Fucosylated TGF-β receptors transduces a signal for epithelial-mesenchymal transition in colorectal cancer cells

Abstract

Background: Transforming growth factor-β (TGF-β) is a major inducer of epithelial-mesenchymal transition (EMT) in different cell types. TGF-β-mediated EMT is thought to contribute to tumour cell spread and metastasis. Sialyl Lewis antigens synthesised by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilised as tumour markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-β is unclear.

Methods: Colorectal cancer cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion and migration assays. The expression and phosphorylation status of TGF-β downstream molecules were analysed by western blot. Fucosylation of TGF-β receptor (TβR) was examined by lectin blot analysis.

Results: Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TβR and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT.

Conclusion: Fucosyltransferase 3/6 has an essential role in cancer cell adhesion to endothelial cells by upregulation of sialyl Lewis antigens and also by enhancement of cancer cell migration through TGF-β-mediated EMT.

Figure 1

Suppression of FUT3 and FUT6 inhibits Smad2 phosphorylation upon TGF-β stimulation. (A and B) Western blot analysis for FUT3 (A) and FUT6 (B). Lysate from Colo205 cells transfected with indicated siRNAs was electrophoresed and immunoblotted with FUT3 and FUT6 antibodies. The same membranes were reprobed with β-actin to verify equal amounts of protein. (C) FUT3 and FUT6 were required for phosphorylation of Smad2. Colo205 cells transfected with random, siRNA for FUT3 (siFUT3), siRNA for FUT6 (siFUT6) or siRNA for FUT3 and 6 (siFUT3+6) were treated with TGF-β for 6 h, then probed with pSmad2. Western blots for β-actin and Smad2 represent equally loaded amounts of protein. The experiments were repeated three times to confirm the results.

Figure 2

Suppression of either FUT3 and/or FUT6 by siRNA resulted in inhibition of cell migration and cell invasion. (A) Wound healing assay for CRC cell lines transfected with siFUT3 and/or siFUT6. Representative photographs for the indicated conditions (upper panel). Wound field surface area was calculated according to the manufacturer's instructions. Lower panel shows the average wound field surface area based on randomly chosen wells. *P<0.05 (NT or random vs. FUT3, FUT6, FUT3+6). (B) Cell invasion assay for CRC cell lines transfected with siFUT3 and/or siFUT6. Left panel shows representative photographs for the indicated conditions. Right panel shows the number of invaded cells that were transfected with the indicated siRNA. These results were confirmed by 3 independent experiments. *P<0.05 (NT or random vs FUT3, FUT6, FUT3+6).

Figure 3

Suppression of FUT3 and/or FUT6 inhibits TGF-β-induced EMT. (A and B) Suppression of EMT by siFUT3 and/or siFUT6. Colo205 (A) and LS180 (B) cells were treated with TGF-β for 3 days after transduction with random siRNA, siFUT3 and/or siFUT6, then probed with Zeb1, Snail and E-cadherin. Western blot for β-actin was used to control for equal protein loading. The relative mean density is presented as the ratio to no treatment (NT). (C) Representative images of Colo205 cells and immunohistochemical staining for E-cadherin with or without TGF-β. Cells were treated with TGF-β for 3 days after transduction with random siRNA, siFUT3, and/or siFUT6, then probed with E-cadherin and visualised by TRITC. The expression of E-cadherin was quantified and presented as the ratio to no treatment (right panel). The results were confirmed by three independent experiments.

Figure 4

Introduction of siFUT3 and/or siFUT6 resulted in inhibition of HSP27 and p38 activation through suppression of TβR-I fucosylation. (A) Phosphorylation of HSP27 and p38 was inhibited by downregulation of FUT3 and/or FUT6. Colo205 cells transfected with random, siFUT3, siFUT6 or siFUT3+6 were treated with TGF-β under the conditions described in the text, harvested and used for western blotting. (B and C) Colo205 cells transfected with random, siFUT3, siFUT6 or siFUT3+siFUT6 were solubilised and immunoprecipitated with TβR-I (B) and TβR-II (C) antibodies. TβR-I and TβR-II-bound proteins were probed with biotinylated-AAL. The relative mean density is presented as the ratio to no treatment (NT). The results were confirmed by three independent experiments.