Abstract
Inhibitor of β-catenin and TCF (ICAT) inhibits β-catenin transcriptional activity by competing with T-cell factor/lymphoid enhancer factor. We documented high ICAT levels in human melanoma cells, in which β-catenin signaling is frequently deregulated, finding a correlation with the capacity to form metastases in nude mice. Ectopic expression of ICAT in melanoma cells did not affect their proliferation but increased cell motility and Matrigel invasion of metastatic cells in a manner relying upon stable ICAT-β-catenin interaction. This effect was associated with conversion of an elongated/mesenchymal phenotype to a round/amoeboid phenotype in the absence of similar effects on elongated morphology of nonmetastatic melanoma cells. Transition from mesenchymal to amoeboid movement was associated with decreased levels of NEDD9 and activated Rac1, a positive regulator of mesenchymal movement. Ectopic ICAT promoted colonization of melanoma cells in the lungs of nude mice, suggesting an increase in metastatic potential. Together, our results showed that by downregulating Rac signaling in metastatic melanoma cells, ICAT increased their invasive motility by promoting a morphologic variation that facilitates a favorable adaptation to their microenvironment.
Highlights
Introduction bCatenin is a multifunctional protein exhibiting various functions depending on its cellular localization and interactions with diverse protein ligands
High Inhibitor of b-catenin and TCF (ICAT) level expression in human melanoma cell lines is associated with formation of tumor metastases in nude mice
Because cell motility and invasive behavior are essential steps of the metastatic process [33], we investigated whether increased ICAT expression could enhance the invasiveness of melanoma cells
Summary
Cell lines The human melanoma cell lines Lu1205 and WM852, obtained from Dr M. Mouse monoclonal against human ICAT (clone 5C6) and phalloidin-FITC antibodies were from Sigma-Aldrich. The standard methods used for cell lysis, Western blotting, affinity precipitation, cell immunofluorescence, and in-gel zymography are described in Supplementary Methods. For time-lapse microscopy, cells were first transiently transfected with ICAT-Flag cDNA constructs, pGFP empty vector as control or siRNAs. Cells were seeded sparsely to minimize cell–cell interactions and migration analysis was performed using an inverted microscope equipped for fluorescence imaging (Life Imaging Services). Inverted Matrigel invasion assays were performed as described previously [25]. Mice were injected intraperitoneally with 10 mL/g of body weight of the D-Luciferin Firefly solution (15 mg/mL; Caliper Life Sciences) and anesthetized before imaging. Single-cell migration and mRNA content differences were assessed using the Mann–Whitney test (two-tailed). All value sets were tested for normality using a Shapiro–Wilk normality test
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