Breast cancer-specific mutations in CK1ε inhibit Wnt/β-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration

Introduction Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1ε). Because CK1ε is a crucial regulator of the Wnt signaling cascades, we determined how these CK1ε mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines. Methods We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1ε mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1ε affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1ε in these processes. Results In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1ε, is involved in positive regulation of the CK1ε activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1ε failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1ε mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1ε mutants acted as loss-of-function in the Wnt/β-catenin pathway, and that CK1ε mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7. Conclusions In summary, these data suggest that the mutations of CK1ε found in breast cancer can suppress Wnt/β-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1ε, which are correlated with decreased phosphorylation activities of mutated forms of CK1ε both in vitro and in vivo, interfere with positive autophosphorylation at Thr 4

Evaluation of Parameters Critical for Observing Nucleic Acids Inside Living Xenopus laevis Oocytes by In-Cell NMR Spectroscopy

In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, we show that isotopically labeled DNA and RNA can be observed inside the eukaryotic environment of Xenopus laevis oocytes by in-cell NMR spectroscopy. One limiting factor for the observation of nucleic acids in Xenopus oocytes is their reduced stability. We demonstrate that chemical modification of DNA and RNA can protect them from degradation and can significantly enhance their lifetime. Finally, we show that the imino region of the NMR spectrum is devoid of any oocyte background signals enabling the detection even of isotopically nonlabeled molecules

Microenvironmental interactions between endothelial and lymphoma cells: a role for the canonical WNT pathway in Hodgkin lymphoma

The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell–cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and β-catenin in cHL cells. Furthermore, LEF-1- and β-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions

Guanine Bases in DNA G-Quadruplex Adopt Nonplanar Geometries Owing to Solvation and Base Pairing

The effect of base pairing and solvation on pyramidalization of the glycosidic nitrogen found in the residues of parallel G-quadruplex with NDB ID UDF062 is analyzed and explained with theoretical calculations. The extent of the pyramidalization depends on the local geometry of the 2′-deoxyguanosine residues, namely on their glycosidic torsion and sugar pucker, which are predetermined by the 3D-architecture of G-quadruplex. Pyramidal inversion of the glycosidic nitrogen found in 2′-deoxyguanosines of G-quadruplex is induced owing to site-specifically coordinated solvent. Different adiabatic structural constraints used for fixing the base-to-sugar orientation of 2′-deoxyguanosine in geometry optimizations result in different extents of pyramidalization and induce pyramidal inversion of the glycosidic nitrogen. These model geometry constraints helped us analyze the effect of real constraints represented by explicit molecular environment of selected residues of the G-quadruplex. The maximal extent of the glycosidic nitrogen pyramidalization found in the high-resolution crystal structure corresponds to the calculation to deformation energy of only 1 kcal mol–1. The out-of-plane deformations of nucleobases thus provide a way for compensating the site-specific external environmental stress on the G-quadruplex

Structural interpretation of J coupling constants in guanosine and deoxyguanosine: modeling the effects of sugar pucker, backbone conformation, and base pairing

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