Cross-Dysregulation of O-GlcNAcylation and PI3K/AKT/mTOR Axis in Human Chronic Diseases

The hexosamine biosynthetic pathway (HBP) and the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway are considered as nutrient sensors that regulate several essential biological processes. The hexosamine biosynthetic pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the substrate for O-GlcNAc transferase (OGT), the enzyme that O-GlcNAcylates proteins on serine (Ser) and threonine (Thr) residues. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and phosphorylation are highly dynamic post-translational modifications occurring at the same or adjacent sites that regulate folding, stability, subcellular localization, partner interaction, or activity of target proteins. Here we review recent evidence of a cross-regulation of PI3K/AKT/mTOR signaling pathway and protein O-GlcNAcylation. Furthermore, we discuss their co-dysregulation in pathological conditions, e.g., cancer, type-2 diabetes (T2D), and cardiovascular, and neurodegenerative diseases

Targeting O-GlcNAcylation to overcome resistance to anti-cancer therapies

International audienceIn cancer cells, metabolic reprogramming is associated with an alteration of the O -GlcNAcylation homeostasis. This post-translational modification (PTM) that attaches O -GlcNAc moiety to intracellular proteins is dynamically and finely regulated by the O -GlcNAc Transferase (OGT) and the O -GlcNAcase (OGA). It is now established that O -GlcNAcylation participates in many features of cancer cells including a high rate of cell growth, invasion, and metastasis but little is known about its impact on the response to therapies. The purpose of this review is to highlight the role of O -GlcNAc protein modification in cancer resistance to therapies. We summarize the current knowledge about the crosstalk between O -GlcNAcylation and molecular mechanisms underlying tumor sensitivity/resistance to targeted therapies, chemotherapies, immunotherapy, and radiotherapy. We also discuss potential benefits and strategies of targeting O -GlcNAcylation to overcome cancer resistance

Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt*

International audienceThe 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: ␣-actinin, valosin-containing protein, inhibitor B kinase, mortalin, tubulin ␤, cytokeratin 8, actin, 14-3-3, 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 coimmunoprecipitates 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. Molecular & Cellular Proteomics 6:114-124, 2007

Tumor necrosis factor receptor-associated death domain protein is involved in the neurotrophin receptor-mediated antiapoptotic activity of nerve growth factor in breast cancer cells

The common neurotrophin receptor p75NTR has been shown to initiate intracellular signaling that leads either to cell survival or to apoptosis depending on the cell type examined; however, the mechanism by which p75NTR initiates its intracellular transduction remains unclear. We show here that the tumor necrosis factor receptor-associated death domain protein (TRADD) interacts with p75NTR upon nerve growth factor (NGF) stimulation. TRADD could be immunodetected after p75NTR immunoprecipitation from MCF-7 breast cancer cells stimulated by nerve growth factor. In addition, confocal microscopy indicated that NGF stimulation induced the plasma membrane localization of TRADD. Using a dominant negative form of TRADD, we also show that interactions between p75NTR and TRADD are dependent on the death domain of TRADD, thus demonstrating its requirement for binding. Furthermore, the p75NTR-mediated activation of NF- B was inhibited by transfection with a dominant negative TRADD, resulting in an inhibition of NGF antiapoptotic activity. These results thus demonstrate that TRADD is involved in the p75NTR-mediated antiapoptotic activity of NGF in breast cancer cells

Nerve growth factor overexpression and autocrine loop in breast cancer cells

We show here that nerve growth factor (NGF), the canonical neurotrophic factor, is synthesized and released by breast cancer cells. High levels of NGF transcript and protein were detected in breast cancer cells by reverse transcription-PCR, Western blotting, ELISA assay and immunohistochemistry. Conversely, NGF production could not be detected in normal breast epithelial cells at either the transcriptional or protein level. Confocal analysis indicated the presence of NGF within classical secretion vesicles. Breast cancer cell-produced NGF was biologically active, as demonstrated by its ability to induce the neuronal differentiation of embryonic neural precursor cells. Importantly, the constitutive growth of breast cancer cells was strongly inhibited by either NGF-neutralizing antibodies or K-252a, a pharmacological inhibitor of NGF receptor TrkA, indicating the existence of an NGF autocrine loop. Together, our data demonstrate the physiological relevance of NGF in breast cancer and its potential interest as a marker and therapeutic target

Nerve growth factor receptors and signaling in breast 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 p75, 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 p75 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 Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

International audienceThe serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA ( ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappa B and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling

Nerve growth factor receptor TrkA signaling in breast cancer cells involves Ku70 to prevent apoptosis.

International audienceThe nerve growth factor (NGF)-tyrosine kinase receptor TrkA plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation, and proliferation. In breast cancer cells, TrkA stimulation results in the activation of cellular growth, but downstream signaling largely remains to be described. Here we used a proteomics-based approach to identify partners involved in TrkA signaling in breast cancer cells. Wild type and modified TrkA chimeric constructs with green fluorescent protein were transfected in MCF-7 cells, and co-immunoprecipitated proteins were separated by SDS-PAGE before nano-LC-MS/MS analysis. Several TrkA putative signaling partners were identified among which was the DNA repair protein Ku70, which is increasingly reported for its role in cell survival and carcinogenesis. Physiological interaction of Ku70 with endogenous TrkA was induced upon NGF stimulation in non-transfected cells, and co-localization was observed with confocal microscopy. Mass spectrometry analysis and Western blotting of phosphotyrosine immunoprecipitates demonstrated the induction of Ku70 tyrosine phosphorylation upon NGF stimulation. Interestingly no interaction between TrkA and Ku70 was detected in PC12 cells in the absence or presence of NGF, suggesting that it is not involved in the initiation of neuronal differentiation. In breast cancer cells, RNA interference indicated that whereas Ku70 depletion had no direct effect on cell survival, it induced a strong potentiation of apoptosis in TrkA-overexpressing cells. In conclusion, TrkA signaling appears to be proapoptotic in the absence of Ku70, and this protein might therefore play a role in the long time reported ambivalence of tyrosine kinase receptors that can exhibit both anti- and eventually proapoptotic activities