Intestinal epithelium-specific Fut2 deficiency promotes colorectal cancer through down-regulating fucosylation of MCAM

Abstract

Background: Our previous study showed that fucosyltransferase 2 (Fut2) deficiency is closely related to colitis. Colitis increases the risk for the development of colorectal cancer (CRC). This study aimed to investigate the effect and underlying mechanism of action of Fut2 in CRC.

Methods: Intestinal epithelium-specific Fut2 knockout (Fut2^△IEC) mice were used in this study. CRC was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS). Immunofluorescence was used to examine the fucosylation levels. Proteomics and N-glycoproteomics analyses, Ulex Europaeus Agglutinin I (UEA-I) affinity chromatography, immunoprecipitation, and rescue assay were used to investigate the mechanism of Fut2 in CRC.

Results: The expression of Fut2 and α-1,2-fucosylation was lower in colorectal tumor tissues than in the adjacent normal tissues of AOM/DSS-induced CRC mice. More colorectal tumors were detected in Fut2^△IEC mice than in control mice, and significant downregulation of melanoma cell adhesion molecule (MCAM) fucosylation was detected in the colorectal tumor tissues of Fut2^△IEC mice. Overexpression of Fut2 inhibited cell proliferation, invasion and tumor metastasis in vivo and in vitro in SW480 and HCT116 cells. Moreover, fucosylation of MCAM may be a mediator of Fut2 in CRC. Peracetylated 2-F-Fuc, a fucosyltransferase inhibitor, repressed fucosylation modification of MCAM and reversed the inhibitory effects of Fut2 overexpression on SW480 cell proliferation, migration, and invasion. Our results indicate that Fut2 deficiency in the intestinal epithelium promotes CRC by downregulating the fucosylation of MCAM.

Conclusions: The regulation of fucosylation may be an potential therapy for CRC, especially in patients with Fut2 gene defects.

Keywords: CRC; Colorectal cancer; Fucosylation; Fut2; MCAM.

Fig. 1

Fut2 is down regulated in CRC and correlates with poor prognosis. A Expression of Fut2 mRNA (including 275 CRC tissues and 45 normal colon tissues) from TCGA database was first analyzed. B Amplification of Fut2 gene copy number associated with good disease-free survival in TCGA cohort (n = 135, log-rank test, P = 0.047). C ROC curves for classifying CRC tissues versus normal colon tissues in the TCGA database. D The proteins level of Fut2 in colon tissues extracted from normal and colorectal cancer patient (n = 4 per group). E The relative mRNA expression of Fut2 in control and colorectal cancer patient colon tissues was detected by qPCR (n = 4 for per group). F The proteins level of Fut2 in colon tissues extracted from control mice and AOM/DSS mice (n = 3 per group). G The relative mRNA expression of Fut2 in control and AOM/DSS mice colon tissues was detected by qPCR (n = 5 for per group). H The typical images of mice colon tissues stained with UEA-I. I The mean gray value of UEA-I in control mice and AOM/DSS mice (n = 5 per group). Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: *p < 0.05, **p < 0.01. (CRC colorectal cancer, TCGA The Cancer Genome Atlas, AOM/DSS azoxymethane/dextran sodium sulfate, UEA-I Ulex Europaeus Agglutinin-I, SEM Standard Error of Mean)

Fig. 2

Fut2 deficiency enhances promotion of AOM/DSS-induced CRC. A Schematic overview of this colon carcinogenesis model. B Body weight change during the disease process. C Survival rate of mice from each group. D Macroscopical changes in colon. Colons were removed at day 70 from treated WT and Fut2^△IEC mice, and representative results from 5 independent animals are shown here. E Tumor numbers of each group. F The typical images of mice colon tissues from treated WT and Fut2^△IEC mice. G The number of tumors corresponding to different tumor sizes of each group. Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: ***p < 0.001, ****p < 0.0001. (CRC colorectal cancer, AOM/DSS azoxymethane/dextran sodium sulfate, WT wild type, SEM Standard Error of Mean)

Fig. 3

Fut2 inhibits cell proliferation of SW480 and HCT116 cells. A The relative mRNA expression of Fut2 in control and fut2 overexpressed SW480 and HCT116 cells was detected by qPCR (n = 3 for per group). B The relative protein expression of Fut2 in control and fut2 overexpressed SW480 and HCT116 cells was detected by western blot (n = 3 for per group). C The typical images of SW480 and HCT116 cells stained with UEA-I. D The plate clone formation experiment of SW480 and HCT116 cells in control and Fut2-overexpression group. E CCK8 assays of SW480 and HCT116 cells in control and Fut2-overexpression group. F The typical images of transwell migration assay in control and fut2 overexpressed SW480 and HCT116 cells. G The typical images of transwell matrigel invasion assay in control and fut2 overexpressed SW480 and HCT116 cells. H, I Wound-healing assay at 48 h in control and fut2 overexpressed SW480 and HCT116 cells. Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: *p < 0.05, ***p < 0.001. (CCK8 Cell Counting Kit-8, SEM Standard Error of Mean)

Fig. 4

Fut2 inhibits tumor proliferation in xenograft model A The typical images of nude mice injected with SW480 and HCT116 cells in control and Fut2-overexpression group. B The tumor weight of in control and Fut2-overexpression group of SW480 and HCT116 cells. C The tumor volume of in control and Fut2-overexpression group of SW480 and HCT116 cells. D The representative image of Ki67 staining of nude mice injected with SW480 and HCT116 cells in control and Fut2-overexpression group. Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: *p < 0.05, **p < 0.01, ****p < 0.0001. (SEM Standard Error of Mean)

Fig. 5

Glycoproteomics reveals glycoproteins that may be associated with Fut2-mediated CRC tumorigenesis A Schematic illustration of our systems biology approach to identify N-glycoproteins mediating the effects of Fut2 on CRC metastasis. B The number glycoproteins with fold change over 2.0 in WT vs. Fut2^△IEC mice by proteomic analysis. C The differentially expressed glycoproteins by GO analysis in WT vs. Fut2^△IEC mice. D The differentially expressed glycoproteins by COG analysis in WT vs. Fut2^△IEC mice. E The differentially N-glycosylation expressed glycoproteins presented by the volcano plot in WT vs. Fut2^△IEC mice. F The relative protein level of MCAM in colon tissues from WT vs. Fut2^△IEC mice by proteomic analysis. G The relative N-glycosylation expression level of MCAM in colon tissues from WT vs. Fut2^△IEC mice by glycoproteomic analysis. Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: ***p < 0.001, ****p < 0.0001. (CRC colorectal cancer, WT wild type, MCAM melanoma cell adhesion molecule, SEM Standard Error of Mean)

Fig. 6

Fut2 interacts with MCAM and mediates the fucosylation of MCAM. A, B Co-immunoprecipitation experiments showed structural interaction between Fut2 and MCAM. C Immunoprecipitation experiments showed the expression of MCAM with or without biotinylated UEA-I in colon tissues of WT and Fut2^△IEC mice. D The typical western blot image of MCAM expression in vector and MCAM overexpression SW480 cell line with Fut2 overexpressed. E Immunoprecipitation experiments showed the expression of MCAM with or without biotinylated UEA-I in control and MCAM overexpression SW480 cell line with Fut2 overexpressed (with or without peracetylated 2-F-Fuc treated). Data are expressed as mean ± SEM. The data come from three independent experiments. (MCAM melanoma cell adhesion molecule, UEA-I Ulex Europaeus Agglutinin-I, WT wild type, 2-F-Fuc 2-fluorofucose, SEM Standard Error of Mean)

Fig. 7

Fut2 inhibits colon cancer via fucosylation of MCAM. A, B The typical image and corresponding statistical results of transwell migration or invasion assay in control and MCAM overexpression SW480 cell line with Fut2 overexpressed (with or without peracetylated 2-F-Fuc treated). C Wound-healing assay at 48 h in control and MCAM overexpression SW480 cell line with Fut2 overexpressed (with or without peracetylated 2-F-Fuc treated). D The CCK8 assay at different time points (24 h, 48 h and 72 h) in control and MCAM overexpression SW480 cell line with Fut2 overexpressed (with or without peracetylated 2-F-Fuc treated). Data are expressed as mean ± SEM. The data come from three independent experiments. In all panels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (MCAM melanoma cell adhesion molecule, CCK8 Cell Counting Kit-8, 2-F-Fuc 2-fluorofucose, SEM Standard Error of Mean)