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p120-catenin (p120) contains a large central armadillo repeat domain, via which it binds to E‑cadherin to stabilize the latter, thereby regulating cell‑to‑cell adhesion. A previous study by our group demonstrated that O‑linked N‑acetylglucosamine (O‑GlcNAc) is involved in the regulation of the interaction between p120 and E‑cadherin. As O‑GlcNAc transferase (OGT) is able to directly bind to the majority of its target proteins, the present study hypothesized that OGT may additionally regulate the formation of the E‑cadherin/catenin complex independent of its catalytic activity. To verify this hypothesis, a catalytically inactive OGT mutant was expressed in H1299 cells, and its effects on the formation of the E‑cadherin/catenin complex were assessed. A cytoskeleton‑binding protein extraction assay confirmed that OGT inhibited the formation of the E‑cadherin/catenin complex independent of its catalytic activity. In addition, co‑immunoprecipitation and pull‑down assays were used to evaluate the interaction between OGT and p120. Immunoblotting indicated that OGT was able to directly bind to p120. To determine the domain of p120 involved in binding to OGT, a series of deletion mutants of p120 were constructed and subjected to protein binding assays by pull‑down assays. Immunoblotting showed that OGT bound to the regulatory and armadillo domains of p120, which might interfere with the interaction between p120 and E‑cadherin. Finally, OGT, p120 and E‑cadherin cytoplasmic domains (ECD) were recombinantly expressed in BL21 (DE3) recombinant E. coli cells, and a glutathione S‑transferase (GST) pull‑down assay was performed to assess the interactions among the purified recombinant proteins. Immunoblotting indicated that maltose‑binding protein (MBP)‑OGT inhibited the binding of His‑p120 to GST‑ECD in a dose‑dependent manner. All of these results suggested that OGT inhibited the formation of the E‑cadherin/catenin complex through reducing the interaction between p120 and E‑cadherin. The present study provided a novel underlying mechanism of the regulation of the interaction between p120 and E‑cadherin, and thus E‑cadherin‑mediated cell‑cell adhesion, which has essential roles in cancer development and progression.

O-GlcNAc is an O-linked ?-N-acetylglucosamine moiety attached to the side-chain hydroxyl of a serine or threonine residue in numerous cytoplasmic and nuclear proteins. In this study, we detected the level of O-GlcNAc in prostate, liver and pancreatic cancer tissues, and found that the global O-GlcNAc modification also known as O-GlcNAcylation, is specifically increased in prostate cancer tissues compared to corresponding adjacent tissues. In addition, we found that global O-GlcNAcylation is increased in prostate cancer cells and not in benign prostatic hyperplasia (BPH) epithelial cells. O-GlcNAc enhanced the anchorage-independent growth and the migratory/invasive ability of prostate cancer cells. More importantly, we provide here, for the first time to the best of our knowledge, direct evidence that increased O-GlcNAcylation induces malignant transformation of nontumorigenic (BPH) cells. Furthermore, our study suggested that inhibiting the formation of the E-cadherin/catenin/cytoskeleton complex may underly the O-GlcNAc-induced prostate cancer progression. Overall, these findings indicated that O-GlcNAcylation is increased in prostate, but not in liver and pancreatic cancer tissues, and that O-GlcNAc can enhance the malignancy of prostate cancer cells.

GlcNAcylation, a dynamic posttranslational modification, is involved in a wide range of biological processes and some human diseases. Although there is emerging evidence that some tumor-associated proteins are modified by GlcNAcylation, the role of GlcNAcylation in tumor progression remains unclear. Here, we show that GlcNAcylation enhances the migration/invasion of breast cancer cells in vitro and lung metastasis in vivo. The decrease of cell surface E-cadherin is the molecular mechanism underlying GlcNAcylation-induced breast cancer metastasis. p120 and β-catenin, but not E-cadherin, are GlcNAcylated; the GlcNAcylation of p120 and β-catenin might play roles in the decrease of cell surface E-cadherin. Moreover, immunohistochemistry analysis indicated that the global GlcNAcylation level in breast tumor tissues is elevated significantly as compared with that in the corresponding adjacent tissues; further, GlcNAcylation was significantly enhanced in metastatic lymph nodes compared with their corresponding primary tumor tissues. This is the first report to clearly elucidate the roles and mechanisms whereby GlcNAcylation influences the malignant properties of breast cancer cells. These results also suggest that GlcNAcylation might be a potential target for the diagnosis and therapy of breast cancer. Cancer Res; 70(15); 6344–51. ©2010 AACR.