Underpinning the need for accurate biomarkers for breast cancer detection is the strong correlation between survival rate and early diagnosis. Whereas stage-I diagnosis results in a near 100% survival rate, the chances are reduced by almost four times at stage IV (1).
Glycosylation networks have massive potential for early detection. Glycogenes and altered glycosylation patterns, including increased sialylation of O-glycans (2), core fucosylation (3), and glycosyltransferase expression levels (4), have been detected in various cancers.
At Northwest University, Xi’an, China, the focus was on bisecting N-acetylglucosamine (GlcNAc) levels (5). This structure consists of GlcNAc linked to a core β-mannose residue via a β1,4 linkage, catalyzed by N-acetylglucosaminyltransferase III (MGAT3).
The research team had previously reported a decrease in GlcNAc and the corresponding glycosyltransferase expression (MGAT3) during tumor-induced epithelial-to-mesenchymal transition (EMT) (6). Considering other studies about the relations between bisecting GlcNAc and cancer (7,8), the team decided to elucidate its involvement in breast cancer. More specifically, they aimed to investigate the change in expression in breast cancer and related glycoproteins.
Using mass spectrometry, the researchers first identified the types of GlcNAc expressed in normal cells and breast cancer cell lines, helping them assess the differential expression of bisecting GlcNAc in breast cancer cells.
Using a series of lectins, they confirmed the differential expression of N-glycan structures in breast cancer. The next step was to identify the glycoproteins bearing bisecting GlcNAc in breast cancer. Mass spectrometry with PHA-E-agarose enrichment was used to identify the glycoproteins expressed in the cell lines. Using bioinformatics software tools, they constructed a heatmap of differential glycoprotein expression and revealed the glycoproteins with the highest differential scores and connectivity with other glycoproteins.
Among the highest-ranked glycoproteins, EGFR stood out because of its strong association with breast cancer. That’s why the research team chose to focus on the bisecting GlcNAc–EGFR pair. Immunofluorescence assays then determined the correlated expression levels of GlcNAc and EGFR while also providing insight into their locations in breast cancer cells.
The final task was to discover the functional role of bisecting GlcNAc in EGFR activity in breast cancer. The researchers achieved this by overexpressing MGAT3—the enzyme responsible for bisecting GlcNAc formation, in vitro and in vivo.
N-glycan Profile: Normal vs. Breast Cancer Cells
The starting point of the study was mapping out the N-glycan expression pattern in breast cancer. Using a human mammary epithelial cell line and three breast cancer cell lines, the ran mass spectrometry and identified 56 N-glycan structures in total. They then calculated the relative proportions of all N-glycans in all cell lines. In parallel with their previous research, they found significant downregulation of bisecting GlcNAc-bearing N-glycans in breast cancer cell lines.
To validate the findings of mass spectrometry, the researchers performed lectin microarray analysis using 37 different lectins that would recognize various glycan structures. The variation in fluorescence intensities, resulting from differential recognition in normal and cancer cells, was translated into a heatmap. Among the 14 lectins with the highest variations, PHA-E was found to correspond to bisecting GlcNAc, which was downregulated in breast cancer cells.
Lectin staining with PHA-E confirmed the suppressed expression of bisecting GlcNAc in breast cancer cells.
Which Glycoproteins Bear Bisecting GlcNAc?
After substantiating the decrease in bisecting GlcNAc expression in breast cancer, the research team explored the set of glycoproteins that formed this specific N-glycan structure. Mass spectrometry with PHA-E-agarose revealed 112 glycoproteins linked to bisecting GlcNAc. A subsequent heatmap showed that there were differences in the glycoprotein profile between normal and breast cancer cells.
Bioinformatics software tools enabled analysis of the functions carried out by these glycoproteins. Notably, they were involved in cell signaling pathways, such as EGF-activated receptor activity. These tools were also used to construct an interaction network between these glycoproteins, illustrating whether a particular glycoprotein was upregulated or downregulated. Another benefit of such network analysis was the ability to show relevant interactions with other glycoproteins, such as integrin β4.
In this interaction network, EGFR stood out not only because of its previously demonstrated role in breast cancer (9) but also because of its connection to other downregulated glycoproteins. In particular, its interaction with integrin β4 could be highlighted, as this interaction was shown to induce chemoresistance in previous studies (10).
Confirming the Expression Changes of EGFR–Bisecting GlcNAc
Shifting the focus to EGFR, the research team aimed to observe the abundance and cellular location of the EGFR-GlcNAc structure in vitro. Immunofluorescence assay confirmed that both EGFR and bisecting GlcNAc were co-localized on the breast cancer cell membrane and that each showed a reduced expression in breast cancer.
How Does Bisecting GlcNAc Affect EGFR Function?
Evaluating the implications of reduced GlcNAc–EGFR expression could help characterize the role of this glycan structure in breast cancer progression. To achieve this, the research team introduced the MGAT3 gene into two breast cancer cell lines so that they would start expressing more bisecting GlcNAc. Disease modeling in various assays showed that the increased GlcNAc expression led to a decreased EGF binding and EGFR autophosphorylation, which in turn suppressed cell proliferation and invasion.
The team successfully reproduced their findings in vivo by injecting nude mice with MGAT3-overexpressing cells, which reduced lung metastasis.
Conclusion and Possible Mechanisms
The collection of results, as well as similar studies, helped the team hypothesize possible mechanisms of how GlcNAc—or a lack thereof—altered breast cancer pathways.
According to the analysis of the suppression mechanism, overall EGFR expression did not change with increased bisecting GlcNAc expression. However, EGFR phosphorylation—the precursor of breast cancer-associated EGFR/ERK signaling—was significantly reduced. Therefore, the results suggested that bisecting GlcNAc induces structural changes in EGFR glycosylation, inhibiting its activity in EGFR/ERK signaling.
Bisecting GlcNAc is known to inhibit the transformation of high-mannose-type N-glycans to complex and hybrid N-glycans, as high mannose N-glycans showed elevated activity in breast cancer patient serum previously (11). The mass spectrometry results of the current study also displayed a significant decrease in fucosylated structures and an increase in high mannose structures, supporting the hypothesis that reduction in bisecting GlcNAc could result in branched N-glycans that maintain integrity and invasiveness in tumor cells.
Overall, the study confirmed the reduction of bisecting GlcNAc levels in breast cancer while identifying EGFR as its target glycoprotein. The altered expression of bisecting GlcNAc suggests it has potential in cancer biomarker discovery, opening a clearer channel to understanding its impact on EGFR-mediated pathways. Future studies can dig deeper into the inhibitory effect of bisecting GlcNAc on EGFR activity. In other words, targeted therapies favoring the expression of bisecting GlcNAc could potentially be employed for the prevention and treatment of breast cancer.
- Miller, Kimberly D., et al. 2019. Cancer treatment and survivorship statistics, 2019. CA Cancer J Clin.
- Sewell, Robert, et al. 2006. The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer. J Biol Chem
- Geng, Fei, et al. 2021. The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients: prognostic implications. Cell Research.
- Sato, Yuichi, et al. 2001. Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors. J Biol Chem.
- Cheng, Lanming, et al. 2020. Bisecting N-Acetylglucosamine on EGFR Inhibits Malignant Phenotype of Breast Cancer via Down-Regulation of EGFR/Erk Signaling. Frontiers in Oncology
- Tan, Zengqi, et al 2014. Altered N-Glycan expression profile in epithelial-to-mesenchymal transition of NMuMG cells revealed by an integrated strategy using mass spectrometry and glycogene and lectin microarray analysis. J Proteome Research
- Sethi, Manveen K., et al 2014. Comparative N-glycan profiling of colorectal cancer cell lines reveals unique bisecting GlcNAc and α-2, 3-linked sialic acid determinants are associated with membrane proteins of the more metastatic/aggressive cell lines.. J Proteome Research
- Allam, Heba, et al 2015. Glycomic analysis of membrane glycoproteins with bisecting glycosylation from ovarian cancer tissues reveals novel structures and functions. J Proteome Research
- Lo, Hui-Wen, Sheng-Chieh Hsu, and Mien-Chie Hung. 2006. EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization. Breast Cancer Research and Treatment
- Huafeng, Jia, et al. 2018. A cross-talk between integrin β4 and epidermal growth factor receptor induces gefitinib chemoresistance to gastric cancer. Cancer Cell International
- de Leoz, ML, et al. 2011. High-mannose glycans are elevated during breast cancer progression. Mol Cell Proteomics