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MGAT1 is a novel transcriptional target of Wnt/β-catenin signaling pathway
Izzet Akiva1, Necla Birgül Iyison2
1Department of Molecular Biology and Genetics, Institute of Graduate Studies in Science and Engineering, Boğaziçi University, İstanbul, Turkey.
Background:
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. 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).
Methods:
MGAT1 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.
Results:
In 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.
Conclusions:
Taken together, we showed for the first time that MGAT is a novel Wnt/β-catenin pathway target that has important implications for tumorigenesis.
Insights
Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) is a novel Wnt/β-catenin pathway target. Upregulation of MGAT1 in hepatocellular carcinoma cells enhances proliferation, migration, and tumor growth, implicating it in tumorigenesis.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The Wnt/β-catenin signaling pathway is crucial for vertebrate development and cancer progression.
- Aberrant Wnt/β-catenin signaling is implicated in hepatocellular carcinoma (HCC).
- Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) was identified as a putative Wnt/β-catenin target in HCC cell lines.
Purpose of the Study:
- To investigate the role of MGAT1 as a Wnt/β-catenin target in HCC.
- To determine the functional significance of MGAT1 in HCC cell proliferation, migration, and tumorigenesis.
Main Methods:
- Wnt/β-catenin pathway activation in Huh7 HCC cells using LiCl and mutant β-catenin.
- Analysis of MGAT1 expression (protein and mRNA) via Western Blotting and qRT-PCR.
- Luciferase reporter assays for MGAT1 promoter activity.
- Cell proliferation, migration (wound healing), and invasion assays.
- In vivo tumor formation studies in SCID mice.
Main Results:
- Wnt/β-catenin pathway activation led to MGAT1 upregulation at transcriptional and post-transcriptional levels.
- β-catenin overexpression enhanced MGAT1 promoter activity.
- MGAT1 overexpression in Huh7 cells increased proliferation and migration.
- MGAT1 overexpression significantly accelerated tumor growth in vivo.
Conclusions:
- MGAT1 is a novel, functionally significant target of the Wnt/β-catenin pathway in HCC.
- MGAT1 plays a critical role in promoting HCC cell proliferation, migration, and tumorigenesis.
- Targeting MGAT1 may offer a therapeutic strategy for HCC.
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