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

Abstract

Increasing evidence supports the crucial role of Epithelial-Mesenchymal Transition (EMT) in cancer invasion and metastasis. N-acetylglucosaminyltransferase V (MGAT5), which is associated with multiantenna glycosylation, can contribute to tumorigenesis, yet its specific role in promoting colorectal cancer (CRC) metastasis remains unclear. Bioinformatics analysis of CRC datasets revealed that elevated MGAT5 expression was associated with EMT and a poor prognosis. In vitro experiments confirmed the pivotal role of MGAT5 as an EMT regulator in CRC cells. MGAT5 overexpression stimulated cell proliferation and migration, while MGAT5 knockdown had the opposite effect. Mechanistically, MGAT5 promoted EMT through multiantenna glycosylation of ZO-1, promoting its ubiquitination and reducing its expression. Clinically, MGAT5 upregulation in the CRC TMA correlated negatively with ZO-1 expression, which is indicative of malignancy and a poor prognosis. This study revealed that MGAT5 promotes EMT in CRC via interactions between multiple antenna glycosylation products and ZO-1 ubiquitination/degradation, indicating that MGAT5 could serve as a promising therapeutic target for CRC.

Graphical Abstract

Background

Colorectal cancer (CRC) is the second most common cause of cancer-related death globally and the third most common cancer overall [1]. Most cancer patients die not from primary tumours but rather from the consequences of metastasis. Approximately 22% of CRC patients suffer from metastatic disease at diagnosis [2], and 19.6% of CRC patients develop metachronous metastases during their disease course [3]. Despite significant advancements in the diagnosis and treatment of CRC, metastasis continues to be the primary driver of high mortality rates and a poor prognosis. Therefore, it is imperative to investigate the fundamental molecules and mechanisms underlying CRC progression to develop new therapeutic approaches that inhibit metastasis and improve treatment outcomes.

EMT is a phenomenon in which cells undergo reduced polarization and disrupted intercellular junctions. As a result, the integrity of the basement membrane is disrupted, causing cells to transform from immobile epithelial cells to mobile mesenchymal cells [4]. During the EMT process, the expression or function of epithelial phenotypic proteins such as tight junction protein 1 (often referred to as ZO-1) and E-cadherin (encoded by CDH1) is reduced. Concurrently, the expression of proteins associated with the mesenchymal phenotype, including fibronectin (FN), N-cadherin (encoded by CDH2), vimentin (VIM), β1, and β3 integrins, is upregulated [5, 6]. Recent research indicates that ZO-1 is essential for the initiation and spread of cancer [7, 8]. During the progression of cancer, a disturbance of ZO-1 can induce alterations in the cellular environment, ultimately facilitating tumour invasion and metastasis [9, 10]. Despite the extensive attention given to the role of EMT in cancer, the critical molecular alterations involved in cancer remain largely unclear.

Numerous studies have been performed on the molecular pathways underlying EMT, some of which have concentrated on modifications to N-glycosylation [11]. The β1,6-GlcNAc-branched multiantenna N-glycan is a commonly found tumour-associated glycan structure. It is generated by the addition of β1,6-GlcNAc to an α1,6-linked mannose through the catalysis of N-acetylglucosaminyltransferase V (MGAT5). Previous studies have shown an increase in β1,6-GlcNAc-branched multiantenna N-glycan levels in CRC tissues stained with PHA-L (Phaseolus vulgaris lectin L.) [12]. An increase in PHA-L corresponds with lymph node metastasis and is an independent prognostic factor for both survival and tumour recurrence [13]. These results are supported by the observation that MGAT5 expression is greater in samples from colorectal adenomas, carcinomas, and liver metastases than in those from comparable mucosa [14]. However, the mechanism through which MGAT5 promotes CRC metastasis has not yet been fully investigated.

In this study, we aimed to explore the potential molecular mechanisms underlying MGAT5-mediated EMT in CRC, with a specific emphasis on elucidating the relationship between MGAT5 and ZO-1. We used the TCGA and GEO databases to investigate the role of MGAT5 in colorectal cancer and to perform a comprehensive analysis of its association with EMT. Subsequently, we engineered colorectal tumour cells with modified N-glycosylation profiles by stably overexpressing or knocking down MGAT5. This allowed us to probe the relationship between MGAT5 and EMT. Furthermore, we utilized immunofluorescence (IF) and coimmunoprecipitation (co-IP) assays to assess the interaction between MGAT5 and ZO-1. Finally, through immunohistochemistry (IHC) and IF experiments, we confirmed the clinical relevance of MGAT5 in CRC, revealing a negative correlation between MGAT5 and ZO-1.

Materials and methods

Datasets and collection

Public data

The RNA transcriptomics and clinical data for 33 distinct cancer types (10,535 samples) were downloaded from The Cancer Genome Atlas (TCGA), and the genomic data for normal tissues (7862 samples) were obtained from Genotype-Tissue Expression (GTEx), all sourced from the University of California Santa Cruz (UCSC) Xena platform (http://xena.ucsc.edu/). The abbreviations used for cancer treatment are presented in Supplemental Table S1. A total of 1206 samples from 30 cell lines and 194 IHC staining datasets sourced from the Human Protein Atlas were used to confirm the specificity of MGAT5 for CRC tissues. Additionally, we obtained two independent microarray datasets from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). One of these datasets, GSE32323, included RNA sequencing data along with clinical information for 17 pairs of cancer and paired normal tissues from colorectal cancer patients, while the other dataset, GSE17537, consisted of 55 CRC samples.

Tissue microarray

The tissue microarray (TMA) obtained from the Shanghai WellBio Technology Co., Ltd. (ZL-Cocsur1241) consisted of a cohort comprising 69 CRC patients, encompassing a total of 69 cancer tissue samples and 55 corresponding para-cancerous tissue samples. Comprehensive histopathological data (Supplemental Table S2), including tumour grade, tumour stage (pT), and distant metastasis status, were available for the entire cohort of 69 CRC patients. Overall survival (OS) was defined as the time from tumour resection to death or the last follow-up date. PFS was defined as the time from tumour resection to recurrence, death, or the last follow-up. For the 69 CRC patients, the median follow-up period was 40.09 months.

Differential gene expression analysis

We performed log2 transformation and t tests on the expression data for these tumour types based on the RNA transcriptome data from 33 different types of tumours in the TCGA database. Differences in the expression of genes between cancer and adjacent normal tissue samples were identified based on a significance threshold of P < 0.05. The data were analysed using R software (https://www.Rproject.org), and box plots were created using the “ggplot2” R package.

Gene set enrichment analysis (GSEA)

GSEA was also conducted to elucidate the functional role of MGAT5 with the reference gene set h.all.v7.2.symbols.gmt. Three datasets were utilized: TCGA pancancer, GSE32323, and GSE17537. Patients with MGAT5 expression in the top 30% and the bottom 30% were subsequently compared. The enrichment score (ES) for the gene sets positively or negatively correlated with MGAT5 was computed via single-sample gene set enrichment analysis (ssGSEA) within the R package 'GSVA'. P < 0.05, |ES|> 1, and FDR < 0.25 indicated statistical significance.

CRC cell lines with MGAT5 overexpression or knockdown

SW-480, DLD1 and 293 T cells were cultured in DMEM supplemented with 10% FBS (10099141C; Gibco) and 1% penicillin/streptomycin (15,140,122; Gibco) at 37 °C in a humidified incubator (HeraCell VIOS, Thermo Fisher) with 5% CO₂. The MGAT5 overexpression plasmids, control plasmids, MGAT5 siRNAs and corresponding negative control (NC) siRNAs were synthesized by Hanbio Biotechnology Co., Ltd. For the construction of the MGAT5 overexpression cell line, the transfer plasmid, envelope plasmid, and packaging plasmids were transfected into 293 T packaging cells by Lipofectamine 3000 (L3000015, Thermo Fisher, USA) according to the manufacturer’s instructions. The culture medium was refreshed 16 h later, and the culture supernatant was collected at 2 and 3 days post transfection. The viruses were harvested by ultracentrifugation. A total of 2 × 10⁵ SW480 or DLD1 cells were seeded into 12-well plates overnight and subsequently infected with lentivirus expressing the MGAT5 gene or a control lentivirus. The transfected cells were screened using puromycin (1 μg/mL) one week later. MGAT5 siRNA or negative control siRNA was preincubated with RNAfit (HB-RF-500; Hanbio Biotechnology) for 20 min before being added to the cell medium at a final concentration of 50 nM. The cells were incubated at 37 °C for 1–3 days, after which the transfected cells were analysed. The sequences of the siRNAs used were as follows:

si-MGAT5: 5ʹ-CCTGGAAGCTATCGCAAAT-3ʹ; and.

siRNA NC: 5ʹ-TTCTCCGAACGTGTCACGT-3ʹ.

Colony formation and EdU proliferation assays

MGAT5-overexpressing or MGAT5-knockdown cells (1000 cells/well) were plated into 12-well plates and cultured for seven days. Subsequently, the cells were fixed in 4% paraformaldehyde (w/v) for 10 min. After washing three times with PBS, staining was performed with crystal violet staining solution (C0121, Beyotime). Images were taken with a KEYENCE BZ-X800 microscope, and colonies with aggregates of ≥ 50 cells were counted.

MGAT5-overexpressing or MGAT5-knockdown cells were seeded into 8-well plates (07–2108, Biologix). Cell proliferation was assessed via 5-ethynyl-2’-deoxyuridine (EdU) (C0078S; Beyotime) incorporation according to the manufacturer’s instructions. The percentage of EdU-positive cells in three random images captured under a KEYENCE BZ-X800 microscope was calculated.

Wound healing

MGAT5-overexpressing or MGAT5-knockdown cells were seeded into 12-well plates and allowed to incubate for 1 day. When the cells reached approximately 90% confluence, the bottom of each well was scratched with a 10 μl pipette tip. After washing with PBS, the culture medium was replaced with serum-free medium for cultivation. Images were captured under a microscope (DMi8, Leica) at the same position at both 0 and 24 h, and the migration area was computed using ImageJ software.

Cell migration

MGAT5-overexpressing or MGAT5-knockdown cells were collected and diluted with serum-free DMEM to a concentration of 1 × 10⁵ cells/mL. The upper chamber (353,097, FALCON) was filled with 200 μL of cell suspension to measure the capacity for migration. The migrated cells were subsequently stained with crystal violet staining solution and observed under a KEYennia BZ-X800 microscope. The number of migrated cells was determined using three randomly selected images of the selected fields.

Quantitative RT–PCR

An EZ-press RNA Purification Kit (B0004D, EZBioscience) was used to extract total RNA from the cells. The concentration of the extracted RNA was measured by a NanoDrop (Thermo Fisher). A PrimeScript™ RT Reagent Kit (RR037A, Takara) was used for reverse transcribing RNA into cDNA. Each reverse transcription reaction contained 500 ng of RNA. TB Green™ Premix Ex Taq™ (RR420A, TAKARA) was used for qPCR, and the data were collected and analysed using an Applied Biosystems PCR System (7500 Real-Time PCR System, Thermo Fisher). The relative expression levels of each gene were determined using the 2^(-ΔΔCt) method, with GAPDH serving as the internal reference.

The sequences of primers used were as follows:

MGAT5, 5′-GCAGACTCTCACACTCAACCTACAC-3′ and 5′-CTGGCAACTTCACCTGTCCTTGG-3′;

ZO-1, 5′-GTGCTGGCTTGGTCTGTTTGC-3′ and 5′-ACGCTGGGTGATAGGGATTTGTG-3′;

E-Cad, 5′-GCCATCGCTTACACCATCCTCAG-3′ and 5′-CTCTCTCGGTCCAGCCCAGTG-3′; and.

N-Cad, 5′-GCTTACACCTATGACCTTGGCTTCG-3′ and 5′-AATTGTTGACCCTGGCACTCTTCTC-3′.

Western blot (WB) analysis

MGAT5-overexpressing or MGAT5-knockdown cells were lysed in lysis buffer (9803 s, CST) supplemented with ProtLytic Protease and Phosphatase Inhibitor Cocktail (P002, NCM). Then, the protein concentration was quantified with a BCA protein assay kit (P0011, Beyotime). Equivalent amounts of proteins were separated by 7.5 or 10% SDS–PAGE, and the resulting proteins were then transferred from the gel to PVDF membranes (Millipore, cat. no. ISEQ00010).

After an hour of incubation with 5% skim milk, primary antibodies against ZO-1 (13,663, CST), E-Cad (3195, CST), MGAT5 (MAB5469, R&D), N-Cad (13,116, CST), ubiquitin (20,326, CST), and PHA-L (B-1115–2, Vector Laboratories) were added to the membranes. The membranes were then incubated with goat anti-rabbit (7074, CST), goat anti-mouse (7076, CST), or streptavidin-HRP (3999, CST) secondary antibodies. GAPDH was used as the loading control (10,494–1-AP; Proteintech). Protein visualization was performed with a chemiluminescence (ECL) detection kit (NcmECL Ultra, P10300, NCM).

Immunofluorescence (IF)

MGAT5-overexpressing cells were treated with or without PNGase F and blocked for one hour using QuickBlockTM Blocking Buffer for Immunol Staining (P0260, Beyotime) after being fixed for ten minutes with 4% paraformaldehyde. The CRC TMA was deparaffinized in xylene and rehydrated in a series of gradually decreasing alcohol concentrations. The EDTA heat-induced antigen retrieval method was used for antigen detection. After 10 min of incubation in 3% H₂O₂, the sections were blocked for 30 min in Beyotime's QuickBlockTM Blocking Buffer (P0260). Then, the cells and TMAs were incubated with primary antibodies (ZO-1, R, 13,663, CST; MGAT5, M, MAB5469, R&D) overnight at 4 °C and incubated with Cyanine5 (A10523, Thermo Fisher) and Texas Red-X-labelled (T-862, Thermo Fisher) secondary antibodies for 1 h. Each step involved washing three times with PBS for five minutes. Finally, the slides were sealed using Antifade Mounting Medium containing DAPI (A4084, UElandy) and then photographed with a Zeiss Tissue Gnostics microscope.

Immunohistochemistry (IHC)

Human N-acetylglucosaminyltransferase V/MGAT5 (M, MAB5469, R&D) and ZO-1 (R, 13,663, CST) antibodies were applied to the CRC TMA. The TMA-stained images were automatically scanned by a Tissue FAXS imaging system (Tissue FAXS Plu, Tissue Gnostics, Zeiss), and the reconstituted virtual slides were subjected to quantitative image analysis using StrataQuest (v. 7.1.1.119). StrataQuest analysis software was used to define the different morphologies of the tissues to distinguish between malignant and nonmalignant regions. Subsequently, intensity values for the different fluorescence channels were obtained for the two regions. By calculating the intensity values for each cell in specific fluorescence channels, the expression levels of target genes in different regions were determined.

Coimmunoprecipitation (co-IP)

MGAT5-overexpressing or MGAT5-knockdown cells were lysed in IP lysis buffer (87,787; Thermo Fisher) containing protease/phosphatase inhibitor cocktail (5872S; CST) for 30 min on ice. After centrifugation for 15 min at 12,000 rpm at 4 °C, the cell lysates were subsequently incubated with primary antibodies (ZO-1, 13,663, CST; Flag, 66,008–4-Ig Proteintech) overnight at 4 °C. The immunocomplexes were subsequently mixed with Dynabeads™ Protein G (Thermo Fisher, 10004D) for 4 h at 4 °C. The immunocomplexes were washed three times with PBST and boiled in SDS‒PAGE Sample Loading Buffer (LT101S, Epizyme Biotech) for WB analysis. ZO-1 was immunoprecipitated for WB detection of MGAT5, PHA-L, ZO-1, and ubiquitin, and Flag (fused with MGAT5) was immunoprecipitated for WB detection of ZO-1 and Flag. The WB detection method was performed as described above.

Statistical analysis

GraphPad Prism 9 was used to conduct the statistical analyses. The error bars in the experiments represent the standard deviation (s.d.). The legends provide information about the number of events and independent experiments. Statistical comparisons between two experimental conditions were conducted using the Mann‒Whitney test or Student's t test for unpaired samples, and the Wilcoxon rank-sum test was used for all paired t tests. Two-way analysis of variance (ANOVA) was used to compare multiple experimental groups. Survival analysis was conducted using Kaplan‒Meier plots. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. p < 0.05 indicated statistical significance.

Results

MGAT5 is upregulated in colorectal cancer tissues and is correlated with both poor survival and EMT

MGAT5 is thought to participate in the metastasis of various cancers due to its aberrant expression, but its role in CRC has not yet been fully elucidated. To explore the potential role of MGAT5 in the development of CRC, we conducted a comprehensive analysis of MGAT5 expression in tumours and corresponding normal tissues across various cancers using TCGA and GTEx gene expression data. The results revealed significant upregulation of MGAT5 mRNA expression in 25 different types of tumours, with a particularly prominent increase observed in CRC, where the median expression level reached 20.6 fragments per kilobase million (FPKM) (Fig. 1A). Next, 1206 samples from 30 cell lines from the Human Protein Atlas (https://www.proteinatlas.org/) revealed that MGAT5 gene expression was highly specific to CRC cell lines (Figure S1A). The immunohistochemical staining data from the Human Protein Atlas further validated the CRC-specific expression of MGAT5 at the protein level, as indicated by the observation that 100% of cells were positive for MGAT5 (11 of 11 samples) (Figure S1B). These findings strongly indicate that MGAT5/GnT-V expression is significantly greater in CRC tissues than in normal colon tissues.

Fig. 1

Upregulation of MGAT5 is correlated with a poor prognosis and EMT in CRC patients. A Dot plots were generated to illustrate the expression profile of the MGAT5 gene across 33 tumour types and corresponding normal tissues from the TCGA database. Each data point represents the sample's expression level. B The overall survival (OS) curves of MGAT5 were plotted for all CRC patients in the TCGA database. C GSEA was conducted on MGAT5 expression in the TCGA dataset. The corresponding enrichment scores and the enriched posttranslational modification pathways are displayed. D Relationships between EMT-related mRNAs in CRC and MGAT5 according to the TCGA database (n = 455). The R values and p values were obtained from Pearson’s correlation analysis. E MGAT5 mRNA expression and EMT-related gene expression were positively correlated according to the hallmark gene sets determined via gene set enrichment analysis (GSEA)

To explore whether MGAT5 expression was correlated with CRC prognosis, a survival analysis of MGAT5^high and MGAT5^low patients with CRC classified by X-tile software was performed. According to the TCGA COAD dataset (n = 284), the OS of patients in the MGAT5^high subgroup was notably shorter than that of patients in the MGAT5^low subgroup (P = 0.01; Fig. 1B). To further elucidate the association between MGAT5 and signalling pathways, GSEA was conducted for each type of cancer based on the transcriptomes of both the normal and tumour tissue samples (top and bottom 30% of the MGAT5 gene sets).

The epithelial–mesenchymal transition (EMT) pathway was enriched in tumours with higher MGAT5 expression levels, and pathways that were frequently enriched (> 6 cancers) are presented in Fig. 1C. Furthermore, we explored the transcriptional correlation between MGAT5 expression and EMT marker expression using data from the TCGA database. MGAT5 was strongly associated with nearly all EMT markers, including CDH1 and CDH2, as well as VIM, ZEB1, TJP1, and ITGB1 (Fig. 1D). To further validate our findings, we performed GSEA using GEO datasets (GSE32323 and GSE17537). This analysis confirmed the significant association between MGAT5 expression and the EMT pathway (Fig. 1E).

Taken together, these results suggest that MGAT5 is elevated in CRC and that its expression is positively correlated with EMT.

MGAT5 promotes the proliferation and migration of CRC cells

To further investigate the regulatory role of MGAT5 in CRC, we constructed two CRC cell lines, SW480 and DLD1, that stably overexpress MGAT5 (SW480 GNT5-OE and DLD1 GNT5-OE cells, respectively). The transfection efficiency was assessed by WB and RT‒qPCR (Fig. 2A, B). We also used small interfering RNA (siRNA) to effectively suppress the expression of MGAT5 (Fig. 3A, B). As expected, compared with the control, the overexpression of MGAT5 promoted the proliferation of CRC cells (Fig. 2C, D). Conversely, knockdown of MGAT5 had the opposite effect on DLD1 and SW480 cell proliferation (Fig. 3C, D). To further evaluate the prometastatic role of MGAT5, we performed wound healing and Transwell migration assays on CRC cells. MGAT5 overexpression significantly promoted the migration of SW480 and DLD1 cells (Fig. 2E–H), whereas MGAT5 downregulation inhibited the invasion and migration of SW480 and DLD1 cells (Fig. 3E–H). Collectively, these results indicate that MGAT5 is a positive regulator of CRC proliferation and metastasis.

Fig. 2

MGAT5 induces proliferation and metastasis in CRC cells. A RT‒qPCR was used to assess the mRNA expression of MGAT5 in MGAT5-overexpressing and vector control cells. B The protein expression of GNT5 and multiantenna level of PHA-L in MGAT5-overexpressing and vector control cells was examined by western blotting and lectin blotting. C Proliferation was detected by a colony formation assay. D The EdU assay was used to compare the proliferation of MGAT5-overexpressing and vector control cells. Scale bar, 100 μm. E, F Transwell migration assays. G, H Wound healing assays of MGAT5-overexpressing and vector control cells. The mean ± SD of three separate experiments is shown by each bar

Fig. 3

MGAT5 knockdown alleviates EMT in CRC cells. A, B The protein expression of GNT5 and multiantenna level of PHA-L in MGAT5-knockdown CRC cells and control cells was examined by western blotting and lectin blotting. C Proliferation was detected by a colony formation assay. D The EdU assay was used to compare the proliferation of MGAT5-knockdown and si-NC cells. Scale bar, 100 μm. E, F Wound healing (E) and Transwell migration (F) assays of MGAT5-knockdown SW480 cells. G, H Wound healing (G) and Transwell migration (H) assays of MGAT5-knockdown DLD1 cells. The mean ± SD of three separate experiments is shown by each bar

MGAT5 plays a pivotal role as an EMT regulator in CRC cells

To determine whether MGAT5 regulates the EMT process in CRC cells, we initially assessed the expression of EMT-related genes in human CRC cells with MGAT5 overexpression or MGAT5 knockdown using quantitative real-time PCR. CDH2 expression was upregulated in cells stably expressing MGAT5, while knockdown of MGAT5 had the opposite effect on the mRNA expression of CDH2. However, neither MGAT5 overexpression nor interference affected the mRNA expression of ZO-1 (Fig. 4A, B). Western blotting assays demonstrated that the upregulation of MGAT5 resulted in reduced expression of E-cadherin and ZO-1 (EMT epithelial markers) and increased expression of N-cadherin (EMT mesenchymal marker) (Fig. 4C, D). In contrast, MGAT5 knockdown had the opposite effect on EMT, as indicated by an increase in the expression of ZO-1 and a decrease in the expression of N-cadherin, as evidenced by Western blotting (Fig. 4E, F). Taken together, these findings indicate that MGAT5 is a positive regulator of CRC EMT.

Fig. 4

MGAT5 induces EMT in CRC cells. A, B The mRNA levels of markers associated with EMT. C, D The protein expression of ZO-1, E-Cad, and N-Cad in MGAT5-overexpressing and vector control cells was detected by western blotting. E, F The protein expression of ZO-1, E-Cad, and N-Cad in MGAT5-knockdown CRC cells and control cells was detected by WB. The mean ± SD of three separate experiments is shown by each bar

MGAT5 enhances the multiantenna glycosylation of ZO-1 and facilitates ZO-1 degradation through ubiquitination

As indicated by the above results, MGAT5 is capable of reducing the expression of ZO-1. However, there are currently no reports on the regulatory role of MGAT5 in relation to ZO-1. Therefore, we used this result as a starting point for our subsequent experiments. First, we used immunofluorescence to detect the expression and localization of MGAT5 and ZO-1. ZO-1 expression was reduced in cells with high MGAT5 expression, and colocalization of ZO-1 and MGAT5 was observed at the cell membrane (Fig. 5A). ZO-1 was readily detected in the immunoprecipitates of FLAG-MGAT5 (Fig. 5B). Similarly, MGAT5 and PHA-L glycosylation were detected in the ZO-1 immunoprecipitated samples (Fig. 5C). Knockdown of MGAT5 expression reduced the amount of the multiantenna N-glycan recognized by PHA-L on the surface of the ZO-1 protein (Fig. 5D). Using the PNGase F enzyme to remove multiantenna N-glycan proteins, we observed an increase in ZO-1 expression (Fig. 5E).

Fig. 5

MGAT5 interacts with ZO-1 and promotes its glycosylation and ubiquitination. A Immunostaining of MGAT5 or ZO-1 in MGAT5-OE CRC cells and control cells. Scale bars, 100 μm. B Lysates from CRC cells expressing FLAG-MGAT5 were immunoprecipitated with an anti-FLAG antibody and examined via WB analysis with the indicated antibodies. C Lysates from CRC cells expressing FLAG-MGAT5 were immunoprecipitated using an anti-ZO-1 antibody, and the corresponding antibodies were subsequently subjected to WB analysis. D Lysates from MGAT5-knockdown CRC cells were immunoprecipitated using an anti-ZO-1 antibody, and the corresponding antibodies were subsequently subjected to WB analysis. E The expression and localization of ZO-1 in MGAT5-overexpressing CRC cells treated with or without PNGase F were assessed using confocal imaging. Scale bars, 10 μm. F SW480-MGAT5 and DLD1-MGAT5 cells were treated with 20 μM CHX alone or in combination with 10 μM MG132 for the indicated durations. WB was used to detect the indicated antibodies. G Lysates from MGAT5-OE and MGAT5-knockdown (H) cells from each group were immunoprecipitated with an anti-ZO-1 antibody, after which ubiquitin expression was detected

Since the above results showed that MGAT5 had no effect on the transcription level of ZO-1, to determine whether this regulation was mediated by protein modification, MGAT5-overexpressing CRC cells were treated with the proteasome inhibitor MG132 (ubiquitin-mediated degradation inhibitor, 10 μM) or CHX (50 μg/mL) for different durations, after which ZO-1 protein expression was measured by western blotting. ZO-1 protein levels decreased markedly within 8 h of CHX treatment in SW480 (MGAT5) cells (Fig. 5F). Co-IP assays revealed that the overexpression of MGAT5 significantly enhanced the ubiquitination of ZO-1 (Fig. 5G). Conversely, knockdown of MGAT5 in CRC cells suppressed ZO-1 ubiquitination (Fig. 5H). Overall, these results suggest that MGAT5 regulates ZO-1 stability via ubiquitination.

Elevated MGAT5 expression in CRC tissues negatively correlates with ZO-1 expression and predicts poor prognosis

To better understand the potential clinicopathological effects of MGAT5, we investigated the expression levels of MGAT5 in human CRC specimens (adjacent and tumour tissue microarray) via IHC staining. The immunohistochemistry (IHC) score confirmed that MGAT5 was more highly expressed in tumour tissues than in adjacent nontumor tissues (Fig. 6A, B). Compared to patients with high MGAT5 expression, those with low MGAT5 expression had noticeably longer overall survival (OS) according to survival curve analysis (Kaplan‒Meier curve) (Fig. 6C). Next, we correlated the response to MGAT5 expression with recurrence and metastasis. Notably, higher MGAT5 expression was closely associated with a greater recurrence rate (Fig. 6D) and greater metastasis (Fig. 6E). To validate the association between ZO-1 and MGAT5, immunofluorescence staining was conducted on colorectal cancer tissues. Similarly, the expression of ZO-1 was significantly reduced in tissues with high MGAT5 expression, indicating a negative correlation between the ZO-1 and MGAT5 protein levels (Fig. 6F). Collectively, these findings demonstrate the upregulation of MGAT5 in human colorectal cancer and its association with ZO-1 downregulation, thereby promoting metastasis and suggesting that MGAT5 plays an oncogenic role in the progression of colorectal cancer.

Fig. 6

Clinical significance of MGAT5 as a potential therapeutic target in tumour EMT. A Representative IHC staining of MGAT5 in CRC patient tissue from the TMA (ZL-Cocsur1241). B MGAT5 protein expression in malignant colorectal cancer (T) and adjacent nonmalignant tissues (N). The data are presented as a box plot. Whiskers show the min-to-max values, n = 69 per group. C Kaplan‒Meier analysis of OS in patients with varying MGAT5 expression levels based on data from selected CRC tissues. D Kaplan‒Meier analyses were performed to assess the associations between MGAT5 expression and RFS. E MGAT5 protein levels in nonmetastatic and metastatic colorectal cancer (T) tissues. The data are presented as a box plot. The whiskers show the min-to-max values. F Immunostaining of the CRC tissue TMA for MGAT5 or ZO-1. Correlations between the mean MGAT5 intensity and the mean ZO-1 intensity in CRC tissue samples (n = 15). Pearson's correlation analysis yielded the R and p values. Scale bar: 100 µm

Discussion

EMT is thought to constitute the first stage of tumour metastasis and invasion. Accumulating data highlight the critical role of EMT in colorectal cancer invasion and metastasis, which occurs at the invasive front of colon carcinoma and is accompanied by selective loss of the basement membrane [15]. Here, we observed a significant increase in MGAT5 expression in colorectal cancer patients, which was closely associated with tumour metastasis and patient survival outcomes. Furthermore, we found that MGAT5 promotes ZO-1 degradation through enhanced ubiquitination, thereby triggering the activation of EMT.

Glycosylation is one of the most prevalent posttranslational modifications (PTMs) that are observed in proteins, among others. Approximately 20% of cellular proteins are thought to undergo glycosylation [16]. Aberrant glycosylation is a distinctive feature of tumour cells [17, 18]. MGAT5, a pivotal N-glycan processing enzyme located in the Golgi apparatus, regulates the glycan structure of cell surface glycoproteins, thereby enhancing cell malignancy and promoting tumour metastasis [19]. MGAT5 is upregulated in various cancers, such as ovarian cancer [20], gliomas [21], gastric cancer [22, 23], and hepatocellular carcinoma[24, 25]. MGAT5 also exacerbates the proliferation and metastasis of breast cancer [26]. We manipulated MGAT5 levels through overexpression and knockdown to explore its potential impact on CRC. Our findings suggest that MGAT5 promotes the proliferation of CRC cells, consistent with the findings of prior research demonstrating that MGAT5 overexpression confers resistance to anoikis, a type of apoptosis induced by the loss of cell–matrix contact [27].

While previous studies have established the role of MGAT5 in promoting CRC proliferation [28, 29], limited research has been conducted on the mechanisms that underlie MGAT5-mediated metastasis in CRC. Furthermore, it is essential to underscore the crucial role of MGAT5 in processes that promote the development of CRC metastasis. Our findings align with those in previous studies, demonstrating that upregulation of MGAT5 in colorectal cancer cells induces morphological changes associated with EMT, promoting migration. This change was accompanied by a decrease in mesenchymal marker expression and an increase in epithelial marker expression. These observations suggest that the role of MGAT5 in colorectal cancer metastasis may be attributed, at least in part, to the regulation of EMT.

Several previous studies have demonstrated the importance of MGAT5 and β1,6-GlcNAc-branched multiantenna N-glycan in modulating interactions between cells and the matrix in epithelial cells. GnT-V promotes the destabilization of E-CAD, which leads to the mislocalization of E-CAD and impairs adherens junctions, ultimately compromising cell‒cell adhesion [30]. The downregulation of MGAT5 in breast cancer cells has been demonstrated to reduce the amount of β1,6-GlcNAc-branched N-glycan on N-CAD, resulting in decreased turnover of cell‒cell adhesions and reduced cell migration [31]. Furthermore, MGAT5 has been identified as a critical regulator of stiffness-induced invasion in GSCs [32]. Our study adds to this body of knowledge by revealing that MGAT5 overexpression contributes to the downregulation of E-CAD and upregulation of N-CAD in colorectal cancer cells, providing insights into its potential role in promoting invasion in this context.

ZO-1 is a 220 kDa membrane scaffold protein that interacts with ZONAB to regulate gene expression, cell proliferation and tight junction formation [33, 34]. The downregulation of ZO-1 has been implicated in tumour development and progression, suggesting that it functions as a tumour suppressor [35,36,37]. Research has suggested that patients with colorectal cancer exhibit impaired intestinal barrier function, as evidenced by a reduction in the levels of the intestinal tight junction proteins ZO-1 and occludin [38, 39]. Similarly, we noted that, compared with that in adjacent tissue, ZO-1 expression in tumour tissue was downregulated, and this change was significantly negatively correlated with MGAT5 expression. MGAT5 overexpression led to a reduction in ZO-1 levels. Further investigation revealed that MGAT5 interacted with ZO-1 and that the downregulation of ZO-1 was attributed to enhanced ubiquitination by MGAT5. Glycosylated proteins may be recognized and targeted for ubiquitination-dependent degradation [40]. The relationship between MGAT5 and ubiquitination has been minimally investigated in the current literature. Nonetheless, our findings present novel evidence underscoring the role of MGAT5 in facilitating colon cancer metastasis via its regulation of ubiquitination. This study has certain limitations, including the need for further confirmation of the interaction between the ubiquitin ligase and ZO-1.

In conclusion, our study illustrates the crucial role of MGAT5 in orchestrating epithelial-mesenchymal transition. MGAT5 is highly expressed in CRC and actively promotes EMT by facilitating the ubiquitination and degradation of ZO-1. These findings contribute to a deeper understanding of colorectal cancer metastasis and reveal potential therapeutic targets for managing malignant colorectal cancer.