The Roles Of O-GlcNAc Modification In Breast Cancer Metastasis

O-GlcNAc, first characterized 20 years ago, is an O-linkedβ-N-acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residue. O-GlcNAc is found on numerous cytoplasm and nucleus proteins. The addition of O-GlcNAc to proteins is catalyzed by O-GlcNAc transferase (OGT) and its removal by O-GlcNAc-selective N-acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, and implies its importance in many basic cellular and disease processes.It has been demonstrated that O-GlcNAc plays important roles in some human diseases, such as diabetes and neurological disorders. Several proteins involved in tumorigenesis and cancer progression have been identified as O-GlcNAc modified proteins, such as p53 and c-Myc. However, the roles of O-GlcNAc in cancer formation and progression have not been investigated. In this study, the roles of O-GlcNAc in breast cancer formation and progression were elucidated and the mechanisms underlying O-GlcNAc induced breast cancer cells migration were investigated.To determine the roles of O-GlcNAc in breast tumor formation and metastasis, we investigated whether suppression of O-GlcNAc in the highly metastatic 4T1 cells could affect their malignances. To suppress the level of O-GlcNAc, the mouse OGT was silenced by lentiviral vector mediated short hairpin-interfering RNAs (shRNAs); on the other hand, the OGN specific inhibitor PUGNAc was used to increase the level of O-GlcNAc. We then examined whether O-GlcNAc could affect 4T1 cells proliferation and anchorage-independent growth in vitro. The results from MTT assay and cell counting assay indicated that the level of O-GlcNAc has no effect on 4T1 cells in culture, and the soft-agar assays demonstrated that O-GlcNAc didn't affect the anchorage-independent growth of 4T1 cells. Then Transwell chambers were used to assay the migration ability of the cells. The results indicated that OGT knockdown significantly suppressed the migration ability in 4T1 cells and PUGNAc treatment enhanced the cell migration ability. An experimental animal model for breast cancer metastasis was used to assay the effect of O-GlcNAc on primary tumor growth and cancer metastasis, the results demonstrated that OGT knockdown in 4T1 cells didn't affect the tumor growth but significantly suppressed the lung metastasis in vivo.Then, the mechanisms underlying O-GlcNAc induced breast cancer cells migration were investigated. WB and immunofluorence were used to assay the expression and localization of E-cadherin. The results indicated that the expression of E-cadherin was enhanced and E-cadherin was recruited to cell-cell contact sites in the OGT knockdown 4T1 cells, which were confirmed by the results from the cells pre-treated by PUGNAc. We found thatβ-catenin and p120 but not E-cadherin were modified by O-GlcNAc, which might be the causal mechanism of the inhibited binding of E-cadherin toβ-catenin and p120, and thus resulted in the suppression of the cell surface E-cadherin. To investigate the roles of E-cadherin in O-GlcNAc-associated breast cancer migration, E-cadherin knockdown and OGT/E-cadherin double knockdown breast cancer cell models were constructed.Migration assay demonstrated that E-cadherin played important roles in O-GlcNAc-associated breast cancer cell migration. ERK1/2 signaling has been implicated in the migration of numerous cell types, and some recent studies indicated that O-GlcNAc modulated ERK1/2 signaling in some cell types. Our results indicated that OGT silencing in 4T1 cells inhibited the activity (phosphorylation) of ERK1/2 and PUGNAc treatment enhanced the phosphorylation of ERK1/2. Then MAPK specific inhibitor PD98058 was used to inhibit the activity of ERK1/2 in breast cancer cells and the migration assays were carried out. The results indicated that PD98058 could markedly inhibit the phosphorylation of ERK1/2 and the cell migration ability in breast cancer cells. Taken together, these results suggested that the inhibition of ERK1/2 signaling could contribute to the modulation of migration by O-GlcNAc.This is the first report that clearly elucidates the roles and mechanisms whereby O-GlcNAc influences the malignant properties of breast cancer cells. These results suggest that the usefulness of O-GlcNAc as a target for the therapy of breast cancer.