N-acetylglucosaminyltransferase V drives colorectal cancer metastasis by facilitating ZO-1 ubiquitination and degradation

In a recent study published in “Cancer Cell International,” Yueping Zhan and others explored the role of N-acetylglucosaminyltransferase V (MGAT5) in colorectal cancer (CRC) and its mechanism in promoting cancer cell metastasis. The publication of this paper is driven by the urgent need to study CRC due to its high mortality and metastatic characteristics, as it is the second leading cause of cancer-related deaths globally. Despite significant progress in diagnosis and treatment in recent years, CRC metastasis remains the primary cause of poor prognosis in patients. Therefore, clarifying the molecular mechanisms driving CRC progression is crucial for developing new therapeutic strategies.

Research Background and Objectives CRC metastasis is the leading cause of high cancer mortality, with approximately 22% of CRC patients diagnosed with metastatic lesions. Researchers are increasingly focusing on the role of epithelial-mesenchymal transition (EMT) in cancer invasion and metastasis. EMT causes cells to lose polarity and disrupt intercellular connections, thereby facilitating the transition of cells from non-motile epithelial cells to highly motile mesenchymal cells. Studies have found that tight junction protein 1 (ZO-1) plays a crucial role in the initiation and spread of cancer, and its functional disorder may alter the cellular environment, ultimately promoting tumor invasion and metastasis.

Research shows that N-glycosylation modification is significant in regulating EMT, and MGAT5 is a key enzyme catalyzing the synthesis of β1,6-GlcNAc-branched multi-antennary N-glycan structures. These multi-antennary N-glycan structures are increasingly expressed in various tumor tissues, including CRC, and their high expression is associated with lymph node metastasis and poorer survival prognosis. However, the specific mechanism of MGAT5 in CRC metastasis is not yet fully understood.

Research Source This study was conducted by the research team at the Clinical Experimental Medicine Center of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, and published in the 2024 issue of “Cancer Cell International.” The main research authors include Yueping Zhan and Chenjun Huang, who significantly contributed to the study design and data analysis.

Research Process The study explored the molecular mechanism of MGAT5 in CRC through a multi-step experimental process, particularly focusing on its relationship with ZO-1. The study used CRC datasets from the TCGA and GEO databases, confirmed via bioinformatic analysis that high MGAT5 expression is associated with EMT and poor prognosis. Subsequently, Researchers validated its function by constructing CRC cell lines overexpressing and knocking down MGAT5.

1. Dataset and Sample Collection:

   • RNA transcriptomics data from the TCGA and GTEx platforms were used to analyze MGAT5 expression levels in various cancers.
   • Two independent microarray datasets (GSE32323 and GSE17537) containing RNA sequencing data and clinical information of CRC samples were obtained from the GEO database.
   • Colorectal cancer tissue microarrays (TMA), including 69 cancer tissues and 55 corresponding adjacent tissues, were acquired from Wellbio Technology, Shanghai.

2. Cell Line Experiments:

   • MGAT5 overexpressing cell lines of SW480 and DLD1 CRC cells were constructed, and MGAT5 expression was knocked down using siRNA. RT-qPCR and Western blot (WB) were used to verify transfection efficiency.
   • Colony formation assay, Edu proliferation assay, wound healing assay, and Transwell migration assays evaluated the impact of MGAT5 on CRC cell proliferation and migration.

3. ZO-1 Expression and Degradation Mechanism:

   • Immunofluorescence (IF) was used to detect the colocalization of MGAT5 and ZO-1, finding that ZO-1 expression decreased in MGAT5 high-expression cells and colocalization of both was observed at the cell membrane.
   • Co-immunoprecipitation (Co-IP) confirmed the interaction between MGAT5 and ZO-1. Further research showed that MGAT5 promotes ZO-1 ubiquitination through multi-antennary N-glycosylation, accelerating ZO-1 degradation.

4. Clinical Validation:

   • In the immunohistochemistry (IHC) of CRC tissue microarrays, MGAT5 expression levels were significantly higher than in adjacent non-cancerous tissues. Kaplan-Meier survival curve analysis showed that patients with high MGAT5 expression had significantly lower overall survival rates than those with low expression.
   • Immunofluorescence experiments further validated significantly reduced ZO-1 expression in MGAT5 high-expression tumor tissues, showing a negative correlation between the two.

Research Results The results showed that MGAT5 is highly expressed in CRC tissues and is significantly associated with EMT pathways and poor cancer prognosis. In SW480 and DLD1 cell lines, MGAT5 overexpression promoted cell proliferation and migration, whereas MGAT5 knockdown exhibited the opposite effect. Further analyses indicated that MGAT5 decreases ZO-1 expression by modifying ZO-1 with multi-antennary N-glycosylation and promoting its ubiquitination degradation, thereby triggering EMT initiation. MGAT5 expression levels negatively correlated with ZO-1 expression in CRC tissues and were closely associated with higher recurrence and metastasis rates.

Conclusion and Significance This study revealed the mechanism of MGAT5 as a key regulatory factor of EMT in CRC, indicating that it promotes CRC cell metastasis by facilitating ZO-1 ubiquitination and degradation. This finding not only deepens the understanding of the CRC metastasis mechanism but also provides a potential target for developing new therapeutic strategies targeting MGAT5, with significant scientific and clinical application value.

Research Highlights - MGAT5 is highly expressed in colorectal cancer and is significantly related to EMT and poor prognosis. - Through multi-antennary N-glycosylation modification and ubiquitination, MGAT5 promotes the degradation of ZO-1 and initiates EMT. - Offers a new perspective on MGAT5 as a potential target for CRC diagnosis and therapy.

This study provides strong support for future CRC therapeutic strategies, indicating that inhibiting MGAT5 might become a method to prevent cancer [The sentence is incomplete, assuming the original text intended to discuss prevention significance].