Inhibition of α(1,6)fucosyltransferase: Effects on Cell Proliferation, Migration, and Adhesion in an SW480/SW620 Syngeneic Colorectal Cancer Model

Abstract

The present study explored the impact of inhibiting α(1,6)fucosylation (core fucosylation) on the functional phenotype of a cellular model of colorectal cancer (CRC) malignization formed by the syngeneic SW480 and SW620 CRC lines. Expression of the FUT8 gene encoding α(1,6)fucosyltransferase was inhibited in tumor line SW480 by a combination of shRNA-based antisense knockdown and Lens culinaris agglutinin (LCA) selection. LCA-resistant clones were subsequently assayed in vitro for proliferation, migration, and adhesion. The α(1,6)FT-inhibited SW480 cells showed enhanced proliferation in adherent conditions, unlike their α(1,6)FT-depleted SW620 counterparts, which displayed reduced proliferation. Under non-adherent conditions, α(1,6)FT-inhibited SW480 cells also showed greater growth capacity than their respective non-targeted control (NTC) cells. However, cell migration decreased in SW480 after FUT8 knockdown, while adhesion to EA.hy926 cells was significantly enhanced. The reported results indicate that the FUT8 knockdown strategy with subsequent selection for LCA-resistant clones was effective in greatly reducing α(1,6)FT expression in SW480 and SW620 CRC lines. In addition, α(1,6)FT impairment affected the proliferation, migration, and adhesion of α(1,6)FT-deficient clones SW480 and SW620 in a tumor stage-dependent manner, suggesting that core fucosylation has a dynamic role in the evolution of CRC.

High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model

“…Another aspect to highlight is the correlation between polylactosamine levels and enhanced invasion and metastasis, as seen in SW620, LS174T, and LoVo CRC cells. 86 In a previous study, we reported increased proliferation, colony formation, and a more mesenchymal phenotype in FUT8knockdown SW480 cells, 28 which is consistent with the current findings of highly branched/polylactosamine N-glycans in SW480 cells defective in FUT8 expression. Contrarily, FUT8attenuated SW620 cells appear to exceed the regulatory capabilities of FucT-8, as previously suggested 28,29 since the currently observed modifications in the antenna profile did not worsen their natural metastatic behavior.…”

“…71 In correspondence with the increased levels of α (2,6)-sialylation in the FUT8-attenuated clones of the SW480 and SW620 lines (Figure 3C), the results of functional assays indicated a reduced migration capacity in our SW480 FUT8knockdown cells. 28 As for the likely presence of bisecting GlcNAc residues in our N-glycome results, a significant reduction in oligosaccharide structures fulfilling the Hex = HexNAc relationship was shown in the FUT8-attenuated clones of the SW620 line. A close relationship has been previously described between the function of FucT-8 and N-acetylglucosaminyltransferase III (GlcNAc-T III) since both act directly on the N-glycan core.…”

“…27 We then developed a core-fucosylation-deficient cellular system in the syngeneic CRC lines SW480 and SW620 by shRNAiknockdown of the FUT8 gene. 28,29 Using this model, we found that FUT8 knockdown in the primary tumor cell line SW480 led to a more mesenchymal phenotype, with increased proliferation and reduced migration and adhesion, 28 as well as enhanced sensitivity to TRAIL-induced apoptosis. 29 Therefore, the role of FucT-8 in CRC remains somewhat uncertain, as a combination of pro-tumor and tumor-suppressive effects have been observed.…”

“…These findings reinforced the benefits of including LCA selection in the generation of FUT8-knockdown clones. 28 Correspondingly, the following MALDI-TOF MS glycome analyses were carried out using the FUT8-silenced LCAtreated cells. Each spot deposited on the MALDI plate provided an MS spectrum with a set of m/z signals that can be related to specific oligosaccharide structures or, if no tandem MS/MS (MS2) fragmentation is performed to elucidate the exact structure, with two or three indistinguishable possibilities.…”

FUT8-Mediated Core Fucosylation Promotes the Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension

“…Increased core fucosylation, along with an increase in salivary acidification and branch glycan structure, is the most frequently occurring cancer-related change among protein glycosylation [10]. The interaction of FUT8 with the proliferation and migration of various tumor cells and its epithelial-mesenchymal transition (EMT) have been studied [61][62][63]. In some cancers, FUT8 expression is significantly higher than in paracancerous tissues or cells and affects cell invasion, metastasis, and drug resistance [12,17,64].…”