Abstract
BackgroundThe Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. To date most of the identified targets of this pathway are shown to harbor tumorigenic properties. We previously showed that Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) enzyme is among the Wnt/β-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7).MethodsMGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/β-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant β-catenin overexpression. Luciferase reporter assay was used to analyze the promoter activity of MGAT1. The mRNA levels of MGAT1 were determined by quantitative real-time PCR from Huh7 cells that were treated with either Wnt agonist or GSK-3β inhibitor. Wound healing and XTT cell proliferation assays were performed in order to determine the proliferation and migration capacities of MGAT1 overexpressing stable Huh7 cells. Finally, xenograft experiments were carried out to measure the tumor formation capacities in vivo.ResultsIn this study we showed that the activation of Wnt/β-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the β-catenin gene (CTNNB1) increased the promoter activity of MGAT1. We applied a set of complementary approaches to elucidate the functional importance of MGAT1 as a vital target of Wnt/β-catenin signaling in Huh7 cells. Our analyses related to cell proliferation and migration assays showed that in comparison to the control cells, MGAT1 expressing Huh7 cells have greater proliferative and invasive capabilities. Furthermore, the stable overexpression of MGAT1 gene in Huh7 cell lines lead to a significant increase in tumor growth rate in Severe Combined Immunodeficient (SCID) mice.ConclusionsTaken together, we showed for the first time that MGAT is a novel Wnt/β-catenin pathway target that has important implications for tumorigenesis.
Highlights
The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis
MGAT1 gene has been included among the candidate Wnt/β-catenin target genes based on the data obtained from Serial Analysis of Gene Expression (SAGE) and Microarray analyses
MGAT1 is upregulated in response to Wnt/β-catenin pathway activation The basal protein levels of MGAT1, along with a few other key proteins of cell proliferation were determined in various cell types, including carcinoma and noncarcinoma cell lines (Fig. 1a)
Summary
The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. The Wnt Signaling pathway is conserved in various organisms from worms to mammals, and play important roles in development, differentiation, cellular proliferation, morphology, motility and fate [1]. The canonical Wnt/β-catenin signaling pathway is initiated by the binding of a Wnt ligand to the Frizzled receptor. It progresses through sequential events leading to the stabilization and translocation of β-catenin into the nucleus where it interacts with the TCF/LEF family of transcription factors in order to activate target gene expression [7]. Several targets of the Wnt/β-catenin pathway were identified as genes that regulate cell proliferation, development and are involved in tumor progression [8]
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