FUT3 facilitates glucose metabolism of lung adenocarcinoma via activation of NF-κB pathway

Abstract

Objective: Fucosyltransferases (FUTs) molecules have been identified to be involved in carcinogenesis of malignant tumors. Nevertheless, the biological function of fucosyltransferases-3 (FUT3) in lung adenocarcinoma (LUAD) malignant phenotype remains unclear. Herein, we investigated the association between FUT3 and LUAD pathological process.

Methods: Immunochemistry, RT-qPCR and western blot assays were conducted to evaluate the expression of FUT3 in LUAD and corresponding adjacent tissues. The prognostic value of FUT3 was assessed via Kaplan‑Meier plotter database. The biological process and potential mechanism of FUT3 in LUAD were conducted via GSEA. Additionally, immunofluorescence and metabolite activity detection were performed to determine the potential role of FUT3 in LUAD glucose metabolism. The active biomarkers associated with NF-κB signaling pathway were detected via western blot. Subcutaneous tumor model was conducted to analyze the effect of FUT3 on tumorigenesis of LUAD.

Results: FUT3 was remarkably upregulated in LUAD tissues compared with adjacent tissues from individuals. FUT3 overexpression may predict poor prognosis of LUAD patients. Knockdown of FUT3 significantly inhibited tumor proliferation, migration and glucometabolic alteration in LUAD cells. Moreover, GSEA demonstrated that elevated FUT3 was positively related to NF-κB signaling pathway. Additionally, in vitro and in vivo assays also indicated that downregulation of FUT3 resulted in the suppression of oncogenesis and glucose metabolism via inactivation of NF-κB pathway.

Conclusion: Our findings demonstrated that FUT3 was involved in glucometabolic process and tumorigenesis of LUAD via NF-κB signaling pathway. FUT3 may be an optimal target for diagnosis and treatment of LUAD patients.

Keywords: FUT3; Glucose metabolism; Lung adenocarcinoma; NF-κB pathway.

Fig. 1

Differential expression and prognostic value of FUT3 in LUAD patients. (a) Pan-cancer analysis of FUT3 expression by Sangerbox database. (b) The FUT3 mRNA levels in LUAD tissues compared with the normal pulmonary tissues from TCGA. (c) Expression of FUT3 in different stages of LUAD by TCGA. (d) The FUT3 mRNA expression of LUAD tissues and adjacent tissues was detected via RT-qPCR. (e) Immunohistochemistry analysis of FUT3 expression in LUAD tissues and corresponding adjacent tissues. (f) FUT3 histological score in tumor tissues and adjacent tissues with immunohistochemistry. (g) Kaplan-Meier analysis of FUT3 expression with first progression (FP) and overall survival (OS) in LUAD patients. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 2

Promoter methylation and function enrichment analysis of FUT3. (a-b) Promoter methylation level of FUT3 in primary LUAD tumors and different stages of LUAD from UALCAN database. (c) Protein interaction network of FUT3 constructed by GeneMANIA. (d-e) Association of FUT3 with glycolysis. (***P < 0.001, ****P < 0.0001)

Fig. 3

FUT3 expression in LUAD cell lines. (a-c) FUT3 mRNA and protein expressions in the normal pulmonary epithelial cell and LUAD cell lines were evaluated by RT-qPCR and western blot. (d-f) RT-qPCR and western blot analysis were performed to detect FUT3 expression after transfection of FUT3 siRNA in H1975 and SPCA-1 cells. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 4

Downregulation of FUT3 inhibited the proliferation and migration of LUAD cell lines. (a-b) EdU staining assay was conducted to evaluate the proliferation of FUT3-downregulated LUAD cells, scale bar 50 μm. (c-d) Transwell migration assay was performed after transfection with si-FUT3 in H1975 and SPCA-1 cells, scale bar 50 μm. (*P < 0.05)

Fig. 5

FUT3 participated in glucose metabolism in LUAD cells. (a) GLUT1 and LDHA immunofluorescence was performed to present the alteration of glucose metabolism in H1975 and SPCA-1 cells after FUT3 knockdown. (b-c) The enzyme activity of G6PDH and LDH was measured in LUAD cells. (*P＜0.05, **P < 0.01, ****P < 0.0001)

Fig. 6

FUT3 was correlated with NF-κB pathway. (a-b) FUT3 related differential gene enrichment analysis was performed via KEGG database and GSEA. (c-e) Essential molecules’ expressions of NF-κB pathway were measured by western blot after transfection with si-FUT3 in H1975 cells and SPCA-1 cells. (**P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 7

Effect of FUT3 on the tumor growth and glucometabolic alteration of Lewis lung carcinoma cells (LLC) in vivo. (a) FUT3 mRNA expression in the C57BL/6J was evaluated by RT-qPCR. (b) Representative images of subcutaneous tumors dissected from C57BL/6J mice in different groups. (c) The growth curve showed the subcutaneous tumor of C57BL/6J mice injected with LLC cells with or without si-FUT3 and Asatone. (d) Comparison of tumor weight from different groups of C57BL/6J mice. (e) Glucometabolic changes in tumor tissues was detected by LDH activity assay. (*P < 0.05, **P < 0.01, ****P < 0.0001)