FUT2 inhibits the EMT and metastasis of colorectal cancer by increasing LRP1 fucosylation

Abstract

Background: Fucosyltransferase 2(FUT2) and its induced α-1,2 fucosylation is associated with cancer metastasis. However, the role of FUT2 in colorectal cancer (CRC) metastasis remains unclear.

Methods: The expression levels and clinical analyses of FUT2 were assessed in CRC samples. Migration and invasion assays, EMT detection, nude mice peritoneal dissemination models and intestinal specific FUT2 knockout mice (FUT2^△IEC mice) were used to investigate the effect of FUT2 on metastasis in colorectal cancer. Quantitative proteomics study of glycosylated protein, UEA enrichment, Co-immunoprecipitation identified the mediator of the invasive-inhibiting effects of FUT2.

Results: FUT2 is downregulated in CRC tissues and is positively correlated with the survival of CRC patients. FUT2 is an inhibitor of colorectal cancer metastasis which, when overexpressed, suppresses invasion and tumor dissemination in vitro and in vivo. FUT2 knock-out mice (FUT2^△IEC mice) develop AMO and DSS-induced tumors and promote EMT in colorectal cancers. FUT2-induced α-1,2 fucosylation impacts the ability of low-density lipoprotein receptor-related protein 1(LRP1) to suppress colorectal cancer invasion.

Conclusions: Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer. Video Abstract.

Keywords: Epithelial-to-mesenchymal transition (EMT); Fucosylation; Fucosyltransferase 2(FUT2); Low-density lipoprotein receptor-related protein-1(LRP1); Metastasis.

Fig. 1

FUT2 and altered protein fucosylation are linked with prognosis of CRC patients. A FUT2 mRNA in CRC tissues and normal tissues in TCGA dataset. B FUT2 expression level in 29 CRC tissues and adjacent non-tumor tissues detected by qRT-PCR. C FUT2 expression level in CRC tissues and adjacent non-tumor tissues detected by western blotting. D Quantification of C. **p < 0.01. E Representative images of UEA-I lectin fluorescence of CRC tissues and adjacent non-tumor tissues. F The overall survival analysis was plotted using GEPIA for patients with CRC

Fig. 2

FUT2 overexpression inhibits CRC cell EMT and invasiveness in vitro. A FUT2 levels in SW480 and HCT116 cells after overexpression of FUT2 were assessed by qRT-PCR and western blot (B). C Quantification of B. *p < 0.05. **p < 0.01. D β-catenin and vimentin levels in SW480 and HCT116 cells after overexpression of FUT2 were assessed by western blot. E Quantification of D. **p < 0.01. F Transwell migration and invasion by SW480 and HCT116 cells transduced with FUT2 or Vector. G–H Quantification of F. *p < 0.05

Fig. 3

FUT2 overexpression decreases in vivo CRC EMT and metastasis. A Representative images of peritoneal metastases of nude mice injected with HCT116 cells transduced with LV-FUT2 or LV-NC. B Quantification of A. **p < 0.01. C FUT2 expression in tumors was assessed by western bloting. D Representative images of IHC staining with N-cadherin, E-cadherin, β-catenin and vimentin antibody in xenograft tumor sections from FUT2 overexpression cells. E Quantification of D. *p < 0.05. **p < 0.01. F H&E-stained images of mouse lungs and livers at end-point. G–H Quantification of F. **p < 0.01. ***p < 0.001

Fig. 4

FUT2 knock-out mice develop AMO and DSS-induced tumors. A FUT2 levels in colon lysates of WT mice and FUT2 knock-out mice were detected by qRT-PCR. B Representative images of UEA-I lectin fluorescence staining revealed α-1,2 fucosylation in WT mice and Fut2^△IEC mice. C Representative images of H&E-stained colon sections after AMO and DSS administered. D–E Quantification of C. *p < 0.05. **p < 0.01. F Representative images of IHC staining with N-cadherin and E-cadherin in colon lysates in colon sections in WT mice and FUT2 knock-out mice after AMO and DSS administered. G FUT2 knock-out increased N-cadherin and vimentin, as shown by Western blotting

Fig. 5

1.2- fucosylated glycoproteins reveals regulators of EMT and metastasis. A GO enrichment analysis of N-fucosylated proteins in WT and Fut2^△IEC mice based on biological processes. B GO enrichment analysis (category of functional categories) of N-fucosylated proteins in WT and Fut2^△IEC mice. C Validation of 1,2 fucosylation in proteins bound with UEA-I related to migration in SW480 cells. D Validation of 1,2 fucosylation in proteins bound with UEA-I related to migration in HCT116 cells. E IP of LRP1 from cell lysates of SW480 cells transduced with LV-FUT2 or LV-NC. F IP of LRP1 from cell lysates of HCT116 cells transduced with LV-FUT2 or LV-NC. G LRP1 immunoprecipitation from cell lysates of HCT116 cells transfected with LV-FUT2 or LV-NC. Anti-LRP1 immunoprecipitates were treated with or without SGN-2FF and blotted with UEA-I or α-LRP1.

Fig. 6

LRP1 is a major mediator of the suppressing metastatic role by FUT2. A Downregulation of LRP1 mRNA in CRC tissues than normal tissues in TCGA dataset. B LRP1 IP on cell lysates of HCT116 cells stably overexpressing FUT2 or control vector and transfected with NC or LRP1 shRNA. Anti-LRP1 immunoprecipitates were blotted with UEA-I or α-LRP1. C Transwell migration and invasion assays by HCT116 cells stably overexpressing FUT2 or control vector and transfected with NC or LRP1 shRNA. D–E Quantification of C. F β-catenin and vimentin levels in HCT116 cells stably overexpressing FUT2 or control vector and transfected with NC or LRP1 shRNA were assessed by western blot. G Quantification of F