Proteomic analysis and abrogated expression of O-GlcNAcylated proteins associated with primary breast cancer
Voraratt Champattanachai1, Pukkavadee Netsirisawan, Parunya Chaiyawat
1Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand. voraratt@cri.or.th
Abstract:
O-GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc-modified proteins might be one of the vital malignant characteristics of cancer.
Insights
O-GlcNAcylation, a protein modification linked to glucose metabolism, is elevated in malignant breast tumors. This suggests aberrant O-GlcNAcylation may drive cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- O-GlcNAcylation is a dynamic post-translational modification (PTM) regulating nuclear and cytoplasmic proteins.
- It is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase, impacting glucose metabolism and disease, including cancer.
Purpose of the Study:
- To investigate the role and extent of O-GlcNAcylation in breast cancer.
- To identify specific O-GlcNAc-modified proteins associated with malignancy.
Main Methods:
- 2D O-GlcNAc immunoblotting and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) were employed.
- OGT knockdown was performed to assess functional impact.
Main Results:
- O-GlcNAcylation levels were significantly increased in malignant breast tumors compared to benign ones, correlating with higher OGT expression.
- LC-MS/MS identified 29 proteins, with seven uniquely associated with O-GlcNAcylation in cancer, including those involved in the Warburg effect, stress response, RNA metabolism, gene expression, and cytoskeleton.
- Decreased O-GlcNAcylation via OGT knockdown inhibited anchorage-independent growth in vitro.
Conclusions:
- Aberrant protein O-GlcNAcylation is closely associated with breast cancer.
- The abnormal modification of identified O-GlcNAc-proteins may represent a critical characteristic of cancer malignancy.
