A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-κB Activation and Apoptosis in P19 Cells

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38MAPK and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38MAPK phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/β phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis

Neurotrophin Receptor-Interacting Melanoma-Associated Antigen Protein Activates Nuclear Factor Kappa Beta Repressing Bone Morphogenic Protein Induced Apoptosis Through Macrophage Migration Inhibitory Factor Expression

Neural progenitor cell apoptosis is regulated in part by activation of the mitogen-activated protein kinase (MAPK) through the bone morphogenic pathway (BMP) pathway independent of classical SMAD signaling. Research has revealed that \u27non-canonical\u27 BMP signaling requires the formation and accumulation of a NRAGE/XIAP/Tab1/Tak1 complex at the BMP receptors present on the cell membrane, culminating in the phosphorylation of Tak1. Phosphorylated Tak1 is then able to activate the MAPK, JNK, and NF-kB pathways. The scaffolding protein NRAGE (neurotrophin receptor-interacting melanoma-associated antigen protein) contains a highly conserved MAGE Homology Domain (MHD), a less well-conserved MHD domain (MHD2), and a unique WQXPXX hexapeptide repeat region. Unlike the other MAGE family of genes whose protein expression is highly specific, NRAGE protein expression is found in most embryonic and adult tissues, suggesting a critical role in basic cellular function. While BMP-4 signaling has previously been linked to NF-KB activation, we wanted to determine if the scaffolding protein NRAGE was required. Using 293HEK cells as a model, the expression of NRAGE was systematically altered as well as other parts of the non-canonical BMP pathway. It was found that BMP-4 required the presence of NRAGE to activate the NF-kB pathway, and the overexpression of NRAGE constitutively activated NF-kB. The consequence of NF-kB activation was the dramatic increase in expression of the anti-apoptotic cytokine, macrophage migration inhibitory factor (MIF). Using P19 cells as a model for neural progenitor apoptosis, it was found that overexpression of a MIF:EGFP protein or stimulation of cells with rMIF decreased the amount of phospho-p53 and decreased the number of Annexin V and 7AAD double positive apoptotic cells. In contrast, the amount of apoptosis was increased when MIF expression was ablated. Overall, the studies herein provide novel insight into how neural progenitors escape BMP-4 induced apoptosis leaving behind a small subset of stem cells

Human NUMB6 induces epithelial-mesenchymal transition and enhances breast cancer cells migration and Invasion.

Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, β-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc