MGAT1 is a novel transcriptional target of Wnt/ß-catenin signaling pathway.

BACKGROUND: The Wnt/ß-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/ß-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/ß-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7). METHODS: MGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/ß-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant ß-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-3ß 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. RESULTS: In this study we showed that the activation of Wnt/ß-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the ß-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/ß-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. CONCLUSIONS: Taken together, we showed for the first time that MGAT is a novel Wnt/ß-catenin pathway target that has important implications for tumorigenesis.

An in vivo RNAi mini-screen in Drosophila cancer models reveals novel potential Wnt targets in liver cancer.

BACKGROUND/AIMS: Aberrant activation of the Wnt/ß-catenin signaling, which arises from the accumulation of mutant ß-catenin in the cell, is one of the most common driving forces in hepatocellular carcinoma (HCC). We previously identified several genes that are regulated on the overexpression of ß-catenin in the HCC cell line that are suggested to be novel Wnt/ß-catenin targets playing effective roles in cancer. The aim of the present study was to elucidate the roles of these putative target genes in tumorigenesis with an in vivo analysis in Drosophila. MATERIALS AND METHODS: We selected 15 genes downregulated in two Drosophila cancer models. RESULTS: The results from the RNAi mini-screen revealed novel roles for the analyzed putative Wnt/ß-catenin target genes in tumorigenesis. The downregulation of the analyzed nine genes led to tumor formation as well as metastasis in Drosophila, suggesting a tumor suppressor function. On the other hand, the knockdown of the other two genes suppressed tumor and metastasis formations and disturbed the development of the analyzed eye tissues, indicating an oncogenic or developmental role for these genes. CONCLUSION: These findings could serve to identify novel subjects for cancer research in order to provide insight into the diagnostic and therapeutic processes of several cancer types.

Identification of IFITM3 and MGAT1 as novel interaction partners of BRI3 by yeast two-hybrid screening.

BRI3 (brain protein I3) is one of the Wnt/ß-catenin pathway target genes as indicated by the results of serial analysis of gene expression (SAGE) and microarray analyses performed in our laboratory. The Wnt/ß-catenin signaling pathway is an evolutionarily conserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, and morphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon ß-catenin activation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) cell lines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell death pathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim of finding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positive clones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells and coimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification of novel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/ß-catenin pathway.