High-mannose glycans are elevated during breast cancer progression

Abstract

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.

Fig. 1.

Strategy used in the analysis of N-linked glycans in the serum samples. N-linked glycans were released from denatured glycoproteins in the serum by PNGase F enzymatic digestion. Ethanol was added to precipitate the proteins, leaving the glycans in the supernatant. Fractionation and purification was done by solid phase extraction using graphitized carbon cartridges. Mass spectra were recorded on a MALDI FT-ICR MS. Symbol representations of glycans (39): N-acetylglucosamine, black square; mannose, gray circle; galactose, white circle; fucose, dark gray triangle; N-acetylneuraminic acid, dark gray diamond.

Fig. 2.

MALDI FT-ICR MS representative profiles of N-linked glycans in mouse and human sera from the 10%, 20%, and 40% acetonitrile (ACN) fractions taken in positive (10% and 20%) and negative (40%) ion modes. Profiles shown are from (A) serum of a tumor-transplanted mouse in its 2nd week and (B) serum of a human with breast cancer. Only the abundant N-linked glycans were annotated. Structures are putative and based on exact mass and when possible, MS/MS. An * indicates that the peak is identical in mass and already annotated in the previous fraction. See Fig. 1 for glycan symbols.

Fig. 3.

Representative MALDI FT-ICR mass spectra of A, mock surgery control and B, tumor-transplanted mouse sera at different time points. m/z 1000–2000 region of 10% acetonitrile fraction taken in the positive ion mode is shown. See Fig. 1 for glycan symbols.

Fig. 4.

Change in intensities from MALDI FT-ICR MS of high-mannose N-linked glycans in sera of an intact control mouse, mock surgery control mouse, and a tumor-transplanted mouse during breast cancer progression. All values relative to Week 0 intensity. Error bars are expressed as standard error of the mean (S.E.) from three spectral scans per mouse sample. Symbol representations of glycans (39): N-acetylglucosamine, blue square; mannose, green circle; glucose, blue circle.

Fig. 5.

N-linked glycans that are statistically different at Week 0 (pretumor) and Week 4 (tumor) in mouse sera of tumor-transplanted mice (C). These same glycans are not statistically different at Week 0 and 4 in mouse sera of both the intact (A) and mock surgery (B) control mice. All glycans except m/z 1809.639 are from the 10% ACN fraction. Glycan m/z 1809.639 is from the 20% ACN fraction. Glycans marked with * have p value < 0.05. Glycans were analyzed using MALDI FT-ICR MS. Error bars are expressed as S.E. See Fig. 1 for glycan symbols.

Fig. 6.

High-mannose glycans in human sera of control (n = 5) and cancer (n = 7) groups. Glycans marked with * have p value < 0.05. Glycans were analyzed using the internal standard method by MALDI FT-ICR MS. Error bars are expressed as S.E. See Fig. 1 for glycan symbols.