Fucosyltransferase 4 shapes oncogenic glycoproteome to drive metastasis of lung adenocarcinoma

Abstract

Background: Aberrant fucosylation plays a critical role in lung cancer progression. Nevertheless, the key fucosyltransferase with prognostic significance in lung cancer patients, the enzyme's intracellular targets, and complex molecular mechanisms underlying lung cancer metastasis remain incompletely understood.

Methods: We performed a large-scale transcriptome-clinical correlation to identify major fucosyltransferases with significant prognostic values. Invasion, migration, cell adhesion assays were performed using lung cancer cells subject to genetic manipulation of FUT4 levels. Genome-wide RNA-seq and immunoprecipitation-mass spectrometry were used to characterize major cellular processes driven by FUT4, as well as profiling its intracellular protein targets. We also performed lung homing and metastasis assays in mouse xenograft models to determine in vivo phenotypes of high FUT4-expressing cancer cells.

Findings: We show that FUT4 is associated with poor overall survival in lung adenocarcinoma patients. High FUT4 expression promotes lung cancer invasion, migration, epithelial-to-mesenchymal transition, and cell adhesion. FUT4-mediated aberrant fucosylation markedly activates multiple cellular processes, including membrane trafficking, cell cycle, and major oncogenic signaling pathways. The effects are independent of receptor tyrosine kinase mutations. Notably, genetic depletion of FUT4 or targeting FUT4-driven pathways diminishes lung colonization and distant metastases of lung cancer cells in mouse xenograft models.

Interpretation: We propose that FUT4 can be a prognostic predictor and therapeutic target in lung cancer metastasis. Our data provide a scientific basis for a potential therapeutic strategy using targeted therapy in a subset of patients with high FUT4-expressing tumors with no targetable mutations.

Keywords: Cancer metastasis; Fucosyltransferase; Glycoproteomics; Lung cancer; Terminal fucosylation.

Fig. 1

Higher levels of FUT4 is associated with poor prognosis in lung adenocarcinoma. (a) Heatmap of RNA-seq transcriptomic analysis for eight α-(1,3)-fucosyltransferases in surgically-resected lung cancer tissues at National Taiwan University Hospital (LUAD, N = 44, and LUSC, N = 37) and from TCGA database (LUAD, N = 517, and LUSC, N = 502), respectively. LUAD: lung adenocarcinoma. LUSC: lung squamous cell carcinoma. (b) Relationships between expression levels of individual fucosyltransferases and overall survival in patients with lung adenocarcinoma. (N = 428, High vs. Low expressions, cutoff=median; Cox proportional hazard models) (c,d) Kaplan-Meier curves of overall survival for patients with lung adenocarcinoma (LUAD) (c) or squamous cell carcinoma (LUSC) (d). Patients were divided into two groups based on the median value of FUT4 expressions. (e) The correlation matrix of all members in the FUT family based on the RNA-seq data of the TCGA lung adenocarcinoma samples. Numbers in the colored boxes represent Pearson correlation coefficients. Red and blue colors denote positive and negative correlations with statistical significance (p < 0.05), respectively. (f) mRNA levels of FUT4 in lung adenocarcinoma tissues compared to those of paired adjacent unaffected tissues from National Taiwan University Hospital (N = 75). Data information: p value in (b–d) was calculated by the log-rank test, and in (f) was calculated by Wilcoxon matched-pairs signed-rank test.

Fig. 2

FUT4 promotes aggressive phenotypes of human lung cancer cells. (a) FUT4 mRNA expressions in various human lung cancer cell lines analyzed by quantitative real-time PCR. All experiments were performed in triplicates and presented as mean ± SEM. LUAD: adenocarcinoma. LUSC: squamous cell carcinoma. (b) FUT4 mRNA (left panel) and protein levels (right panel) in A549 human lung cancer cells transduced with FUT4 (A549_vector, A549_FUT4^med, A549_FUT4^high) measured by quantitative real-time PCR and western blots. (c) Dot plots showing relative invasion ability of A549 cells measured by matrigel-based transwell invasion assays. Representative images of cells with DAPI nuclear stains in the lower chambers are shown in the right panels. Relative invasion ability was calculated using the cell number in the lower chamber of the transwell system for each clone compared to that of the vector control. (d) Dot plots showing migration velocity of A549 cells measured by single-cell tracking assays under a fluorescence microscope. Data were analyzed using Metamorph® software. The paths of cell migration were delineated in the right panels using pseudo-colors. (e) FUT4 mRNA (left panel) and protein levels (right panel) in CL1–0 human lung cancer cells overexpressed with FUT4 (CL1–0_vector, CL1–0_FUT4) measured by quantitative real-time PCR and western blots. (f) Dot plots showing relative invasion ability of CL1–0 cells measured by matrigel-based transwell invasion assays. Representative images of cells with DAPI nuclear stains in the lower chambers are shown in the right panels. Relative invasion ability was calculated using the cell number in the lower chamber of the transwell system for each clone compared to that of the vector control. (g) Dot plots showing the migration ability of CL1–0 cells measured by transwell migration assays. Relative migration ability was calculated using the cell number in the lower chamber of the transwell system for each sample compared to that of the vector control. (h) Diagram of in vivo extravasation and lung colonization assay following injection of lung cancer cells into the right ventricle of C57BL/6 mice. Mice were sacrificed 30 mins after intracardial injection. Whole lung perfusion with normal saline was performed to remove blood and cells not adhered to the pulmonary vasculature. (i) Numbers of lung cancer cells with FUT4 over-expression (left panel, A549_FUT4^high; right panel, CL1–0_FUT4) adhered to the vascular walls or retained in the lung tissues following intracardial injection were visualized under Zeiss Axio Observer microscope. Scale bar in c, d, f, and g; 100 μm. Scale bar in i ; 1 mm. p value in (b-d) was calculated by one-way ANOVA with Dunnett's test, and in (e, f, g, and i) was by Mann-Whitney test. All experiments were performed in three biological replicates and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: not significant.

Fig. 3

FUT4 potentiates lung homing ability and drives in vivo metastasis of lung cancer cells. (a) Whole animal imaging of in vivo tumor metastasis in nude mice with the IVIS® Spectrum imaging system at 24 hours following tail vein injections of A549 lung cancer cells pre-stained with Cyto-ID^TM long-term dyes. Quantification of signal radiances of metastatic foci in the dorsal and ventral sides of animals was graphed on the right panel. (b) Numbers of metastatic foci in the lungs of nude mice at 28 days after tail vein injection of lung cancer cells with FUT4 overexpression (A549_FUT4^med, A549_FUT4^high). Pictures of lung nodules from one representative mouse in each group are also shown on the right. (c) Numbers (percentages) of mice with spontaneous lung metastases in NOD/SCID mice bearing subcutaneous tumors of A549_vector, A549_FUT4^med, and A549_FUT4^high lung cancer cells. Data are summarized from three independent mouse experiments. (d) Representative hematoxylin & eosin (H&E) stained images of lung tissues from mice bearing subcutaneous xenograft lung tumors from (c). Scale bar in (b): 5 mm. Scale bar in (d): 0.1 mm. Data information: p value in (a, b) was calculated by one-way ANOVA with Dunnett's test. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. All experiments were performed in three biological replicates and presented as mean ± SEM.

Fig. 4

FUT4 mediates the activation of membrane trafficking and oncogenic pathways. (a) Top 15 positivelyenriched gene sets in genome-wide RNA-seq data of A549_FUT4^high and CL1–0_FUT4 cells versus respective vector controls. Enriched gene set data from Gene Set Enrichment Analysis (GSEA) with a nominal p value less than 0.05 and false discovery rate (FDR) less than 0.25 are presented. (b) GSEA enrichment plots of top 8 positively enriched gene sets in A549_FUT4^high and CL1–0_FUT4 cells, including membrane trafficking, cell cycle, TGFβ signaling, RNA processing, hypoxia, metastasis, EGF, and MAPK signaling pathways. (c) Immunofluorescent imaging analysis of cytoskeleton and cell morphology of A549 lung cancer cells with various levels of FUT4 over-expression (A549_vector, A549_FUT4^med, and A549_FUT4^high). Scale bar: 50 μm (left panel) and 20 μm (right panel). (d) Western blot analyses of EMT marker proteins in A549 lung cancer cells with various levels of FUT4 over-expression. (e) Heatmap of RNA-seq transcriptomes for the leading-edge genes of KEGG_pathway_in_cancer from GSEA analyses of TCGA lung adenocarcinoma. Top sidebars denote the mutation status of EGFR and expression levels of FUT4 grouped with the median value cut-off. p value in (a, b) was calculated by one-way ANOVA with Dunnett's test. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. Experiments were performed in three biological replicates and presented as mean ± SEM.

Fig. 5

FUT4 induces aberrant fucosylation of intracellular transport and signaling proteins. (a) LC-MS²/MS³ analysis of N-glycomic profiles of A549 and CL1–0 lung cancer cells with FUT4 overexpression. A significant increase in fucosyl LacNAc (m/z 638) contributed mostly by Le^x, concomitant with a decrease in sialyl LacNAc (m/z 825) was registered in both cell lines. (b) Flow cytometric analysis of cell surface glycans, Lewis x (Le^x), sialyl Lewis x (sLe^x), and Lewis y (Le^y) in FUT4-overexpressing A549 lung cancer cells. Left panels, representative flow cytometric dot plots of Le^x, sLe^x, and Le^y expressions are shown. Right panels, dot plots showing percentages of cells expressing individual surface glycans in three biological replicates. (c) Experimental diagram of immunoprecipitation (IP) with anti-Le^x antibody followed by liquid chromatography-mass spectrometry (LC/MS-MS). (d) Venn diagram comparing the numbers of identified proteins in FUT4-overexpressing cells vs. vector controls in A549 (left panel) and CL1–0 (right panel) cells. Pie charts show percentages of increased or decreased fucosylated proteins in FUT4-overexpressing cells. (e) Bar graphs reveal the percentages of FUT4 acceptor proteins (gray bars) in the core enriched genes of top 8 positively enriched gene sets upon FUT4 overexpression. The numbers above the bars denote the numbers (numerator) of identified proteins by anti-Le^x IP-MS and the numbers (denominator) of core enriched genes in individual gene sets. (f) Bar graphs show the relative abundance of fucosylated membrane trafficking-related proteins in A549_FUT4^high and CL1–0_FUT4 cells compared to their respective vector controls. The proteins also identified in RNA-seq GSEA analysis are marked with asterisks below.

Fig. 6

FUT4 enhances metastasis-related signaling via the fucosylation of cascade proteins. (a) Immunoprecipitation (IP) with anti-Le^x antibody followed by tandem mass spectrometry (LC-MS/MS) in A549_FUT4^high and CL1–0_FUT4 cells reveals key mediator proteins (in pink) in oncogenic signaling cascades including EGF, TGFβ, WNT and HIPPO pathways. (b) Protein networks showing protein-protein interactions between FUT4 acceptor proteins bearing higher levels of Le^x antigens in A549_FUT4^high cells relative to A549_vector cells. The data were analyzed by Cytoscape (ver. 3.6.1) using the GeneMania application (ver. 3.4.1). (c) Western blot analyses of signaling cascade proteins in the EGFR pathway in A549_FUT4^highversus A549_vector cells at 0, 0.5, 1, 2, and 6 hrs following the addition of 10 ng/mL EGF. (d) Quantifications of signal intensities in (c) for phospho-EGFR, phospho-ERK, and phospho-Smad2 from three biological replicates. (e) Western blot analyses of signaling cascade proteins in the TGFβ signaling pathway in A549_FUT4^highversus A549_vector cells at 0, 0.5, 1, 2, and 6 hrs following the addition of 5 ng/mL TGFβ. (f) Quantifications of signal intensities in (e) for phospho-TGFβ receptor I, phospho-ERK, and phospho-Smad2 from three biological replicates. p value in (d, f) was calculated by one-way ANOVA with Dunnett's test. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. All experiments were performed in three biological replicates and presented as mean ± SEM.

Fig. 7

Genetic depletion of FUT4 diminishes the aggressive phenotypes of human lung cancer cells. (a) FUT4 mRNA levels in CL1–5 lung cancer cells transfected with FUT4 shRNA (#751, #753, #792) measured by quantitative real-time PCR. (b) Dot plots showing relative invasion ability of CL1–5 cells with FUT4 knockdowns measured by Matrigel-based transwell invasion assays. Representative images of cells with DAPI nuclear stains in the lower chambers are shown on the right. (c) Dot plots showing relative migration ability of CL15 cells with FUT4 knockdowns. Relative migration ability was calculated using the cell number in the lower chamber of the transwell system for each clone compared to that of the scramble control. Representative images of cells with DAPI nuclear stains in the lower chambers are shown on the right. (d) Numbers of lung cancer cells with FUT4 knockdowns (CL1–5_shFUT4) adhered to the vascular walls or retained in the lung tissues following intracardial injection visualized under Zeiss Axio Observer microscope. Scale bar: 5 mm. p value was calculated by the Mann-Whitney test. *** p < 0.001. (e) Numbers of metastatic foci in the lungs of nude mice at 28 days after tail vein injection of lung cancer cells with FUT4 knockdown (CL1–5_shFUT4). Pictures of lung nodules from one representative mouse in each group are shown in the right panels. (f) Diagram of in vivo lung cancer metastasis assay via tail vein injection in nude mice subject to treatments with EGFR or TGF-β inhibitors. (g) Dot plots showing relative metastatic abilities of A549_vector or A549_FUT4^high lung cancer cells in nude mice receiving 0.25 mg/kg Afatinib or 15 mg/kg LY2157299 treatment. Numbers of metastatic foci in the lungs were counted and normalized to the numbers in the mock (normal saline)-treated groups. p value in (a, b, c, g) was calculated by one-way ANOVA with Dunnett's test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and in (d, e) was calculated by Mann-Whitney test. ns: not significant. Data in (a–f) were performed in three biological replicates and presented as mean ± SEM.