O-GlcNAcylation Plays An Essential Role In Breast And Lung Cancer Progression

O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation of specific substrates has been shown to modulate diverse protein functions, including protein-protein interactions, protein turnover, subcellular localization and changes in activity. O-GlcNAcylation is involved in a wide range of biological processes, such as signal transduction, transcription, cell cycle progression, and metabolism. O-GlcNAcylation not only has a role in normal biological processes, but its faulty regulation is also involved in some human diseases such as diabetes, neurologic disorders and cardiovascular disease. Also, the effect of O-GlcNAcylation in diabetes, neurologic disorders and cardiovascular was studied extensively. Several tumor-associated proteins have also been identified as O-GlcNAcylated proteins. For instance, c-Myc is O-GlcNAcylated mainly on Thr58, which is a known hotspot for mutation in lymphoma and is a major GSK3 phosphorylation site. The O-GlcNAcylation of c-Myc at Thr58 could competitively inhibit phosphorylation and thus suppress the proteasome-mediated degradation of c-Myc. In addition, the p53 protein is a transcription factor essential for the prevention of cancer formation, and O-GlcNAcylation at Ser149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Although the effects of O-GlcNAcylation on some tumor-associated proteins have been elucidated, the roles of O-GlcNAcylation in cancer progression have not been investigated. In this study, the effects of O-GlcNAcylation on the malignant properties of breast and lung cancer and the molecular mechanisms underlying O-GlcNAcylation-mediated breast and lung cancer migration and metastasis are investigated.First of all, to learn the correlation between O-GlcNAcylation and cancer formation and metastasis, the O-GlcNAcylation level in tumor tissues and the corresponding metastatic lymph nodes tissues are detected by immunohistochemistry analysis. The results indicate that O-GlcNAcylation is markedly enhanced in breast and lung tissues compared with that in the adjacent tissues. This indicates O-GlcNAcylation is positive correlation with tumor formation and metastasis.Secondly, the effects of O-GlcNAcylation on the progression of breast and lung cancer are studied. The O-GlcNAcylation level in breast and lung cells is suppressed by lentiviral-mediated OGT silencing and elevated with OGA-specific inhibitor PUGNAc. Although OGT silencing and PUGNAc treatment do not affect the proliferation of breast and lung cancer cells, O-GlcNAcylation increase enhances the colony formation ability. OGT silencing significantly inhibits breast and lung caner cells migration, whereas OGA inhibition markedly increases cell migration. Also, the elevation of O-GlcNAcylation suppresses intercellular adhesion of cancer cells. The effects of O-GlcNAcylation on the tumor formation and metastasis of breast cancer cells are further examined in vivo by the mouse model. Consistent with the results in vitro, OGT silencing dramatically reduces the number of visible metastatic nodules on the lung surface of tumor-bearing mice but does not have a significant effect on primary tumor weight. These results show O-GlcNAcylation promotes cancer progression.Last, the molecular mechanisms underlying O-GlcNAcylation-mediated breast and lung cancer migration and metastasis are explored. Taking into account the effect of O-GlcNAc in intercellular adhesion and the important role of E-cadherin in cancer malignance, the expression and distribution of the components of E-cadherin/catenin complex were examined. OGT silencing significantly increases E-cadherin expression of breast and lung caner cells, whereas OGA inhibition markedly decreases the expression of E-cadherin. To determine whether O-GlcNAcylation altered cancer cell migration and metastasis via E-cadherin, E-cadherin is silenced in mouse breast caner cells. Cell migration assays showed that the migration ability was negatively correlated with the expression of E-cadherin. Afterward, Ctrl and E-cadherin silencing cells were treated by PUGNAc and PUGNAc markedly enhanced the migration of Ctrl cells but only slightly enhanced the migration of E-cadherin silencing cells. Furthermore, cell migration assays results of OGT/E-cadherin double-knockdown cells are the same as the OGA inhibitor. Then we investigate the role of E-cadherin in the suppression of lung metastasis induced by OGT silencing in mouse breast cancer cells. The results indicate that silencing of E-cadherin in OGT knockdown cells do not affect the primary tumor weights but significantly compensated for the inhibition of lung metastasis induced by OGT knockdown. Therefore, a conclusion is made that the decrease of E-cadherin is one of the main mechanisms underlying the O-GlcNAcylation-induced cancer cell migration and metastasis. Additionally, to clarify the mechanisms underlying the decrease of cell surface E-cadherin induced by O-GlcNAcylation, we first detect the transcriptional expression of E-cadherin in OGT silencing and PUGNAc-treated cells. The changes of O-GlcNAcylation level have no effect on E-cadherin mRNA expression. However, we find O-GlcNAcylation affects the E-cadherin degradation. OGT silencing significantly increases the half-life of E-cadherin, whereas PUGNAc treatment markedly decreases the half-life of E-cadherin. So, O-GlcNAcylation induces the decrease of E-cadherin by promoting the degradation rate. The O-GlcNAcylation of E-cadherin,β-catenin, and p120 is detected by O-GlcNAcylation-specific antibodies (RL2 and CTD110.6) and sWGA-agarose (sWGA recognizes terminalβ-GlcNAc residue). O-GlcNAcylation of E-cadherin could not be detected by using these methods. p120 could be stained by RL2 but not by CTD110.6. Additionally, p120 could be precipitated by sWGA-agarose and the precipitation was diminished by 500 mmol/L of GlcNAc. O-GlcNAcylation of p120 is further identified by the metabolic methods. As previously reported, O-GlcNAcylation ofβ-catenin was detectable by RL2, CTD110.6, and sWGA. OGT silencing enhances the protein interaction between E-cadherin and P120, but notβ-catenin , whereas PUGNAc suppresses the interaction. Therefore, O-GlcNAcylation of p120 may play important roles in its binding to E-cadherin and E-cadherin degradation. This study is the first report to evidently elucidate the roles and mechanisms whereby O-GlcNAcylation influences the malignant properties of cancer cells. Additionally, these results suggest that O-GlcNAcylation may be a potential target for diagnosis and therapy of cancer.