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  • N-Benzylacetamidine hydrobromide Examination of Wnt signalin

    2019-08-13

    Examination of Wnt signaling via, for example, complexes of frizzled (Fz) and lipoprotein-receptor-related protein (LRP) receptors has been hindered by difficulties in producing Wnt ligands due to their inherent hydrophobicity [20]. This bottleneck was recently bypassed by constructing highly soluble synthetic dimeric Wnt agonists composed of the C-terminal domain of Dickkopf-related protein (DKK)1 binding to LRP5/LRP6 and a scFv binding to Fz1, 2, 5, 7 or 8, or the B12 protein from Bacillus halodurans binding to Fz5 or 6 [20]. Of note, DKK1 is a Wnt antagonist but becomes an agonist in the heterodimeric fusion proteins (heterodimerizers) (Figure 1). Consequently, these synthetic Wnt agonists induce LRP/Fz receptor dimerization and activation of signal transduction. Furthermore, using adenoviral expression of synthetic Wnt agonists in mice, a cooperative role of Wnt signaling and R-spondin (RSPO) ligands in improving intestinal stem-cell niche regeneration was demonstrated by histological analysis showing villus hyperproliferation and lengthening 20, 21. Wnt depletion was rescued using these synthetic Wnt agonists resulting in the maintenance of Lgr5+ intestinal stem N-Benzylacetamidine hydrobromide and crypt formation [21]. Thus, this study demonstrated clear implications of Wnt and RSPO ligands in the synergistic control of tissue regeneration. Indeed, synthetic cytokines can surpass their endogenous counterparts in many regards, including receptor affinity, activity, and stability, and may even display novel selected functions [12]. Moreover, their activity may not be limited to specific cells or cell types. Thus, synthetic cytokines are advantageous in that they can activate all cells expressing appropriate corresponding receptors.
    Synthetic Cytokines for Cell-Targeted Therapies Cytokines may be promising drugs in cancer therapy, increasing cancer cell apoptosis either directly or indirectly, via immunotherapy. However, cytokines are highly pleiotropic and may exert opposite effects on target cells of different origins; thus, often preventing their systemic administration due to severe side effects 22, 23. It is therefore desirable to design synthetic cytokines for cell-targeted therapies, which are generally considered to be more effective and leading to fewer side effects than nontargeted therapies.
    Constitutively Active Cytokine Receptor Variants A variety of disease-causing gain-of-function receptor mutants have been identified in many structural domains of all cytokine receptors classes (Figure 2, Table S1 in the supplemental information online). Receptor tyrosine kinases are frequently mutated in cancer (https://cancer.sanger.ac.uk/cosmic), among them the human epithelial growth factor receptor (EGFR, HER, and ErbB) family [65]. An EGFR mutant (EGFRvIII) with deletion of 268 amino acids in the extracellular domain is constitutively active, and WT EGFR plays a critical role in activating EGFRvIII [66]. One study showed that activated WT EGFR promoted EGFRvIII activation and tyrosine phosphorylation by demonstrating that EGFRvIII contributed to its own activation upon induction of heparin-binding (HB)-EGF expression; a ligand for WT EGFR [67]. Moreover, approximately 40% of HER2-positive tumors express a constitutively active C-terminal fragment of HER2 (p95Her2), which forms cysteine-disulfide-bridged homodimers [68]. Complete extracellular truncations have not been described for other cytokine receptors in humans, suggesting that this might not generally lead to constitutive receptor activation. Activating short in-frame deletions in the homologous loop (β3-αC) of the EGFR (ΔELREA/delE746-A750, ΔLREAT, and ΔLRE) and HER2 (ΔLRENT) kinase domains has also been identified in patient samples of varying tumor types [69]. Expression of EGFR β3-αC deletions (ΔELREA and ΔLREAT) in human cells has resulted in increased receptor phosphorylation and pERK levels compared with WT EGFR, and heterodimerization with other HER family members is required for activation of HER2. Accordingly, cotransfection of human cells with HER2 (ΔLRENT) and HER3 induced a significant increase in HER3 activation compared with WT HER2 [69]. HER4 is unique among this family because receptor activation by ligand binding induces ectodomain shedding within the juxtamembrane region by a disintegrin and metalloproteinase (ADAM) 17 and subsequent processing by the γ-secretase, with release of the intracellular domain of HER4 (4ICD) acting as a transcriptional coactivator to regulate STAT5 target genes 70, 71. 4ICD is required for estrogen-stimulated growth of human breast cancer cells in vitro [72]. The majority of HER4 mutations are located in exons encoding the extracellular domain of the receptor [73] and constitutive activity has been demonstrated after transient transfection of human cells N-Benzylacetamidine hydrobromide [74].