Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFRmediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.sialic acid | glycoproteomics | glycan sequencing | click chemistry | mass spectrometry
The up-regulation of fucosyltransferase 8 (FUT8), the only enzyme catalyzing α1,6-fucosylation in mammals, has been observed in several malignant cancers including liver, ovarian, thyroid, and colorectal cancers. However, the pathological role and the regulatory mechanism of FUT8 in cancers remain largely unknown. In the current study, we report that the expression of FUT8 is up-regulated in nonsmall cell lung cancer (NSCLC) and correlates with tumor metastasis, disease recurrence, and poor survival in patients with NSCLC. Knocking down FUT8 in aggressive lung cancer cell lines significantly inhibits their malignant behaviors including in vitro invasion and cell proliferation, as well as in vivo metastasis and tumor growth. The results of glycoproteomic and microarray analyses show that FUT8 globally modifies surface antigens, receptors, and adhesion molecules and is involved in the regulation of dozens of genes associated with malignancy, suggesting that FUT8 contributes to tumor progression through multiple mechanisms. Moreover, we show that FUT8 is up-regulated during epithelial-mesenchymal transition (EMT), a critical process for malignant transformation of tumor, via the transactivation of β-catenin/lymphoid enhancer-binding factor-1 (LEF-1). These results provide a model to illustrate the relation between FUT8 expression and lung cancer progression and point to a promising direction for the prognosis and therapy of lung cancer.TGF-beta | E-cadherin | fucose F ucosylation, the transfer of fucose from GDP-fucose to glycoconjugates such as glycoproteins and glycolipids, is catalyzed by a family of enzymes called fucosyltransferases (FUTs). So far, 13 FUTs are known to be encoded by the human genome, including FUT1 to 11, protein O-fucosyltransferase 1 (POFUT1), and POFUT2. Through these FUTs, fucoses could be attached to N-, O-, and lipid-linked glycans through an α1,2-(by FUT1 and 2), α1,3-(by FUT3 to 7 and FUT9 to 11), α1,4-(by FUT3 and 5), or α1,6-(by FUT8) linkage, or directly link to the serine/threonine residues of EGF-like or thrombospondin repeats (by POFUT1 and 2, respectively) (1, 2). In mammals, fucosylated glycans have pivotal roles in many aspects of biological processes such as lymphocyte homing, immune responses, fertilization, and development (3). Moreover, aberrant fucosylation, which results from the deficiency or overexpression of FUTs, is associated with a variety of human diseases, including cystic fibrosis, leukocyte adhesion deficiency type II, and cancers (3, 4).Unlike other FUTs, which are functionally redundant, FUT8 is the only enzyme responsible for the α1,6-linked (core) fucosylation by adding fucose to the innermost GlcNAc residue of an N-linked glycan. A growing body of evidence indicates that core fucosylation is important for regulating protein functions. For example, deletion of the core fucose from the Fc region of IgG1 greatly improves its binding affinity to Fcγ receptor IIIa, which in turn enhances antibody-dependent cell-mediated cytotoxicity for over 50 folds (5, 6). Co...
Regulatory T cells (Treg) have been shown to prevent the development of allergic asthma; however, the role of Treg in asthma with established airway remodeling is unknown. To address this, we exploited an OVA-induced chronic asthma mouse model wherein Treg were adoptively transferred to the mice at chronic stage of the model. We found that among the structural alterations of airway remodeling, Treg selectively reduced the vessel numbers in both peritracheal and peribronchial regions and the lung parenchyma. Extracellular matrix deposition, mucus metaplasia, muscular hyperplasia, and vasodilation, as were also induced by chronic allergen challenge, were not affected by Treg. TUNEL staining of the lung sections revealed an increased endothelial cell (EC) apoptosis in mice receiving Treg transfers compared with their asthmatic counterparts. By using Matrigel angiogenesis assays, we showed that Treg inhibited EC angiogenesis both in vitro and in vivo. Treg preferentially expressed Notch ligand DLL4, and an anti-DLL4 blocking Ab abrogated the inhibitory effect of Treg on EC tube formation. In vivo, decreased airway and lung vessel numbers as well as ameliorated airway hyperresponsiveness after Treg transfers were reverted when Treg-derived DLL4 signal was blocked by the anti-DLL4 Ab. Our findings demonstrate a novel function of Treg whereby Treg down-regulate remodeling angiogenesis via proapoptotic DLL4-Notch signaling, and suggest a therapeutic potential of Treg in alleviating airway hyperresponsiveness of chronic asthma.
Abstractγδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.
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.
Our study demonstrates that GM2AP might serve as potential diagnostic and prognostic biomarkers in patients with lung cancer.
Multi-kinase framework promotes proliferation and invasion of lung adenocarcinoma through activation of dynamin-related protein 1
Aberrant fucosylation plays critical roles in lung cancer progression. Identification of the key fucosyltransferase as a therapeutic target may refine lung cancer management.Here, we identified a terminal α1,3-fucosyltransferase, FUT4, as the key prognostic predictor for lung adenocarcinoma through a transcriptomic screen in lung cancer cohorts. Overexpression of FUT4 promoted lung cancer invasion, migration, epithelialto-mesenchymal transition and cell adhesion in vitro, which can be reversed by genetic depletion of the enzyme. Notably, knockdown of FUT4 markedly curtailed lung colonization and distant metastases of lung adenocarcinoma cells in mouse xenograft models. Moreover, immunoprecipitation-mass spectrometry with anti-Lewis x, a major fucosylated glycan generated by FUT4, revealed increased fucosylation on cascade proteins of multiple oncogenic signalings including epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) pathways with concomitant transcriptional activation. The malignant phenotype provoked by FUT4-mediated fucosylproteomic networks can be pharmacologically diminished as treating FUT4-high expressing cells with EGFR inhibitors showed reduced metastatic capacity in vivo. Collectively, FUT4 represents a promising therapeutic target in lung cancer metastasis. Our data highlight the potential for integration of glycomics into precision medicine-based therapeutics.
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