Review

Gastric cancer: adding glycosylation to

the equation
Salome S. Pinho1,2, Sandra Carvalho1,2, Ricardo Marcos-Pinto2,3, Ana Magalhaes1,
Carla Oliveira1,4, Jianguo Gu5, Mario Dinis-Ribeiro6,7, Fatima Carneiro1,4,8,
Raquel Seruca1,4, and Celso A. Reis1,2,4
1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr Roberto Frias s/n, 4200-465 Porto,
Portugal
2
Institute of Biomedical Sciences of Abel Salazar (ICBAS), University of Porto, Rua de Jorge Viterbo Ferreira n.8 228, 4050-313
Porto, Portugal
3
Department of Gastroenterology, Centro Hospitalar do Porto, Largo Prof. Abel Salazar, 4099-001 Porto, Portugal
4
Medical Faculty, University of Porto, Alameda Prof. Hernani Monteiro 4200-319 Porto, Portugal
5
Division of Regulatory Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan
6
Department of Gastroenterology, Portuguese Oncology Institute of Porto, Rua Dr. Antonio Bernardino de Almeida, 4200-072
Porto, Portugal
7
Centre for Research in HealthTechnologies and Information Systems (CINTESIS), Medical Faculty, Alameda Prof. Hernani
Monteiro 4200-319 Porto, Portugal
8
Department of Pathology, Hospital S.Joao, Alameda Prof. Hernani Monteiro, 4200-319 Porto, Portugal

Gastric cancer has a high incidence and mortality, so proliferation, invasion, metastasis, and angiogenesis
there is a pressing need to understand the underlying [2,3]. In the process of gastric carcinogenesis, glycans have
molecular mechanisms in order to discover novel bio- been shown to be involved in various steps, such as, cell
markers. Glycosylation alterations are frequent during pathogen interactions, cellcell and cellmatrix interac-
gastric carcinogenesis and cancer progression. This re- tions, cell differentiation, cancer cell migration, invasion
view describes the role of glycans from the initial steps and metastasis [2,4]. The understanding of glycans role in
of the carcinogenesis process, in which Helicobacter these steps sets the ground for the development of novel
pylori adheres to host mucosa glycans and modulates cancer diagnostic and prognostic biomarkers, as well as
the glycophenotype, as well as how glycans interfere novel pharmaceutical agents that target these molecules.
with epithelial cell adhesion by modulating epithelial Thus, deciphering the GC cell glycans code will help to
cadherin functionality in gastric cancer progression. Oth- provide cutting-edge knowledge with potential applica-
er mechanisms regulating gastric cancer malignant be- tions for the improvement of GC clinical management.
havior are discussed, such as increased sialylation This review provides a comprehensive view of the role of
interfering with key signaling pathways and integrin glycans in cancer with a particular focus on GC. We
glycosylation leading to an invasive phenotype. Applica- address the initial steps of the process of gastric carcino-
tions of these glycosylation alterations in the clinical genesis, in which Helicobacter pylori uses glycans to adhere
management of gastric cancer patients are discussed. and modulates the host gastric mucosa glycophenotype for
chronic infection. We also present how glycans interfere
Glycosylation in gastric cancer: understanding its with basic mechanisms involved in cellcell adhesion,
functional roles modulating the functionality of epithelial cadherin (E-cad-
Cancer is a major cause of death worldwide, remaining an herin), a key protein involved in the genesis and progres-
extremely important health problem [1]. Gastric cancer sion of gastric carcinoma. Furthermore, we present other
(GC) is the second leading cause of cancer-related death major mechanisms that modulate the malignant behavior
worldwide, affecting close to one million people per year [1]. of gastric carcinoma, such as the glycosylation (see Glos-
Glycobiology has become a focus of research in cancer sary) of integrins and the expression of sialylated glycans,
biology with several implications for the clinic. Glycans which interferes with key signaling pathways and leads to
are major components of several biomolecules, including the acquisition of an invasive phenotype. Finally, we dis-
glycoproteins, glycosphingolipids, and proteoglycans cuss important applications of glycosylation alterations as
expressed by cells and tissues. Glycans have been shown biomarkers to improve the clinical management of cancer
to be involved in various pathophysiological steps of tumor patients.
development and progression, regulating tumor cell
Gastric cancer: epidemiology, clinicopathological
Corresponding author: Reis, C.A. (celsor@ipatimup.pt).
Keywords: cancer; gastric cancer; glycosylation; e-cadherin; integrins; biomarkers.
features, and clinical management
GC represents a high burden in terms of incidence and
1471-4914/$ see front matter
2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2013. cancer-related mortality, being the fourth most common
07.003 malignancy in the world and the second leading cause of
664 Trends in Molecular Medicine, November 2013, Vol. 19, No. 11
Review Trends in Molecular Medicine November 2013, Vol. 19, No. 11

Glossary carcinogenesis pathway has been shown to be related to

the host characteristics, such as host genetic polymor-
Endoplasmic reticulum-associated degradation: is a protein degradation
pathway in the endoplasmic reticulum that has a central clearance function phisms of pro-inflammatory interleukins [9,10] and mole-
in the cell. Is a cellular pathway that targets misfolded proteins and mutant cules involved in adhesion of the bacteria [1113], as well
proteins for ubiquitination and subsequent degradation by a protein-degrading
complex called the proteosome.
as genetic variations of the bacteria [such as differences in
Familial intestinal gastric cancer (FIGC): an intestinal-type gastric cancer the H. pylori virulence factors cytotoxin-associated gene A
syndrome with familial aggregation. The criteria of the International Gastric (cagA) and vacuolating cytotoxin A (vacA)] [9] (Figure 1).
Cancer Linkage Consortium for FIGC vary accordingly with countries with high
incidence of FIGC or low incidence of FIGC. The germline genetic defect
Other risk factors have been described, such as diet [14]
underlying the disease remains unknown. and familial aggregation [15]. Almost half of the world
Glycosylation: a post- and co-translational modification process in which population is infected with H. pylori, and if occurring, GC
carbohydrates (glycans, saccharides, or sugars) are covalently attached to
proteins, lipids, or other organic molecules. It is a tightly regulated process that
is clinically diagnosed four or more decades later. During
occurs in the endoplasmic reticulum and Golgi compartment of all eukaryotic this period, a prolonged precancerous process takes place,
cells. This process depends on the availability of nucleotide sugar donors and represented by a cascade of events with the following
on the coordinated action of a large number of enzymes (glycosyltransferases
and glycosidases). The majority of mammalian proteins synthesized in the sequential histopathologic stages: chronic active non-atro-
endoplasmic reticulum undergo glycosylation. There are two main classes of phic gastritis, multifocal atrophic gastritis, intestinal
protein glycosylation: N- and O-glycosylation. metaplasia (IM) (precancerous conditions), dysplasia (pre-
Hereditary diffuse gastric cancer (HDGC): an inherited form of diffuse-type
gastric cancer that represents a rare but incurable autosomal-dominant gastric cancerous lesion), and carcinoma [16] (Figure 1). The
cancer syndrome with high penetrance. It is a highly invasive type of tumor clinical guidelines for the management of these precan-
with early onset gastric cancer (diagnosed before 45 years of age).
cerous conditions and lesions on the stomach recommend a
Approximately 45% of families with HDGC have germline alterations in the
E-cadherin (CDH1) gene. This early gastric cancer is located beneath an intact schedule for endoscopic follow-up of patients with exten-
mucosal surface, and therefore early detection is extremely difficult. Prophy- sive precancerous multifocal conditions [17,18]. Recently,
lactic total gastrectomy is usually advised after the age of 20 and before the age
of 40. In 1999, the International Gastric Cancer Linkage Consortium (IGCLC)
a new hereditary syndrome has been identified, gastric
proposed specific criteria to define HDGC. Families with aggregation of gastric adenocarcinoma and proximal polyposis of the stomach
cancer and an index case with diffuse IGCLC, but not fulfilling the IGCLC criteria (GAPPS), which is characterized by the autosomal domi-
for HDGC, are termed familial diffuse gastric cancer (FDGC).
Mucins: a family of glycoproteins with a high content of serine, threonine, and
nant transmission of fundic gland polyposis, including
proline residues, and numerous O-linked glycans. Mucins can be membrane- areas of dysplasia or intestinal-type gastric adenocarcino-
bound or secreted onto mucosal surfaces. Mucins are high-molecular-weight ma restricted to the proximal stomach (Figure 1). Genetic
and heavily glycosylated proteins constituting an important interface between
many epithelial surfaces of the body and the exterior environment. The main
defects behind this syndrome have not been elucidated yet
characteristic of mucins is their ability to form gels. [19].
N-glycosylation: this is characterized by the addition of the glycan chains to an Unlike the intestinal type of GC, the diffuse subtype
aspargine residue of the nascent protein in a consensus sequence Asn-X-Ser/
Thr, where X can be any amino acid with the exception of proline. The resulting (representing nearly 30% of GC cases) tends to occur in
N-glycan structures are generally classified into three principal categories: high younger individuals, mainly females, and frequently
mannose, complex, and hybrid types (see Figure 3 in main text). depicts hereditary forms [20,21]. In contrast to the inci-
O-glycosylation: a stepwise process characterized by the covalent linkage of
glycan chains to the hydroxyl group of a serine or threonine. The initial step of dence of the intestinal subtype, which is steadily decreas-
O-glycosylation is controlled by a family of GalNAc transferases that transfer ing in most countries, the incidence of the diffuse subtype
an initial N-acetylgalactosamine (GalNAc) residue. No consensus sequence
is stable [5]. Diffuse-type GC develops without the inter-
defines an O-linked glycosylation site. The initial GalNAc can be extended,
branched and elongated by several glycosyltransferases. mediate steps of glandular atrophy and IM. It is charac-
terized by poorly cohesive cells, organized in cords or
micro-glands, as well as discohesive cells that may widely
cancer death in both sexes worldwide [1,5]. The global invade the gastric wall, some of which may be of the
incidence of GC has been declining over the past few signet ring cell type. Diffuse-type GC displays a major
decades, but the total number of cases is expected to rise molecular abnormality: defective intercellular adhesion
as a result of the ageing population [5]. Most cases of GC mainly resulting from E-cadherin dysregulation [22,23]
are sporadic in nature, but approximately 10% display (Figure 1). In most cases, this is due to abnormal expres-
familial clustering and only 13% of these are predicted sion of E-cadherin [an adhesion molecule encoded by the
to be hereditary [6] (Figure 1). human cadherin 1, type 1, E-cadherin (CDH1) gene]
Several GC classifications have been proposed over the [20,21,23]. Germline alterations (mostly mutations) of
past decades. The most commonly used are the World the CDH1 gene are the genetic cause of hereditary diffuse
Health Organization and the Lauren classifications, the gastric cancer (HDGC) [24].
latter describing two main histological subtypes, intestinal In countries without screening programs for early de-
and diffuse, that display different clinicopathological pro- tection of GC, most cases are diagnosed at an advanced
files and distinct epidemiological settings [7] (Figure 1). stage. Most patients die during the first year after diagno-
The intestinal subtype of GC represents nearly 70% of sis, even if submitted to costly and aggressive therapy [25].
the cases and predominates in high-risk areas, being more The five-year survival rates of GC remain extremely poor
frequently observed in older male patients [8]. The carci- (around 10%) [26]. The diagnosis of GC at advanced disease
nogenesis process leading to the intestinal subtype of GC stage increases the risk of relapse after surgical treatment.
is considered to be gradual and stepwise, leading to au- Consequently, GC remains a huge concern in clinical prac-
tonomously growing tumors with glandular structure [8]. tice, and therefore new biomarkers and molecular tools are
The H. pylori infection is a pivotal risk factor for gastric urgently needed in order to improve the success of early
carcinogenesis, with tumor development occurring only in diagnosis, prognosis, and therapeutic options for GC
a subset of individuals. The progression of the gastric patients and their relatives.
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Gastric carcinoma 90% sporadic forms

10% familial clustering (13% hereditary forms)
Diuse type (30%) Intesnal type (70%)
Normal gastric mucosa Helicobacter pylori Normal gastric mucosa
Weeks
CagA; VacA Supercial gastris
BabA; SabA
Years
Sporadic gastric cancer

Host suscepbility
Hyperplasc/dysplasc changes Environmental factors Polymorphisms
Chronic atrophic gastris
Years
Diet IL-1 T/T
Smoking IL1-RN *2/*2 Intesnal metaplasia
TNF- -A -308*A
Years
Dysplasia

Diuse-type carcinoma Intesnal-type carcinoma

(Sporadic) (Sporadic)

E-cadherin dysfuncon
Hereditary gastric cancer

Diuse-type carcinoma GAPPS (Intesnal-type ca.)

(Hereditary) (Hereditary)

In situ carcinoma Dysplasia

pagetoid spread

Proximal (fundic gland)

Normal mucosa polyposis

TRENDS in Molecular Medicine

Figure 1. The two pathways of gastric cancer. There are two main histological subtypes of gastric cancer, the intestinal subtype (70% of cases) and the diffuse subtype (30%
of cases), which display different clinicopathological profiles and occur in distinct epidemiological settings. Sporadic forms account for most cases (90%), whereas
hereditary forms account for only 13% of cases. Abbreviations: BabA, blood group antigen-binding adhesin; GAPPS, gastric adenocarcinoma and proximal polyposis of
the stomach; IL, interleukin; SabA, sialic acid-binding adhesin; TNF-a, tumor necrosis factor-a.

Glycosylation in normal gastric mucosa and gastric H. pylori is a major trigger of gastric carcinogenesis, and
carcinogenesis the colonization of the gastric mucosa depends on bacterial
The gastric mucosa displays a set of mucins that are the attachment to the gastric mucus layer and epithelial cells.
most frequent carriers of O-glycans in higher eukaryotes This binding is mediated by bacterial outer-membrane
[27]. In healthy gastric mucosa, the superficial foveolar proteins, named adhesins, which specifically bind to host
cells express the membrane-associated mucin 1 (MUC1) glycosylated receptors (Figure 2). Blood group antigen-
and the secreted MUC5AC, which are the major compo- binding adhesin (BabA) binds to the fucosylated blood
nents of the gastric mucus layer. The deeper gastric glands group antigens H-type 1 and Lewis b [35,36], which are
express the secreted MUC6 [2830]. MUC5AC is accompa- present in the MUC5AC mucin of gastric epithelial cells of
nied by co-expression of type 1 Lewis a and Lewis b healthy secretor individuals. Some H. pylori strains,
antigens, whereas MUC6 expression is associated with named generalists, admit modification of Lewis b with
the presence of type 2 Lewis x and Lewis y antigens AB glycan determinants, whereas specialist strains only
[30,31] (Figures 2 and 3). recognize the naked form of this epitope [37]. BabA was
Genetic polymorphisms in glycosyltransferase genes also shown to bind Globo H and Globo A, which are blood
lead to different expression profiles of gastric mucosa group O and A determinants, respectively, on type 4 core
histo-bood group antigens. For instance, the fucosylated chains [38]. Individuals infected with strains that harbor a
H-type 1 antigen is only expressed in the foveolar epitheli- functional BabA adhesin present a higher risk of develop-
um of secretor individuals and the difucosylated Lewis b ing more severe gastric lesions, including IM and gastric
antigen is only expressed in secretor and Lewis-positive adenocarcinoma [39].
individuals [32]. Secretor individuals constitute 80% of the The gastric glycosylation patterns define the H. pylori
Caucasian population and express a functional secretor tropism, and bacteria are mainly found at the surface of
fucosyltransferase 2 (FUT2) enzyme; Lewis-positive indi- mucous cells, where there is co-expression of MUC5AC and
viduals represent 90% of the Caucasian population and type 1 fucosylated Lewis antigens. By contrast, coloniza-
express the Lewis FUT3 enzyme [33]. Genetic polymor- tion of the deeper gastric glands where MUC6 is expressed
phisms of the genes encoding these enzymes are associated is rare. This distribution is explained by the presence of
with different host susceptibilities to infection by H. pylori unique O-glycan structures with terminal a1,4-linked N-
[11,34]. acetylglucosamine (a1,4GlcNAc) residues in the MUC6
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backbone. Terminal a1,4GlcNAc presents a natural antimi- cadherin dysfunction occurs in intra-epithelial pre-inva-
crobial activity by inhibiting the synthesis of a-glucosyl sive lesions, such as in situ carcinoma and pagetoid spread
cholesterol, an important constituent of the H. pylori cell of signet ring cells in the setting of HDGC (caused by CDH1
wall, and therefore suppressing bacterial growth [40]. In germline mutations) [20,58] (Figure 1). These lesions dis-
addition, a1,4GlcNAc expression was recently shown to play decreased or absent expression of E-cadherin. Similar
suppress tumor-promoting inflammation. Mice that lack pre-invasive lesions of GC have not been identified in the
the enzyme responsible for a1,4GlcNAc biosynthesis sporadic setting. In the intestinal subtype of GC, altera-
(A4gnt / mice) develop gastric adenocarcinoma in the ab- tions in E-cadherin expression occur predominantly at
sence of H. pylori infection, demonstrating that a1,4GlcNAc later stages of cancer progression (Figure 1). These altera-
loss is sufficient for cancer initiation in this model [41]. tions in E-cadherin expression have been associated with
In healthy conditions, gastric mucosa expresses most- poor clinical outcome of GC patients [23,59].
ly neutral fucosylated glycans, but H. pylori infection and Several molecular mechanisms explain the abnormal E-
the associated host inflammatory response can induce a cadherin expression in GC at the genetic and epigenetic
remodeling of the gastric glycophenotype with de novo level (Box 1), and also at the post-translational level
expression of sialylated antigens, including sialyl-Lewis through glycosylation.
x and sialyl-Lewis a. H. pylori has been shown to induce The mature E-cadherin protein contains a single trans-
an overexpression of b3-N-acetylglucosaminyltransfer- membrane domain, a cytoplasmic domain, and an extracel-
ase-5 (b3GnT5), leading to increased biosynthesis of lular domain, which comprises five repeated subdomains
sialyl-Lewis x [42]. H. pylori binding to inflamed tissue (EC1EC5) [60]. The extracellular domain mediates the
is mainly mediated by sialic acid-binding adhesin (SabA) Ca2+-dependent homophilic/homotypic cellcell adhesion.
[43]. The E-cadherin ectodomain has four potential N-glycosyla-
During gastric carcinogenesis, aberrant cell-surface gly- tion sites: two in EC4 (Asn554 and Asn566) and two in EC5
cosylation is frequently observed in precancerous condi- (Asn618 and Asn633). These N-glycan sites were found to be
tions, such as IM, which shows major alterations of mucin crucial for E-cadherin folding, expression, and biological
expression with marked de novo expression of the intesti- functions [61,62]. Cellcell adhesion is further accomplished
nal MUC2 mucin [44] and aberrant expression of the through the molecular interaction between the E-cadherin
simple mucin-type antigen sialyl-Tn (Figure 3) in goblet cytoplasmic domain and catenins (b-catenin, g-catenin,
cells [45,46]. Recently, plasminogen with sialyl-Tn was p120 catenin, and a-catenin), which couple cadherin
identified in the serum of patients with gastritis and IM, to the actin cytoskeleton [60]. The interaction with p120
suggesting its possible application as a biomarker for non- catenin has been shown to regulate E-cadherin internaliza-
invasive clinical screening and diagnosis [47]. tion, preventing its endocytosis and promoting its stability
In GC, various types of glycosylation alterations have at the cell surface [63]. E-cadherincatenin complex is the
been described and shown to play key roles in modulating major component of the adherens junctions (AJs), and its
cancer cell behavior (Figure 2). These molecular mecha- stability is regulated by E-cadherin glycosylation [64]. Fur-
nisms are discussed in detail in the following sections. thermore, the engagement of E-cadherin in homophilic
interactions can act as a mechanosensor, triggering the
Cellular adhesion in homeostasis and gastric cancer: activation of signal transduction pathways that are impor-
E-cadherin as a key molecule tant in tissue organization [65,66] and in which glycosyla-
Cellular adhesion determines the polarity of cells, partici- tion modifications play a role.
pating in the spatial and functional tissue organization of
epithelial cells [48,49]. Cellcell and cellmatrix adhesion Glycosylation as a modulator of gastric cancer cellular
are involved in various processes of tissue morphogenesis behavior
and in the maintenance of mechanical integrity and nor- Role of glycosylation as a regulator of E-cadherin-
mal cell physiology [50]. Integrins and E-cadherin are mediated gastric tumor development and progression
fundamental adhesion molecules with key roles in cell Although epigenetic and structural alterations of E-cad-
extracellular matrix (ECM) and cellcell interactions, re- herin may be considered instrumental for protein down-
spectively, being essential for normal tissue architecture. regulation or inactivation (Box 1), cancer cells have been
Alterations of glycosylation in these key molecules inter- shown to exhibit additional mechanisms that contribute to
fere dramatically with their functions and can modulate the acquisition of the malignant phenotype.
physiological and pathological processes. In GC, E-cadherin immunostaining does not always
One of the major regulators of epithelial tissue morpho- indicate the absence of expression (which is compatible
genesis and homeostasis is the cellcell adhesion molecule with epigenetic or structural alterations), but frequently
E-cadherin [51,52]. The critical importance of E-cadherin shows a redistribution from the cell membrane into the
in normal development is demonstrated by the lethality of cytoplasm (aberrant E-cadherin expression). Around 70%
E-cadherin knockout mice at an early stage in embryogen- of all GC cases showing E-cadherin aberrant expression do
esis [53]. not harbor CDH1 structural alterations (mutations, loss of
In the process of carcinogenesis, alterations in cellcell heterozygosity, or hypermethylation) [23] (Box 1).
and cellmatrix adhesion have a central role [50,54,55]. Recent evidence has shown that glycosylation can inter-
The downregulation or inactivation of E-cadherin-mediat- fere with E-cadherin functions in normal and pathological
ed cellcell adhesion may occur in early steps of cancer conditions. E-cadherin N-glycosylation seems to be essen-
development [5557]. Specifically, in diffuse-type GC, E- tial for its correct folding and transport to the cell surface
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(A)

Normal Gastris

Epithelial cells

Extracellular matrix (ECM)

Sle x

3GnT5
E-cadherin
Integrin

ECM

(B)
Invasive carcinoma

GnT-III
GnT-V

GnT-III ST6 Gal I

ST6 GalNAc I
GnT-V
ST3 Gal IV

Key:
- Membrane-associated glycoprotein - -catenin

- Glycolipid - -catenin

- H. pylori - -catenin

- Mucin gel layer - EPLIN

- F-acn - p120-catenin

TRENDS in Molecular Medicine

Figure 2. Schematic representation of the glycosylation phenotype in normal gastric mucosa and gastritis (A), and the glycosylation alterations in gastric cancer cells (B). (a)
In normal gastric mucosa, the epithelial cells display normal cellcell adhesion mediated by epithelial cadherin (E-cadherin) glycosylated with stabilizing glycoforms: the
bisecting N-acetylglucosamine (GlcNAc) N-glycan structures. The cellextracellular matrix (ECM) interaction is mediated by integrins that are also modified with bisecting
GlcNAc glycan structures. The mucin gel layer covering the gastric mucosa plays an important role in the colonization of Helicobacter pylori. Normal gastric cells express
fucosylated glycans such as Lewis b antigen, which serves as a ligand for H. pylori adhesion and infection, leading to the gastritis process. H. pylori binding is mediated by
bacterial adhesins, such as blood group antigen-binding adhesin (BabA), which specifically binds to Lewis b, and sialic acid-binding adhesin (SabA), which binds to sialyl-
Lewis x antigens expressed during inflammation of the gastric mucosa. H. pylori has been shown to induce an overexpression of b3GnT5, leading to increased biosynthesis
of sialyl-Lewis x. (b) During the gastric carcinogenesis process, the gastric epithelial cells undergo several changes in glycosylation that affect the malignant behavior of
cancer cells. E-cadherin expression induces N-acetylglucosaminyltransferase-III (GnT-III)-mediated glycosylation, which confers advantages over GnT-V-mediated
(Figure legend continued on the bottom of the next page.)

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Terminal oligosaccharide structures/Lewis angens

H-type 1 Lewis a Lewis b Sialyl Lewis a

R R R R
2 3 3 3 3
4 2 3 4 4

H-type 2 Lewis x Lewis y Sialyl Lewis x

R R R
2 4 4 3 2 4 3 3 4 3 R

Simple-mucin-type carbohydrate angens

STn
Tn

Ser/Thr Ser/Thr
2 6

N-glycans

High mannose Hybrid Complex

6 6 6
Bisecng Branched
2 2 2 4 4 4

2 3 6 2 3 6 2 2
2 2 2 2
6
3 6 3 6 3 6 4
3 6 3 6
4 4 4
4 4
4 4 4
6 4 4

Asn Asn Asn
Asn Asn

Symbolic representaons of monosaccharide

Key: Galactose (Gal) N-acetylgalactosamine (GalNAc) Fucose (Fuc)

Mannose (Man) N-acetylglucosamine (GlcNAc) Sialic acid (Neu5Ac)

TRENDS in Molecular Medicine

Figure 3. Summary of important glycan structures expressed in gastric tissues. Schematic representation of the most common glycans structures expressed in gastric tissues
either in normal or in pathological conditions. These glycan structures include terminal Lewis and sialylated Lewis structures; simple mucin type glycan structures, such as Tn
and sialyl-Tn; and the N-glycans, particularly the complex types: bisecting N-acetylglucosamine (GlcNAc) and the b1,6GlcNAc branched structures. Abbreviation: R, core
oligosaccharide.

[62]. Elimination of N-glycans at Asn633 was demonstrated Cytoplasmic O-glycosylation of E-cadherin (O-GlcNAc)
to affect E-cadherin expression, targeting it for endoplasmic was shown to block its cell surface transport, precluding
reticulum-associated degradation [62]. The regulation of the binding to p120 catenin, which results in reduced intercel-
recycling and trafficking pathways of E-cadherin has been lular adhesion [69,70]. N-glycosylation of E-cadherin has
demonstrated to be controlled by glycosylation [67,68]. also been reported to affect E-cadherin-mediated signaling

glycosylation. The modification of E-cadherin and integrins with bisecting GlcNAc structures is associated with suppression of tumor cell invasion. On the contrary, the
modification of E-cadherin with b1,6GlcNAc branched structures promotes dysfunction of E-cadherin-mediated cellcell adhesion, leading to an invasive phenotype. This
malignant phenotype is also due to an increased modification of integrins with the branched N-glycan structures that leads to an increased interaction with the ECM. The
overexpression of sialylated glycan structures, such as sialyl-Lewis x and sialyl-Tn, has also been shown to contribute to the aggressive phenotype of gastric tumor cells.
Changes in the expression of sialyltransferases and an induction of sialyl-Lewis x overexpression in gastric cancer cells alters the activation of signaling pathways, leading
to an invasive phenotype. Abbreviations: EPLIN, epithelial protein lost in neoplasm; b3GnT5, b3-N-acetylglucosaminyltransferase-5.

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Box 1. Genetic/epigenetic regulation of E-cadherin expression in gastric cancer

Loss or abnormal expression of the epithelial cadherin (E-cadherin) frequent somatic event affecting the CDH1 gene is promoter
gene [cadherin 1, type 1, E-cadherin (CDH1)] and protein in neoplastic methylation that leads to complete somatic inactivation of the CDH1
cells generally causes them to turn into the invasive components of gene [either as a single event (50%) or concomitantly with loss of
several human cancers, including gastric cancer (GC) [60]. Hereditary heterozygosity at the CDH1 locus (19%)] [139,140]. Primary HDGC
diffuse gastric cancer (HDGC) is the archetypical example of E- tumors that lack germline CDH1 mutations show exclusively CDH1
cadherin loss significance and is caused by heterozygous germline promoter hypermethylation in 50% of the cases. Among patients with
CDH1 point mutations in about 40% and large deletions in 4% of all FIGC, 17.0% of tumors showed hypermethylation [23].
families with this syndrome [24,136]. In the sporadic context, a recent MicroRNAs (miRNAs) have also emerged as a new layer of CDH1
comprehensive analysis showed that CDH1 somatic structural altera- gene regulation [141]. Members of the miR-200 family of miRNAs
tions were found in 10% of all GC cases: 10.9% within the sporadic have been implicated in the regulation of E-cadherin expression
setting and 9.7% among familial cases [23]. Importantly, the worst through direct targeting of the E-cadherin transcriptional repressors
patient survival rate among all GCs analyzed was seen in patients with zinc finger E-box-binding homeobox 1 (ZEB1) and ZEB2. Inhibition of
tumors carrying CDH1 structural alterations belonging to familial endogenous miR-200 expression levels led to a relief of ZEB1 and
intestinal gastric cancer (FIGC) families [23]. ZEB2 repression, a reduction of E-cadherin mRNA expression levels,
In all GCs, epigenetic silencing through CDH1 promoter hyper- and, consequently, a mesenchymal-like morphology and increased
methylation has been described, with frequencies varying from 50% cell migration [142]. Apart from the miR-200 family, miR-9 and miR-
to 83% in diffuse tumors and from 6.25% to 50% in intestinal tumors 101 have also been implicated in the network of E-cadherin regulation
[137,138]. Recently, a large GC study revealed CDH1 somatic [137]. miR-101 has been shown to be downregulated in GC in
hypermethylation in 20.7% of GC cases: 18.4% of the cases within comparison with normal gastric mucosas and, at least in 65% of GC
the sporadic setting and 26.4% within the familial setting. Particularly cases, this downregulation was due to deletions and/or microdele-
among HDGC patients carrying germline CDH1 mutations, the most tions at miR-101 genomic loci.

pathways, particularly the activation of the extracellular- [64]. Concomitantly with this, GC cells acquire a fibroblas-
regulated kinase pathway [71] (which has also been toid/mesenchymal appearance compatible with an EMT
described for neural cadherin [72]), suggesting that glyco- phenotype induced by GnT-V [87]. In addition, GnT-V-
sylation may influence the mechanotransduction role of mediated glycosylation on E-cadherin has been shown to
E-cadherin. Moreover, the E-cadherin N-glycans have been interfere with the molecular assembly and stability of AJ.
shown to affect E-cadherins intercellular adhesive func- The b1,6GlcNAc branched N-glycans on E-cadherin lead to
tions [73] by interfering with the molecular assembly and an impairment of b-catenin and p120 catenin recruitment,
maturity of the AJ, and consequently the assembly of tight disturbing the stability of AJs, which affects the cellcell
junctions [61,7476]. adhesion capability of gastric tumor cells [64] (Figure 2).
During malignant transformation, E-cadherin displays Moreover, b1,6GlcNAc branched N-glycans on cadherins
substantial alterations of its glycans [77] (Figure 2). E- can also affect the tyrosine phosphorylation of catenins
cadherin controls its own glycosylation by promoting ex- associated with increased cell migration and invasion
pression of N-acetylglucosaminyltransferase-III (GnT-III), [88,89]. GC cell line models overexpressing GnT-V showed
which confers a stabilizing E-cadherin glycosylation at the an increased metastatic capability when injected in athymic
cell membrane, increasing cellcell adhesion [78,79] nude mice [90]. In the clinical setting, gastric carcinoma cells
(Figure 2). This beneficial glycosylation of E-cadherin with (diffuse type) exhibiting an aberrant E-cadherin expression
bisecting GlcNAc N-glycans, catalyzed by GnT-III, is asso- without E-cadherin epigenetic or structural alterations
ciated with a delayed turnover of E-cadherin at the cell showed an increased modification with b1,6GlcNAc
surface and an inhibition of E-cadherin endocytosis [64,80]. branched structures [64].
E-cadherin modified with bisecting GlcNAc N-glycans con- In summary, other than epigenetic and structural
tributes to the increased recruitment of catenins and in- alterations, modification of the glycosylation of E-cadherin
creased AJ stability. This glycosylation type of E-cadherin can remarkably disturb its normal function by promoting
promotes increased intercellular adhesion and a downregu- tumor cell development and progression, constituting a
lation of intracellular signaling pathways involved in cell potential biomarker for clinical applications.
motility [81], supporting its contribution to tumor suppres-
sion (Figure 2). In addition, this E-cadherin N-glycoform Integrins and gastric cancer: role of glycan modifications
also contributes an epithelial phenotype that prevents the in integrin-mediated cell spreading and migration
epithelial-to-mesenchymal transition (EMT) process Integrins are the main link between a cell and the ECM,
[82,83]. The modification of E-cadherin with bisecting N- playing essential roles in cancer invasion and metastases.
glycoforms was found to have an important role in the They consist of a- and b-subunits. Each subunit has a large
suppression of human gastric carcinoma progression [64]. extracellular region, a single transmembrane domain and
Conversely, when E-cadherin is glycosylated by GnT-V, a short cytoplasmic tail (except for b4 integrin). The most
there are major effects on the dysregulation of its functions general feature of integrins is that their interaction with
in a GC context. GnT-V is known to be upregulated in gastric their ligand can activate intracellular signaling pathways
carcinoma [84], leading to biosynthesis of the b1,6GlcNAc and cytoskeletal formation (outside-in signaling). Another
branched N-glycans and contributing to cancer cell invasion important feature of integrins is inside-out signaling, in
and metastases [85,86]. GnT-V overexpression in a GC cell which intracellular signals received by integrins or other
line model induces significant alterations on E-cadherin receptors in turn activate their extracellular domain and
cellular expression, with a delocalization from the cell mem- contribute to the assembly of the ECM [91,92]. Therefore
brane into the cytoplasm (aberrant E-cadherin expression) glycosylation modifications of integrins play major roles in
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the interaction between the cell and the ECM by interfer- [104] (Figure 2). Recently, the overexpression of sialyl-
ing with biological functions, including cell spreading, Lewis x in cell lines transfected with ST3 b-galactoside
migration, and signal transduction. a-2,3-sialyltransferase 4 (ST3GAL4) was shown to in-
Integrins are glycoproteins and major carriers of N-gly- crease the cells invasive potential both in vitro and in vivo
cans. a5b1 integrin, which is one of the best-characterized [105]. This alteration of cell behavior was shown to be
integrins, is modulated with N-glycans that are required for mediated by the activation of c-Met receptor tyrosine
ab heterodimer formation and integrinmatrix interactions kinase and its downstream targets, such as focal adhesion
[93,94]. Indeed, the integrin cannot bind to its substrate or kinase and Src proteins, as well as the cell division control
be normally transported to the cell surface in the presence of protein 42 (Cdc42), Rac1 and RhoA GTPases [105]. These
the N-glycosylation inhibitor tunicamycin and deglycosyla- results demonstrate that the expression of sialyl-Lewis x in
tion mutants [9597]. Additionally, alterations on N-glycans membrane-associated and secreted glycoconjugates of GC
of a5b1 integrin could significantly contribute to tumor cells can cause major alterations in receptor tyrosine
formation, invasion, and metastases. Cells transformed kinases and intracellular signaling pathways controlling
with the oncogenic Ras gene showed an enhanced cell epithelial cell invasion behavior, and therefore they play a
spreading on fibronectin (FN) owing to an increase of key role in the aggressiveness of GC cells.
b1,6GlcNAc branched glycans in a5b1 integrins [98]. Simi- Another sialylated glycan frequently expressed in GC is
larly, the characterization of carbohydrate moieties of a3b1 the sialyl-Tn antigen [106] (Figure 2), which is recognized
integrin from non-metastatic and metastatic human mela- as an indicator of poor prognosis [107,108]. ST6GalNAc-I is
noma cell lines showed that b1,6GlcNAc branched struc- the major enzyme controlling the expression of sialyl-Tn
tures were highly expressed on a3b1 integrin from antigen in GC [109,110]. Sialyl-Tn expression in GC cells
metastatic cells compared with a3b1 integrin from non- has been shown to induce major morphological and cell
metastatic cells [99]. In a GC context, overexpression of behavior alterations, including decreased cellcell aggre-
GnT-V leads to severe peritoneal dissemination in athymic gation, altered ECM adhesion, and increased migration
mice and increased cellular migration owing to the in- and invasion in vitro [111]. These data indicate that the
creased expression of matriptase [90]; as well as due to sialyl-Tn antigen is able to modulate a malignant pheno-
alterations on E-cadherin-mediated cellcell adhesion [64] type, inducing a more aggressive cell behavior.
(as discussed above), but also through the specific modifica- Overall the alterations of expression of terminal sialy-
tion of a3b1 integrin with b1,6GlcNAc-branched N-glycans, lated antigens underlie key molecular events associated
which promotes increased cell migration [89] (Figure 2). with gastric tumor cellcell and cellmatrix interactions,
In contrast to GnT-V, the overexpression of GnT-III in signaling activation, migration, invasion, and metastases.
GC cells inhibited a3b1 integrin-mediated cell migration
by directly counteracting the effect of GnT-V-mediated Glycosylation as a tool for the clinical management of
glycosylation on a3b1 integrin [89] (Figure 2). As a result, cancer patients
GnT-III inhibits GnT-V-induced cell migration in GC cells. One of the major concerns of cancer clinical management
Two mechanisms have been proposed for the inhibition of (and GC in particular) is to improve the early diagnosis and
cell motility and invasion in GC cells on overexpression of the successful rate of the therapeutic strategies. New
GnT-III: an enhancement in E-cadherin-mediated cellcell approaches for cancer early diagnosis and treatment,
adhesion [64] (as discussed above) and the downregulation and new biomarkers for risk stratification are urgently
of integrin-mediated cellECM adhesion [89]. These needed, and glycans can be a source for such applications
results indicate that GnT-III counteracts GnT-V and (Box 2).
strongly suggest that remodeling of glycosyltransferase- Currently, most of the traditional cancer serological
modified N-glycan structures either positively or negative- markers, such as CEA and CA19-9 (for GC), CA125 (for
ly modulates cell adhesion and migration in a GC context ovarian cancer), and CA15-3 (for breast cancer), are based on
[64,73,100,101]. detection of glycoconjugates (glycoproteins and glycolipids)
Consistently, overexpression of GnT-III resulted in an shed from the tumor cells into the bloodstream [112114].
inhibition of a5b1 integrin-mediated cell spreading and
migration, and the phosphorylation of the focal adhesion
Box 2. Outstanding questions
kinase [102]. The affinity of the binding of integrin a5b1 to
FN was significantly reduced as a result of the introduction  Could specific glycosylation alterations be a source of novel
of a bisecting GlcNAc to a specific N-glycosylation site on biomarkers for improving the clinical practice in oncology? Are
they sensitive and specific for the cancer condition?
the a5 subunit [103].
 Could the specific detection of glycoforms on key proteins in
Overall these observations suggest that glycosylation serum and/or tissues contribute to the early diagnosis and
has a critical role in GC through modulation of key patho- determination of prognosis of cancer patients? Can we easily
physiological steps in gastric tumor genesis and progres- detect the glycomarkers during the routine screening of patients,
sion, such as cellcell and cellECM interactions. such as in gastric biopsies and/or in blood samples?
 How can we use tumor-associated glycans to improve cancer
therapy? Can we specifically modify glycan biosynthesis in cancer
Sialylated glycans and gastric cancer: modulating tumor cells? Can we modulate the activity of glycosyltranferases and the
cell signaling and behavior biosynthesis of tumor-associated glycans to control aggressive
GC cells frequently display high levels of terminal sialy- cancer cell behavior? Can we use proteins bearing tumor-
lated glycans such as sialyl-Lewis x, which has been asso- associated glycoforms as a target for immunotherapy ap-
proaches?
ciated with venous invasion and poor disease prognosis
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 Review Trends in Molecular Medicine November 2013, Vol. 19, No. 11

However, the reduced specificity and sensitivity of these carbohydrate determinants sialyl-Lewis x and Sda (a
serological assays for early detection of cancer is driving a blood group carbohydrate antigen) on specific proteins
search for novel biomarkers. In fact, the detection of specific may constitute interesting markers in the clinical setting,
glycoforms of a certain protein could contribute to the particularly as biomarkers for cancer cell biological be-
establishment of a biomarker with higher specificity for havior [122].
early detection of cancer or for diagnosis at a precancerous Glycosylation modifications are also good targets for
stage [115117]. For example, fucosylated a-fetoprotein (L3 cancer therapy. In the case of GC, targeting deleterious
fraction) was approved by the US Food and Drug Adminis- glycosylation pathways, such as synthesis of the
tration as a marker for early detection of hepatocellular b1,6GlcNAc branched N-glycans or terminal sialylation
carcinoma (HCC); it appears in serum at the stage of liver with sialyl-Lewis x, appears to be a promising approach
cirrhosis just before the onset of HCC, being therefore with potential treatment applications. Inhibition of the
considered the best approved marker in patients with biosynthesis of specific glycan structures can be achieved
HCC [118,119]. Recently, altered O-glycosylation (sialyl- using synthetic compounds, and these are attractive tools
Tn antigen) has been detected in circulating serum plas- for modulation of cancer cell behavior [123,124]. Swain-
minogen in patients with IM and gastric carcinoma [47]. sonine, an inhibitor of the Golgi protein a-mannosidase
Such alterations detected in early stages of the carcinogen- II, which blocks the synthesis of complex type N-glycans,
esis process might have valuable applications in the early has been demonstrated to have antitumor properties in
diagnosis setting. In addition, recent reports have proposed vitro [125], in vivo [126], and in patients with advanced
fucosylated haptoglobin as a novel biomarker for pancreatic malignancies [127]. However, its efficacy in the clinical
and colon cancer [120,121]. setting has been shown to be limited [128]. Additional
In line with this, the specific modification of E-cadherin specific compounds targeting specific glycosyltrans-
or integrins with the deleterious b1,6GlcNAc branched ferases expressed in specific cells might constitute a
structure might also be considered a potential biomarker source of important tools for modulating cancer cell be-
for selecting at-risk patients for clinical surveillance. It is havior contributing to cancer therapy.
tempting to suggest the assessment of the pattern of E- Furthermore, anticancer vaccines targeting tumor-as-
cadherin/integrin glycosylation in a gastric biopsy sample sociated carbohydrate antigens provide another appealing
as a biomarker for the early diagnosis and prognosis of option for cancer treatment; these have major advantages,
GC. Furthermore, in combination with the current diag- as they can be designed to incorporate only those elements
nostic procedures, the clinical search of specific glyco- required for a desired immune response [129131]. Vari-
forms in key proteins, both in tissues and/or in serum, ous studies have shown that passive immunotherapy using
is of utmost importance to improve early diagnosis, antibodies directed to glycoform-specific targets, such as
determination of prognosis, and risk stratification of can- MUC1 mucin, expressed in tumor cells can be effective in
cer patients (Figure 4). Several studies are now being inducing an antibody-dependent cell-mediated cytotoxicity
conducted in order to assess aberrant glycoforms of spe- [132]. Moreover, various active immunotherapy studies
cific proteins as candidate glyco-markers for improving targeting glycoproteins expressed in tumor cells have been
clinical practice in oncology. In addition, the terminal tested and are under clinical evaluation [133].

Carcinoma
Epithelial cells

Extracellular matrix (ECM) Invasive carcinoma cells

Glycan markers

Blood vessel
Diagnosis
Treatment

TRENDS in Molecular Medicine

Figure 4. Glycans as potential biomarkers in the clinical setting. The alteration of cellular polarity and topology of cancer cells can lead to the shedding of glycoconjugates
bearing important tumor-associated glycoforms into the bloodstream. These might constitute valuable molecular markers that can be detected in serological assays and
used to improve the early diagnosis, prognosis, risk stratification, and surveillance of cancer patients.

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Review Trends in Molecular Medicine November 2013, Vol. 19, No. 11

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