Hua-Bei Guo

N-Acetylglucosaminyltransferase V Expression Levels Regulate Cadherin-associated Homotypic Cell-Cell Adhesion and Intracellular Signaling Pathways

A common glycan alteration in transformed cells and human tumors is the highly elevated levels of N-linked β(1,6)glycans caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). Here, we define the involvement of GnT-V in modulation of homotypic cell-cell adhesion in human fibrosarcoma HT1080 and mouse NIH3T3 cells. Increased GnT-V expression resulted in a significant decrease in the rates of calcium-dependent cell-cell adhesion. Reduced cell-cell adhesion was blocked by function-blocking antibody against N-cadherin and abrogated by pre-treatment of cells with swainsonine, demonstrating the involvement of N-cadherin in the cell-cell adhesion and that changes in N-linked β(1,6)glycan expression are responsible for the reduction in rates of adhesion, although this reduction could be mediated by the altered N-linked glycosylation of glycoproteins other than N-cadherin. Overexpression of GnT-V had no effect on the levels of cell surface expression of N-cadherin; however, it did cause a marked enhancement of both β(1,6) branching and poly-N-acetyllactosamine expression on N-cadherin. GnT-V overexpression resulted in decreased N-cadherin clustering on the cell surface induced by anti-N-cadherin antibody and affected the outside-in signal transduction pathway of ERK mediated by N-cadherin. Overexpression of GnT-V sensitized stimulation of tyrosine phosphorylation of catenins by growth factors and expression of v-src, which is consistent with its reduction of cell-cell adhesion. In vitro, GnT-V-overexpressing cells showed increased motility concomitant with increased phosphorylation of catenins. Moreover, GnT-V-deficient embryo fibroblasts from GnT-V homozygous null mice (GnT-V-/-) express N-cadherin and showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V+/- mice. These levels of adhesion were inhibited significantly by transient expression of GnT-V, confirming the hypothesis that levels of GnT-V can regulate cadherin-associated homotypic cell-cell adhesion. Aberrant N-linked β(1,6) branching that occurs during oncogenesis can, therefore, lessen cell-cell adhesion, contributing to increased cellular motility and invasiveness.

Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase.

Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked β(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.

Regulation of Homotypic Cell-Cell Adhesion by Branched N-Glycosylation of N-cadherin Extracellular EC2 and EC3 Domains

The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked β(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412–52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some β(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion- mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherin-catenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling.

Specific posttranslational modification regulates early events in mammary carcinoma formation

The expression of an enzyme, GnT-V, that catalyzes a specific posttranslational modification of a family of glycoproteins, namely a branched N-glycan, is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V, we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation, an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover, her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors, evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls, and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that, in turn, regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset.

Knockdown of GnT-Va expression inhibits ligand-induced downregulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis

Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcbeta(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes.

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Background: GnT-V levels are implicated in regulating cancer stem cells and tumor development. Results: GnT-V levels via altered Wnt signaling regulate the compartment of colon cancer stem cells and tumor formation. Conclusion: GnT-V levels modulate Wnt signaling that regulate colon adenoma progression. Significance: A specific post-translational modification regulates Wnt signaling and colon cancer progression. Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apcmin/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apcmin/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.

Loss of expression of N‐acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins

We isolated mouse embryo fibroblasts (MEFs) from N‐acetylglucosaminyltransferase Va (GnT‐Va) knockout mice and studied the effects of loss of expression of GnT‐Va on asparagine‐linked glycans (N‐glycan) synthesis and the gene expression of groups of glycosyltransferases and galectins. Loss of GnT‐Va expression caused aberrant expression of several N‐glycan structures, including N‐linked β(1,6) branching, poly‐N‐lactosamine, bisecting N‐acetylglucosamine (GlcNAc) and sialic acid. Using quantitative reverse transcriptase‐PCR (qRT‐PCR), altered gene expression of several groups of glycosyltransferases and galectins was observed in GnT‐Va null MEFs, supporting the observed changes in N‐glycan structures. These results suggest that genetic disruption of GnT‐Va ultimately resulted in altered MEFs gene expression and decreased tumor progression associated with loss of GnT‐Va observed may result in part from a combination of effects from these altered gene expressions.

Expression of the Vacuolar H+-ATPase 16-kDa Subunit Results in the Triton X-100-insoluble Aggregation of β1 Integrin and Reduction of Its Cell Surface Expression

Vacuolar H+-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H+-ATPase via one of its transmembrane domains, TMD4, strongly associates with β1 integrin, affecting β1 integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of β1 integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose α5β1 integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of β1 integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of β1 integrin (premature form) and, consequently, the level of the 130-kDa form of β1 integrin (mature form). Further experiments showed that the normal levels of association between the premature β1 integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of β1 integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits β1 integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional β1 integrin.

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Background: Her-2-induced mammary tumor onset is significantly delayed in GnT-V knock-out mice. Results: The gene expression of the Pcdhβ cluster is up-regulated in her-2-induced tumors with GnT-V deletion. Conclusion: Up-regulation of the Pcdhβ cluster is one of the mechanisms for the reduced her-2-mediated tumorigenesis resulting from GnT-V deletion. Significance: Our findings shed new light on the molecular mechanisms of the effects of GnT-V on mammary tumorigenesis. Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

N-acetylglucosaminyltranferase VB expression enhances β1 integrin- dependent PC12 neurite outgrowth on laminin and collagen

N‐acetylglucosaminyltransferase VB (GnT‐VB, ‐IX) is a newly discovered glycosyltransferase expressed exclusively in high levels in neuronal tissue during early development. Its homolog, GnT‐V, is expressed in many tissues and modulates cell–cell and cell–matrix adhesion. The ability of GnT‐VB to regulate cell–matrix interactions was initially investigated using the rat pheochromocytoma PC12 neurite outgrowth model. PC12 cells stably transfected with GnT‐VB consistently showed an enhanced rate of nerve growth factor (NGF)‐induced neurite outgrowth on collagen and laminin substrates. Levels of TrkA receptor phosphorylation and downstream ERK activation induced by NGF were not influenced by GnT‐VB expression. No significant difference was observed in the rate of neurite outgrowth when cells were cultured on non‐coated culture dishes, indicating that integrin–ECM interaction is required for the stimulatory effects. Neurite outgrowth induced by manganese‐dependent activation of β1 integrin on collagen and laminin substrates, however, showed a significant increase in neurite length for the PC12/GnT‐VB cells, compared with control cells, suggesting that the enhancement is most likely mediated by alteration of β1 integrin–ECM interaction by GnT‐VB. These results demonstrate that GnT‐VB expression can modulate the rate of neurite outgrowth by affecting β1 integrin–ECM interaction.