cases are diagnosed when the cancer is confined, however, pointing to the critical need for early diagnostics for ovarian cancer. Identifying the changes that occur in the glycome of ovarian cancer cells may provide an avenue to develop a new generation of potential biomarkers for early detection of this disease. We performed a glycotranscriptomic analysis of endometrioid ovarian carcinoma using human tissue, as well as a newly developed mouse model that mimics this disease. Our results show that the N-linked glycans expressed in both nondiseased mouse and human ovarian tissues are similar; moreover, malignant changes in the expression of N-linked glycans in both mouse and human endometrioid ovarian carcinoma are qualitatively similar. Lectin reactivity was used as a means for rapid validation of glycan structural changes in the carcinomas that were predicted by the glycotranscriptome analysis. Among several changes in glycan expression noted, the increase of bisected N-linked glycans and the transcripts of the enzyme responsible for its biosynthesis, GnT-III, was the most significant. This study provides evidence that glycotranscriptome analysis can be an important tool in identifying potential cancer biomarkers.

Figure 1

Transcript analysis of enzymes acting in the N-linked pathway for normal ovary. (A) N-linked glycosylation pathway with enzymes included in analysis numbered as follows: 1, MGAT1; 2, MAN2A1 (Man II) or MAN2A2 (Man IIx); 3, FUT8; 4, MGAT2; 5, MGAT3; 6, MGAT4a or MGAT4b; 7, MGAT5. (B) Relative transcript levels for normal human ovary tissue, average Ct for two pooled cases. Error bars represent the SD from the mean for triplicate Ct values. (C) Relative transcript levels for normal mouse ovary, average Ct for six pooled normal ovaries. Error bars represent the SD from the mean for triplicate Ct values.

Figure 2

Comparative analysis of normal and endometrioid ovarian carcinoma. (A) Relative transcript abundance for mouse normal and ovarian tumors plotted on a log scale. Error bars represent the SD from the mean for triplicate Ct values. (B) Relative transcript abundance for human normal and ovarian tumors plotted on a log scale. Error bars represent the SD from the mean for triplicate Ct values.

Figure 3

Oligosaccharides determinants for lectin binding affinity. Examples of structures with important binding determinants from each lectin circled.

Figure 4

Lectin blot analysis of glycoproteins from mouse and human endometrioid ovarian carcinoma. (A) Glycoproteins extracted from mouse endometrioid ovarian tumors (lanes 2, 4, 6, and 8) or normal mouse ovary (lanes 1, 3, 5, and 7) that were nonadherent to Con A were separated on 4–12% Bis-Tris gels before transfer to PVDF membrane and detection using biotinylated lectins and streptavidin–HRP. Panel below shows the densitometry analysis of bands from normal (NL) or ovarian tumor (OT) in the 49–250 kDa range from the blots shown above with normal set at 1.0 for comparison. Fold increase was adjusted for lectin pull-down inputs based on the levels of ERK2 on a 10% input blot (data not shown). (B) Glycoproteins nonadherent to Con A from human endometrioid ovarian cancer cases (711, 741, and 471) and normal human ovary (NL) were separated on 4–12% Bis-Tris gels before lectin blot detection as described. The panel below represents the densitometry results for glycoproteins 49–250 kDa relative to normal set at 1.0. Increases relative to normal were adjusted for input using ERK2 analysis form 10% input blots (data not shown).