Yanyang Zhao

Functional roles of N-glycans in cell signaling and cell adhesion in cancer

Glycosylation is one of the most common post‐translational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structures are associated with many physiological and pathological events, including cell growth, migration, differentiation, tumor invasion, host–pathogen interactions, cell trafficking, and transmembrane signaling. Emerging roles of glycan functions have been highly attractive to scientists in various fields of life science as they open a field, “Functional Glycomics”, that is a comprehensive study of the glycan structures in relation to functions. In particular, the N‐glycans of signaling molecules including receptors or adhesion molecules are considered to be involved in cellular functions. This review will focus on the roles of glycosyltransferases involved in the biosynthesis of N‐glycan branching and identification of cell surface receptors as their target proteins. We also suggest that the modulation of N‐glycans of those receptors alters their important functions such as cell signaling and cell adhesion which are implicated in cancer invasion and metastasis. (Cancer Sci 2008; 99: 1304–1310)

Branched N-glycans regulate the biological functions of integrins and cadherins

Glycosylation is one of the most common post‐translational modifications, and approximately 50% of all proteins are presumed to be glycosylated in eukaryotes. Branched N‐glycans, such as bisecting GlcNAc, β‐1,6‐GlcNAc and core fucose (α‐1,6‐fucose), are enzymatic products of N‐acetylglucosaminyltransferase III, N‐acetylglucosaminyltransferase V and α‐1,6‐fucosyltransferase, respectively. These branched structures are highly associated with various biological functions of cell adhesion molecules, including cell adhesion and cancer metastasis. E‐cadherin and integrins, bearing N‐glycans, are representative adhesion molecules. Typically, both are glycosylated by N‐acetylglucosaminyltransferase III, which inhibits cell migration. In contrast, integrins glycosylated by N‐acetylglucosaminyltransferase V promote cell migration. Core fucosylation is essential for integrin‐mediated cell migration and signal transduction. Collectively, N‐glycans on adhesion molecules, especially those on E‐cadherin and integrins, play key roles in cell–cell and cell–extracellular matrix interactions, thereby affecting cancer metastasis.

N-Acetylglucosaminyltransferase III Antagonizes the Effect of N-Acetylglucosaminyltransferase V on α3β1 Integrin-mediated Cell Migration

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of β1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that α3β1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in β1,6-GlcNAc-branched N-glycans on integrin α3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in β1,6-GlcNAc-branched N-glycans on the α3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the α3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.

Deletion of Core Fucosylation on α3β1 Integrin Down-regulates Its Functions

The core fucosylation (α1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently α1,6-fucosylation has been further reported to be very crucial by the study of α1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on α3β1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in α3β1 integrin-mediated events has been studied by using Fut8+/+ and Fut8–/– embryonic fibroblasts, respectively. We found that the core fucosylation of α3β1 integrin, the major receptor for laminin 5, was abundant in Fut8+/+ cells but was totally abolished in Fut8–/– cells, which was associated with the deficient migration mediated by α3β1 integrin in Fut8–/– cells. Moreover integrin-mediated cell signaling was reduced in Fut8–/– cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of α3β1 integrin.

N-Glycosylation of the β-Propeller Domain of the Integrin α5 Subunit Is Essential for α5β1 Heterodimerization, Expression on the Cell Surface, and Its Biological Function

The N-glycosylation of integrin α5β1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on theα5 integrin. Removal of the putative N-glycosylation sites on the β-propeller, Thigh, Calf-1, or Calf-2 domains of the α5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1–5) on the β-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the α5 subunit carrying only three potential N-glycosylation sites (3–5 sites) on theβ-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Δ3–5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the β-propeller domain of the α5 subunit is essential for heterodimerization and biological functions of α5β1 integrin and might also be useful for studies of the molecular structure.

Cell-Cell Interaction-dependent Regulation of N-Acetylglucosaminyltransferase III and the Bisected N-Glycans in GE11 Epithelial Cells INVOLVEMENT OF E-CADHERIN-MEDIATED CELL ADHESION

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and α1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

Microrna 130b Suppresses Migration and Invasion of Colorectal Cancer Cells through Downregulation of Integrin β1

MicroRNA 130b (miR-130b) is significantly dysregulated in various human tumor types. In this study, using a microarray assay, we characterized the upregulation of miR-130b expression in colorectal cancer (CRC) specimens. However, there is limited knowledge about the roles of aberrant miR-130b expression in CRC. Our studies in CRC cells demonstrated that miR-130b significantly decreases cell migration and invasion, but it has no evidently effects on cell proliferation and apoptosis. In the overexpression miR-130b CRC cells and the CRC specimens, we observed a decreased level of integrin β1 protein, which is considered as a key molecule involved in cell motility. The targeting of the 3′-UTR region of integrin β1 gene by miR-130b was revealed using a luciferase reporter assay. The regulation of integrin β1 by miR-130b was further shown using the miR-130b mimics and the inhibitor of miR-130b. The impaired motility of the miR-130b overexpression cells is recovered partly by the expression of integrin β1 lacking the 3′-UTR. Additionally, the knockdown of integrin β1 also gives rise to a decrease in cell migration and invasion, which is similar to the impeded motility due to overexpression of miR-130b in CRC cells. Furthermore, the inverse expressions of miR-130b and integrin β1 were observed in CRC specimens. In summary, these data demonstrate that miR-130b downregulates its target-integrin β1, leading to the impaired migration and invasion of CRC cells.

A gain-of-function mutation in Tnni2 impeded bone development through increasing Hif3a expression in DA2B mice.

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.

MicroRNAs as diagnostic and prognostic biomarkers in colorectal cancer

MicroRNAs (miRNAs) are key regulators involved in various tumors. They regulate cell cycle, apoptosis and cancer stemness, metastasis and chemoresistance by controlling their target gene expressions. Here, we mainly discuss the potential uses of miRNAs in colorectal cancer (CRC) diagnosis. We also shed light on the important corresponding miRNA targets and on the major regulators of miRNAs. Furthermore, we discuss miRNA activity in assessing the prognosis and recurrence of CRC as well as in modulating responsiveness to chemotherapy. Based on the various pro-oncogenic/anti-oncogenic roles of miRNAs, the advantages of a therapeutic strategy based on the delivery of miRNA mimics are also mentioned. Together, miRNA seems to be an excellent tool for effectively monitoring and targeting CRC.

Transforming growth factor (TGF) β1 acted through miR-130b to increase integrin α5 to promote migration of colorectal cancer cells.

Transforming growth factor (TGF)-β1 is a significant stimulator of tumor invasion and metastasis. More recently, it has been found that TGF-β1 acts through microRNAs to regulate their target genes to promote cancer progresses. However, such similar regulation is rarely reported in colorectal cancer (CRC). Here, we observed a decrease in TGF-β1 expression in CRC specimens, compared with matched adjacent normal tissues. In parallel, there was an increase in miR-130b characterized in the same samples by microarray assay. Further, treatment of CRC cells with TGF-β1 caused a significant decrease in the expression of miR-130b and an increased CRC cell migration. Luciferase reporter assay revealed that miR-130b directly targeted the 3′ untranslated region (3′UTR) region of integrin α5 gene, which encodes a key molecule involved in cell motility. Subsequently, in the overexpression of miR-130b CRC cells, we observed a decreased level of integrin α5 protein. The regulation of integrin α5 by miR-130b was further shown using the miR-130b mimics and inhibitor of miR-130b. And, knockdown miR-130b with inhibitor in the overexpression of miR-130b CRC cells recovered integrin α5 expression and integrin α5-mediated cell motility. Moreover, the inverse relevance between miR-130b and integrin α5 was also observed in CRC specimens. At last, the enhancement of integrin α5 in TGF-β1-treated cells can be reversed partly when rescuing miR-130b expression. Together, our findings suggested that TGF-β1 acted through miR-130b to promote integrin α5 expression, resulting in the enhanced migration of CRC cells.