Dysfunctions in Wnt signaling increase β‐catenin stability and are associated with cancers, including colorectal cancer. In addition, β‐catenin degradation is decreased by nutrient‐dependent O ‐GlcNAcylation. Human colon tumors and colons from mice fed high‐carbohydrate diets exhibited higher amounts of β‐catenin and O ‐GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O ‐GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β‐catenin. By ETD‐MS/MS, we identified 4 O ‐GlcNAcylation sites at the N terminus of β‐catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β‐catenin reduced O ‐GlcNAcylation by 75%. Interestingly, elevating O ‐GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β‐catenin stability. Analyses of β‐catenin O ‐GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β‐catenin degradation rate. Finally, inhibiting O ‐GlcNAcylation decreased the β‐catenin/α‐catenin interaction necessary for mucosa integrity, whereas O ‐GlcNAcase silencing improved this interaction. These results suggest that O ‐GlcNAcylation regulates not only the stability of β‐catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O ‐GlcNAcylation of β‐catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.—Olivier‐Van Stichelen, S., Dehennaut, V., Buzy, A., Zachayus, J.‐L., Guinez, C., Mir, A‐M., El Yazidi‐Belkoura, I., Copin, M.‐C., Boureme, D., Loyaux, D., Ferrara, P., Lefebvre, T. O ‐GlcNAcylation stabilizes β‐catenin through direct competition with phosphorylation at threonine 41. FASEB J. 28, 3325–3338 (2014). www.fasebj.org

Drug resistance related to aberrant glycosylation in colorectal cancer Drug resistance related to aberrant glycosylation in colorectal cancer

Nerve growth factor (NGF) has long been known for its effects on neuronal cell survival and differentiation. This prototypical neurotrophic factor stimulates neurons through two distinct classes of membrane receptors: the TrkA tyrosine kinase receptor, and the tumor necrosis factor receptor family member p75NTR, also known as the common neurotrophin receptor. Somewhat surprisingly, there is a growing body of evidence indicating that NGF is also a major stimulator of breast cancer cell growth. Both the survival and proliferation of breast cancer cells are strongly stimulated by NGF, mediated by TrkA and p75NTR respectively, utilising signaling pathways similar to those described for neurons. In addition, although NGF is produced by breast cancer cells, it is not in normal breast epithelial cells, giving rise to an autocrine stimulation of tumor growth. Therefore, NGF receptors and signaling are thus looking increasingly promising as potential drug targets for breast cancer.

The serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: α -actinin, valosin-containing protein, inhibitor κB kinase, mortalin, tubulin β, cytokeratin 8, actin, 14-3-3σ, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt co-immunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling.