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SummaryThe roles of Aβ low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions ofSplitCrelabeled Aβ-LTMRs in this regard. Genetic ablation of SplitCre-Aβ-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of SplitCre-Aβ-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which Aβ-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of Aβ-LTMRs for treating mechanical hyperalgesia.

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the

Expression of galectin-3 in WT-β4-integrin expressing MDA-MB435S cells treated with galectin-3 shRNA or control shRNA.

Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N-glycans was found to be colocalized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc-branching formation on β4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo. The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N-glycans promoted β4-integrin–mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N-glycan–mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N-glycan–mediated tumorigenesis. These findings provide a novel role for N-glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3–N-glycan complex.Implications: N-Glycosylation of β4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3–N-glycan complex. Mol Cancer Res; 16(6); 1024–34. ©2018 AACR.

Angular contact ball bearings are widely used in the field of rotating machinery due to their obvious advantages such as relatively good positioning accuracy, high speed rotating performance and low cost, which have already become the most important transmission components. The heterogeneous thermal deformation caused by the high speed effect of bearing parts will lead to excessive noise and even gluing, which can further significantly reduce the machining accuracy of machine tool. Therefore, it is vital to improve the deformation resisting capability and operational reliability of the whole system. For this purpose, this paper presents a reliability model for computerized numerical control (CNC) lathe spindle by considering thermal effect. A five-degree-of-freedom quasi-static model considering thermal deformation is firstly proposed to calculate contact load and contact angle. Then the transient thermal network method is used to solve the temperature value of multi-node spindle-bearing system, and the validity of the proposed model is verified by experiments at different speeds. Next the modified first-order and second-moment method (FOSM) is used to calculate the reliability and reliability sensitivity of CNC lathe spindle deformation model considering thermal effect. Finally, the constrained nonlinear optimization method for the reliability model is proposed and applied to CNC lathe spindle. The results show that the reliability of the optimized model is significantly improved and the reliability robustness is enhanced.

Wear of rolling elements can produce a huge impact on the mechanical properties of screw-nut joint, and is one of primary causes resulting in the accuracy degradation or even failure of kinematic pairs. Many mechanical models have been constructed to investigate the vibration performance of rolling elements. But the wear influence is considered scarcely because of the complicacy of loading status and wear process. In this study, a novel wear prediction model can be proposed for screw-nut joint to overcome the above difficulty. An ultimate stiffness model can be constructed on basis of the wear model. The influences of running distance, external forces, preload, and structural parameters on wear performance are researched. A dynamic model of screw-nut joint with varying stiffness is constructed to carry out a detailed study on its vibration performance, particularly taking into account the coupling effect between wear and vibration. The influences of wear on the axial stiffness and vibration characteristics are studied based on the proposed model. Moreover, the change rules of axial stiffness and dynamic response under variable preloads are researched. The results indicate that the choice of parameters can produce an appreciable impact on the wear rate. The wear can result in the losses of stiffness and critical displacement of screw-nut joint. Although increasing the preload can improve the stiffness and the critical displacement, it also aggravates the wear degree. When the relative displacement between nut and screw is greater than the critical displacement, the balls with preload and axial force in opposite directions will be out of action, and the monotonic property of stiffness will also change. Due to the monotonicity of stiffness is not consistent before and after the critical displacement point, the amplitude-frequency curve presents a jumping phenomenon.

Bearings are the core components in various kinds of rotating machineries. With the increasing demand of reliable bearings in precision equipment, it is of great significance to analyze the motion error of bearings. In this article, a nonlinear dynamic model of angular contact ball bearings installed in pairs is constructed to describe its response characteristics. The definition of a failure mode in matched bearings is that the dynamic response of each inner race is larger than the allowed axial runout. This article introduces a feasible approach to evaluate the reliability of positioning precision of matched bearings with random geometric parameters. The statistical moments of dynamic response are calculated using stochastic perturbation method. The probability distribution function of state function relating to positioning precision is approached by Edgeworth series, from which the reliability and sensitivity are obtained. A pair of 7206B bearings is taken as an application instance of the proposed method. Monte Carlo simulation is employed to provide a benchmark on which to verify the precision and efficiency of the proposed method. In addition, the effects of mean values and variances of random geometric parameters on the positioning precision are analyzed, respectively.

Fucosyltransferase 8 (FUT8) and β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) are glycosyltransferases that catalyze α1,6-fucosylation and α2,6-sialylation, respectively, in the mammalian N-glycosylation pathway. They are aberrantly expressed in various human diseases. FUT8 is non-glycosylated but is responsible for the fucosylation of ST6GAL1. However, the mechanism for the interaction between these two enzymes is unknown. In this study, we show that serum levels of α2,6-sialylated N-glycans are increased in Fut8−/− mice, whereas the mRNA and protein levels of ST6GAL1 are unchanged in mouse live tissues. The level of α2,6-sialylation on IgG was also enhanced in Fut8−/− mice along with ST6GAL1 catalytic activity increase in both serum and liver. Moreover, it was observed that ST6GAL1 prefers non-fucosylated substrates. Interestingly, increased core fucosylation accompanied by a reduction in α2,6-sialylation, was detected in rheumatoid arthritis patient serum. These findings provide new insight into the interactions between FUT8 and ST6GAL1.

The N-glycosylation of integrin α5β1 is involved in multiple cell biological functions. Our group previously reported that the N-glycosylation of the Calf-1,2 domain on α5 subunit (S3-5,10-14) was important for its inhibitory effect on EGFR signaling through regulating α5-EGFR complex formation. In this follow-up study, we provide evidence that the N-glycosylation on integrin β1 subunit suppress cell growth by promoting its association with EGFR under fibronectin (FN)-coated conditions. Expression of wild-type (WT) β1, but not the N-glycosylation mutant S4-6 β1, which contains fewer N-glycans, inhibited EGFR signaling and cell proliferation after cell adhesion to FN. Furthermore, consistent restoration of the N-glycans on sites 1-3 of β1 reinstated the inhibitory effects. Mechanistically, the N-glycosylation mutant of β1 (S4-6+1-3) inhibited the EGFR response upon EGF stimulation via facilitating the α5β1-EGFR complex formation. Moreover, we identified the N-glycosylation of sites 10-14 on α5 and 1-3 on β1 were most important for EGFR signaling. Taken together, these data indicate that α5S3-5+10-14β1S4-6+1-3 mutant represents the minimal N-glycosylation required for its regulation on EGFR signaling and cell proliferation, providing a plausible mechanism for the crosstalk between with α5β1 and EGFR.

The α2,3-sialylation of N-glycans is considered important but complicated because the functions of the three β-galactoside α2,3-sialyltransferases, ST3GAL3, ST3GAL4, and ST3GAL6, could be compensating for one another. To distinguish their specific functions, we established each individual knockout (KO) cell line. Loss of either the ST3GAL3 or ST3GAL6 genes decreased cell proliferation and colony formation, as opposed to the effect in the ST3GAL4 KO cells. The phosphorylation levels of ERK and AKT were significantly suppressed in the ST3GAL6 KO and ST3GAL3 KO cells, respectively. The cell aggregations were clearly observed in the KO cells, particularly the ST3GAL3 KO and ST3GAL6 KO cells, and the expression levels of E-cadherin and claudin-1 were enhanced in both those cell lines, but were suppressed in the ST3GAL4 KO cells. Those alterations were reversed with an overexpression of each corresponding gene in rescued cells. Of particular interest, the α2,3-sialylation levels of β1 integrin were clearly suppressed in the ST3GAL4 KO cells, but these were increased in the ST3GAL3 KO and ST3GAL6 KO cells, whereas the α2,3-sialylation levels of EGFR were significantly decreased in the ST3GAL6 KO cells. The decrease in α2,3-sialylation increased the α2,6-sialylation on β1, but not EGFR. Furthermore, a cross-restoration of each of the three genes in ST3GAL6 KO cells showed that overexpression of ST3GAL6 sufficiently rescued the total α2,3-sialylation levels, cell morphology, and α2,3-sialylation of EGFR, whereas the α2,3-sialylation levels of β1 were greatly enhanced by an overexpression of ST3GAL4. These results clearly demonstrate that the three α2,3-sialyltransferases modify characteristic target proteins and regulate cell biological functions in different ways.

Fucosylation of mucins, the main macrocomponents of the mucus layer that protects the digestive tract from pathogens, increases their viscoelasticity and shear stress resistance. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here we show that the levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients compared to normal individuals. Overexpressing FUT8, as observed in UC, in mucin-producing HT29-18N2 colonic cell line increases trafficking of MUC1 to plasma membrane and secretion of MUC2/MUC5AC. FUT8 depletion (FUT8 KD), instead, causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to shear stress compared to mucins secreted by FUT8 KD cells. These data fit well with the Fut8−/− mice phenotype, which are protected against UC. Fut8−/− mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins compared to Fut8+/+ mice. Together, these data reveal that FUT8 optimizes the viscoelastic properties of the extracellular mucous by controlling the quantities of mucins secreted.SIGNIFICANCE STATEMENTMucins, the major components of the mucous barrier that protects our body from pathogens, are heavily glycosylated proteins. Changes their amounts and properties will alter the viscosity of mucous. Here we show that FUT8, a glycosylation enzyme of the Golgi apparatus, can control the viscosity of secreted mucins. Mucin secreting cells of the distal colon express FUT8, but their levels are altered in Ulcerative colitis patients. As a result, mucous produced by these cells is easily washed away, which exposes them to pathogens. We suggest that a defective mucous production is the main cause of initial inflammation observed in disease. Our findings help in understanding how cells control the quality of mucins and provide a means to prevent Ulcerative colitis.

Assembly process designers typically confront the challenge of seeking information out of large volumes of non-structured files with a view to supporting the decision-making to be made. It is a leading concern that embedding data in text documents can hardly be retrieved semantically in order to facilitate decision-making with timely support. For tackling this gap, we propose in this paper a knowledge graph-based approach used to merge and retrieve information decided to be relevant within an engineering context. The proposed approach is to establish a multidimensional integrated assembly resource knowledge graph (ARKG) based on the structure of function-structure-assembly procedure-assembly resource, and this multidimensional integrated structure can well accomplish the retrieval of related knowledge. The upper semantic framework of ARKG is established by the assembly resource ontology model, which is a semantic-type framework involving multiple domains of knowledge to create instantiated data reflecting the full profile of the assembly resource for obtaining structured data of ARKG while avoiding the data redundancy problem. The ARKG method is validated through assembly scenario of the aircraft, and the results show the effectiveness and accuracy of the ARKG used by the assembly process designer in the assembly process design phase for retrieving the target knowledge of the assembly resources.

Transmembrane-4 L-six family member-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate tumor cell behaviors. However, the function and mechanism of TM4SF1 in esophageal squamous cell carcinoma (ESCC) metastasis remains unclear. Here, we find that TM4SF1 expression is increased and positively correlated with clinical TNM stage, N classification, differentiation, tumor size, and poor prognosis in ESCC patients. Interestingly, we demonstrate that TM4SF1 promotes ESCC cell adhesion, spreading, migration, and invasion, but not cell proliferation, in a laminin-dependent manner by interacting with integrin α6. Mechanistically, the TM4SF1/integrin α6/FAK axis signal pathway mediates cell migration under laminin-coating condition. Inhibiting FAK or knocking down TM4SF1 can attenuate TM4SF1-mediated cell migration and lung metastasis. Clinically, the TM4SF1/integrin α6/FAK axis positively correlates with ESCC. Altogether, these findings reveal a new mechanism of TM4SF1 in promoting ESCC metastasis via binding to integrin α6 and suggest that the cross-talk between TM4SF1 and integrin α6 may serve as a therapeutic target for ESCC.

Wear between balls and races has significant effects on the dynamic characteristics of bearing, which is the main reason to cause bearing failure. Some existing contact stiffness models were established to study the dynamic characteristics of bearing. However, the wear of bearing has been rarely investigated due to the complexities of contact load and wear mechanism. This paper presents a new dynamic wear simulation model of angular contact ball bearings mounted in pairs to solve this problem. A final contact stiffness model is established based on the wear model. The effects of running distance, horizontal load, preload, initial contact angle, number and diameter of balls on wear performances are analyzed. A generalized time-varying and piecewise-nonlinear dynamic model of angular contact ball bearings is established to perform an accurate investigation on its dynamic characteristics, especially considering the coupling effects of wear and rolling contact. The effects of wear on the contact stiffness and nonlinear dynamic characteristics are analyzed according to the dynamic model. Additionally, the variations of the contact stiffnesses and frequency responses with different preloads are discussed and the results indicate that parameter selection has significant effects on the wear and nonlinear response.

Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA–mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3β1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3β1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3β1.

Pancreatic cancer (PC) is characterized by rapid progression and a high mortality rate. The current treatment is still based on surgical treatment, supplemented by radiotherapy and chemotherapy, and new methods of combining immune and molecular biological treatments are being explored. Despite this, the survival rate of PC patients is still very disappointing. Therefore, clarifying the molecular mechanism of PC pathogenesis and developing precisely targeted drugs are key to improving PC prognosis. As the most common β subunit of the integrin family, integrin β1 has been proved to be closely related to the vascular invasion, distant metastasis, and survival of PC patients, and treatment targeting integrin β1 in PC has gained initial success in animal models. In this review, we summarize the various signaling pathways by which integrins are involved in PC, focusing on the roles of integrin β1 in the malignant behaviors of PC. Additionally, recent studies regarding the feasibility of integrin β1 as a diagnostic and prognostic biomarker in PC are also discussed. Finally, we present the progress of several integrin β1-based clinical trials to highlight the potential of integrin β1 as a target for personalized therapy in PC.

Background Pancreatic carcinoma is one of the deadliest malignant diseases, in which the increased expression of α1,6-fucosyltransferase (FUT8), a sole enzyme responsible for catalyzing core fucosylation, has been reported. However, its pathological roles and regulatory mechanisms remain largely unknown. Here, we use two pancreatic adenocarcinoma cell lines, MIA PaCa-2 and PANC-1 cells, as cell models, to explore the relationship of FUT8 with the malignant transformation of PDAC. Methods FUT8 knockout (FUT8-KO) cells were established by the CRISPR/Cas9 system. Cell migration was analyzed by transwell and wound-healing assays. Cell proliferation was examined by MTT and colony-formation assays. Cancer stemness markers and spheroid formations were used to analyzed cancer stemness features. Results Deficiency of FUT8 inhibited cell migration and proliferation in both MIA PaCa-2 and PANC-1 cells compared with wild-type cells. Moreover, the expression levels of cancer stemness markers such as EpCAM, CXCR4, c-Met, and CD133 were decreased in the FUT8-KO cells compared with wild-type cells. Also, the spheroid formations in the KO cells were loose and unstable, which could be reversed by restoration with FUT8 gene in the KO cells. Additionally, FUT8-KO increased the chemosensitivity to gemcitabine, which is the first-line therapy for advanced pancreatic cancer. Conclusions FUT8-KO reduced the cell proliferation and migration. Our results are the first to suggest that the expression of FUT8 is involved in regulating the stemness features of pancreatic cancer cells. General significance FUT8 could provide novel insights for the treatment of pancreatic carcinoma.

Systemic lupus erythematosus (SLE) is a complex multi-system autoimmune disease. Detection of anti-nuclear antibodies (ANA) is fundamental for the diagnosis of SLE. In the present study, we found that the level of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) is markedly up-regulated on immunoglobulin G (IgG) in the sera of SLE patients detected by Aspergillus oryzae lectin (AOL) blot. In sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA), the core fucosylation level was also found significantly increased in the sera from SLE patients with a higher ANA titer. To establish a rapid and sensitive laboratory test for the diagnosis of SLE, we prokaryotically expressed AOL and C3-D1-C3-D2-C3 of protein G (SpG3), and generate AOL-conjugated colloid gold immunochromatographic strips (ICS). The detection limit of core fucosylated IgG was 10 μg/mL for AOL-conjugated colloid gold ICS. As well as indirect immunofluorescence, the AOL-conjugated colloid gold ICS showed reliable results in the serum of 39 SLE patients. Our results indicated that the AOL-conjugated colloid gold ICS could serve as a rapid test for the detection of ANA and suspected cases of SLE.

The fucosylated carbohydrate moieties on intestinal epithelial cells (IECs) are involved in the creation of an environmental niche for commensal and pathogenic bacteria. Core fucosylation catalyzed by fucosyltransferase 8 (Fut8) is the major fucosylation pattern on the N-glycans of the surface glycoproteins on IECs, however, the role of IECs core fucosylation during infection remains unclear. This study was conducted to investigate the interaction between IECs core fucosylation and gut microbiota, and the effects of this interaction on protecting Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) infection. Firstly, the Fut8+/+ and Fut8+/- mice were infected with S. Typhi. The level of IECs core fucosylation and protein expression of intestinal mucosa were then detected by LCA blot and Western blot, respectively. The gut microbiota of Fut8+/+ and Fut8+/- mice before and after S. Typhi infection was assessed by 16S rRNA sequencing. Our results showed that core fucosylation was ubiquitous expressed on the intestinal mucosa of mice and had significant effects on their gut microbiota. Fut8+/- mice was more susceptive to S. Typhi infection than Fut8+/+ mice. Interestingly, infection of S. Typhi upregulated the core fucosylation level of IECs and increased the abundances of beneficial microorganisms such as Lactobacillus and Akkermansia spp. Further in vitro and in vivo studies demonstrated that Wnt/β-catenin signaling pathway mediated the elevation of IECs core fucosylation level upon infection of S. Typhi. Taken together, our data in this study revealed that the IECs core fucosylation plays an important role in protecting against S. Typhi infection via up-regulating the biological antagonism of intestinal microbiota. IMPORTANCE In this study, the importance of epithelial core fucose was explored through studies of mucosal surface-microbe interactions. We provide the first confirmation that the defective of Fut8 gene in mice resulted in susceptibility to S. Typhi infection, and the Wnt/β-catenin pathway is involved in the activation of Fut8 transcription in IECs.

As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8+ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8+/+ OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8-/- OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8-/- CTL, resulted in enhanced Fut8-/- CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.

The epithelial cell adhesion molecule (EpCAM) is one of the most frequently and intensely expressed of tumor-associated antigens, but the role that EpCAM plays in the proliferation, adhesion and migration properties of cancer cells remains unclear. In the present study, we screened several tumor cell lines and found that colorectal cancer CW-2 and epidermoid carcinoma A431 cells expressed relatively higher levels of EpCAM. In order to assess the biological functions of EpCAM expression in cell adhesion and migration, we established a knock out (KO) of EpCAM genes in both of these types of cancer cells via a CRISPR/Cas9 system. The elongated cell morphology was converted to a rounded morphology in the EpCAM-KO cells. These cells showed decreases in cell proliferation and migration into extracellular matrix proteins, as well as decreases in cellular signaling elements such as phosphorylated focal adhesion kinase (FAK), AKT and ERK. Moreover, the cell growth and the colony formation abilities were significantly decreased in EpCAM-KO cells. Importantly, co-immunoprecipitation analysis revealed that EpCAM associated with integrin β1. Also, the expression levels of integrin α5 were decreased in EpCAM-KO cells, compared with that in the wild-type cells. Taken together, these data clearly demonstrate that EpCAM associates with integrin β1 to regulate FAK/ERK signaling pathways in controlling cell adhesion, migration and proliferation via extracellular matrix adhesion, which provides novel mechanisms for EpCAM-mediated biological functions and cancer phenotypes.

Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N-glycans was found to be colocalized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc-branching formation on β4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo. The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N-glycans promoted β4-integrin–mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N-glycan–mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N-glycan–mediated tumorigenesis. These findings provide a novel role for N-glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3–N-glycan complex. Implications: N-Glycosylation of β4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3–N-glycan complex. Mol Cancer Res; 16(6); 1024–34. ©2018 AACR.

Chitosan oligosaccharides (COS) have been shown to regulate various cellular and biological functions. The aim of this study was to investigate the antimetastatic potency of COS and the underlying mechanism. Here, we established a stably N-acetylglucosaminyltransferase V (GnT-V)-overexpressed MCF10A cell line. As expected, GnT-V overexpression greatly promoted cell migration in the transfectants by using wound healing assay. However, the induction in the cell migration was significantly suppressed by an addition of COS. Curiously, COS inhibited the protein expression of GnT-V in a dose dependent manner. Consistent with that, the reactivities with datura stramonium (DSA) and leuko-agglutinating phytohemagglutinin (L4-PHA) lectins, which specifically recognize branched N-acetylglucosamine (GlcNAc) structure, were also suppressed by COS. Taken together, these results demonstrated COS inhibited breast epithelial cell migration through down-regulation of GnT-V and its products, branched N-glycans, indicating that COS may serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.

Background Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. Methods Distribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin–ligand interactions were analyzed using ligand blotting and ELISA. Results Megalins expressed on renal proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by α1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoform megalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoform megalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8–/– mouse kidneys, had significantly decreased binding activity. Conclusions N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin–RBP interactions. General significance Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity.