| O-GlcNAc 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. In the previous study, our group has demonstrated that O-GlcNAc plays essencial roles in breast cancer metastasis; however, the underlying mechanism remains to be unclear.Akt (protein kinase B, PKB) is a serine/threonine protein kinase, a target of phosphoinositide 3-kinase (PI3K). In stimulated cells, inactive Akt is recruited to the membrane by PtdIns-3,4,5-P3, the lipid product of PI3K, and subsequent phosphorylation by upstream kinases locks the enzyme into the active conformation. Akt then relocalizes to either the cytosol, the nucleus, or other cellular compartments, where it phosphorylates numerous substrates. Akt has three isoforms in the cells: Akt1, Akt2 and Akt3. Akt, especially Akt1, plays important roles in cancer growth, apoptosis, migration and metastasis. Here the study discusses that the role of PI3K/Akt signalling in O-GlcNAc-mediated murine breast cancer metastasis, which might provide theoretical basis and valid target for diagnosis and therapy of breast cancer.O-GlcNAc level in 4T1 cells is suppressed by OGT silencing (4T1-shOGT1,4T1-shOGT2) and is elevated with OGA specific inhibitor PUGNAc. Immunoblotting and immunoprecipitation assay show that OGT silencing significantly enhances phosphorylation in Akt Thr308 and Akt1 Ser473, Akt2 Ser474; while PUGNAc treatment distinctly decreases the phosphorylation, accompanied with the change of O-GlcNAcylation of Akt. Transwell assay indicates that LY294002 (a specific inhibitor of PI3K) treatment notably restores the migration of 4T1-shOGT1. These results suggest that O-GlcNAc-mediated breast cancer metastasis might be related to PI3K/Akt signalling.The functions that Akt performs in many varieties of breast cancer cells have been shown to be significant different: Akt enhances metastasis in some tumor cells, while inhibits metastasis in others. To validate the above result, RNAi technology is utilized to establish two Akt1 silencing breast cancer cell lines (4T1-shAkt1-1, 4T1-shAkt1-2). It is found that Akt1 silencing remarkably attenuates the migration of murine breast cancer cells in vitro and lung metastasis in vivo without influencing the tumor growth and cell proliferation. These data demonstrate that Akt1 playes an important role in breast cancer metastasis. However, these findings also indicate that the regulation of Akt activity could be partly involved in the mechanism underlying the role of O-GlcNAc in breast cancer metastasis, and there may be other signaling pathways, through interaction with PI3K/Akt, participating in modulating breast cancer metastasis. The relationships among O-GlcNAc, PI3K/Akt and breast cancer metastasis are complicated, so the relevant molecular mechanism requires to be further investigated.Altogether, the roles of O-GlcNAc and Akt in breast tumorigenesis are explored by molecular and cell biology methods in this study. Our findings provide new insights into the mechanism by which O-GlcNAc and Akt are involved in breast cancer metastasis, suggesting the usefulness of O-GlcNAc and Akt as potential targets for the therapy of breast cancer. |
