Rôle de ICAT (inhibitor of β-catenin and TCF4) dans le développement normal et pathologique des mélanocytes

Abstract : Β-catenin is the central protein of the Wnt signaling pathway, which is involved in many physiological processes including normal melanocyte development. Few direct negative regulators of β-catenin have been described so far and their physiological role is still unclear. One of them, ICAT (inhibitor of β-catenin and TCF-4) has been identified in a yeast two-hybrid screen by using the Armadillo (ARM) region of β-catenin as bait. This small protein of 81 amino-acids, encoded by the highly conserved CTNNBIP1 gene, binds to β-catenin through ARM repeats 5 to 12, preventing its interaction with TCF4 and thus, repressing TCF/LEF-β-catenin transcriptional activity in vitro. Total invalidation of ICAT in mouse leads to premature death and cranio-facial anomalies, suggesting an important role in neural-crest cell differentiation. In order to determine the possible role of ICAT in melanoblasts development, ICAT -/- embryos, on a Dct::LacZ background were obtained. We observed that the total invalidation of the ICAT gene does not affect the number of melanoblasts and their localization at embryonic stages E13.5, E15.5 and E18.5, indicating that ICAT alone does not play a significant role in the proliferation and migration of melanoblasts during mouse development. The Wnt signaling pathway is deregulated in 30% of melanomas, so we found it essential to study the role of ICAT in the formation and progression of melanoma. We observed that high ICAT levels in a panel of human melanoma cell lines correlated with their capacity to form metastases in nude mice and was associated, in some melanoma patients, with reduced distant metastasis-free survival. Ectopic expression of ICAT in various melanoma cell lines had no effect on their proliferation but increased their in vitro motility in 2D and matrigel invasion in 3D. These in vitro effects required stable protein interaction between ICAT and β-catenin. At the cellular level, ICAT promoted switching of metastatic melanoma cells from an elongated/mesenchymal towards a round/amoeboid phenotype but did not affect the elongated morphology of non-metastatic melanoma cells. Transition from a mesenchymal to an amoeboid movement was associated with decreased production of the scaffold protein NEDD9 and decreased levels of Rac1-GTP, a positive regulator of mesenchymal movement. In vivo, ectopic ICAT expression increased lung colonization by melanoma cells in nude mice. Our results indicate that ICAT-induced down-regulation of Rac signaling can increase motility and invasiveness of metastatic cells by promoting morphological variability allowing tumor cells to adapt to their microenvironment. We show, for the first time, that ICAT is a new potential modulator of melanoma cells invasion. Based on a three-dimensional study of ICAT in complex with β-catenin we understood how ICAT competes with members of the TCF/LEF family at the nuclear level. By site directed mutagenesis, we demonstrated that D66, F71 and E75 residues (C-terminal domain of ICAT) are involved in the competition with LEF1 but are not essential in the formation of a stable ICAT/β-catenin complex, while Y15, K19, V22 residues (N-terminal domain) are essential for the formation of this complex.
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Mélanie Domingues. Rôle de ICAT (inhibitor of β-catenin and TCF4) dans le développement normal et pathologique des mélanocytes. Médecine humaine et pathologie. Université René Descartes - Paris V, 2014. Français. ⟨NNT : 2014PA05T011⟩. ⟨tel-01160394⟩

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