Download Aiming at the sweet side of cancer: Aberrant glycosylation as

Cancer Letters 352 (2014) 102–112
Contents lists available at ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
Mini-review
Aiming at the sweet side of cancer: Aberrant glycosylation as possible
target for personalized-medicine
Vered Padler-Karavani ⇑
Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv 69978, Israel
a r t i c l e
i n f o
Article history:
Received 18 August 2013
Received in revised form 9 October 2013
Accepted 9 October 2013
Keywords:
Personalized-medicine
Sialic acids
Neu5Gc
Antibodies
Cancer
a b s t r a c t
One of the frontiers in cancer personalized-medicine aims at glycosylation. Cells are covered with a dense
sugar coat of glycolipids, glycoproteins and free glycans. In cancer, the characteristic cell surface glycosylation is frequently transformed due to altered expression of glycan-modifying enzymes. This often
leads to aberrant expression of sialic acids (Sia) that cap glycan-chains. Additionally, dietary intake of
the non-human Sia N-glycolylneuraminic acid (Neu5Gc) leads to natural metabolic-glycoengineering of
human carcinomas that accumulate and express Neu5Gc. This Sia provokes a polyclonal anti-Neu5Gc
xeno-autoantibodies response that can exacerbate cancer. This review highlights cancer-associated
changes in Sia expression and their potential for personalized-theranostics.
Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Cancer is a complex heterogeneous disease and the leading
cause of death worldwide. Selective biological therapies have
emerged to effectively treat certain types of cancers or to target
specific determinants that are expressed by many different tumor
types [1,2]. Yet patients do not respond uniformly to a given therapy reflecting their inherent heterogeneity. Technological advances in genomics paved the way towards matching a subset of
patients with unique genetic signatures that are most likely to benefit from a certain therapy [3–6], thus leading to individually tailored treatments [6–8]. Other high-throughput technologies have
also emerged to expand personalized-medicine beyond genomics
including proteomics, pharmacogenomics, nutrigenomics [6,8,9]
and more recently glycomics [10,11].
Carbohydrates play a major role in cancer and circulating or cell
surface tumor-associated carbohydrate antigens (TACA) serve as
diagnostics markers [12,13]. TACA represent a modified version
of the carbohydrates normal expression, which frequently involve
altered expression of sialic acids. While this has been extensively
described in the literature, recent research suggests a novel group
Abbreviations: Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic
acid; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucoseamine; Man, mannose; Fuc, fucose; GlcA, glucoronic acid; IdoA, iduronic acid;
Xyl, xylose.
⇑ Address: Department of Cell Research & Immunology, The George S. Wise
Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: +972 3 640
9016; fax: +972 3 642 2046.
E-mail address: [email protected]
0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.canlet.2013.10.005
of TACA that arise from the consumption of the dietary non-human
sialic acid, N-glycolylneuraminic acid (Neu5Gc). This sugar metabolically incorporates into cells like a ‘Trojan horse’ replacing the
human sialic acid thereby generating neo-TACA that become
immunogenic. Since Neu5Gc is a consumed dietary sugar it may
provide a unique opportunity for personalized-medicine that likely
relates to the individual’s dietary habits. To fully appreciate its potential, this assay will first review current literature of glycosylation and its changes in cancer focusing on aberrant sialic acid
expression (Sections 2–4). Subsequently, current knowledge on
the role of Neu5Gc and the related anti-Neu5Gc antibodies in cancer will be described (Section 5), followed by discussion of their
potential as novel personalized-cancer theranostics (Section 6).
2. Cell-surface glycosylation
Cell surface glycosylation is universal to all living cells reflecting
their physiological state, and perfectly positioned to mediate adhesion and motility, as well as intracellular signaling events [14,15].
Monosaccharide units serve as building blocks that are synthesized
via covalent glycosidic-linkages into chains termed glycans (oligosaccharides and polysaccharides) [16]. Each monosaccharide is
transferred from activated sugar-nucleotide donor to the acceptor
molecule in a stepwise fashion. The combined action of various enzymes (i.e., glycosyltransferases and glycosidases) leads to diverse
glycan structures [17]. These can exist either as free forms or conjugated to proteins and lipids (Fig. 1) and include: (1) glycoproteins
with complex and branched N-linked glycans (conjugated to
Asparagine) and/or with O-linked glycans (conjugated to Serine
V. Padler-Karavani / Cancer Letters 352 (2014) 102–112
103
Fig. 1. Common glycans on human cells. Examples of the major glycoconjugates classes are depicted, as described in the main text.
or Threonine) that are abundant on mucins; (2) glycosylphosphatidylinositol (GPI)-anchored proteins; (3) glycosaminoglycans
(GAGs) either as linear free polysaccharides (such as hyaluronan)
or attached to Serine residues of proteoglycans (such as heparan
sulphate and chondroitin sulphate); and (4) glycolipids, which consist of glycans linked to ceramide. In addition to cell surface glycans, nuclear and cytoplasmic proteins can be modified with Olinked N-acetylglucosamine (O-GlcNAc; conjugated to Serine). OGlcNAcylation occurs in the cytoplasm and modifies intracellular
proteins, regulating their activity together with phosphorylation
[18]. In light of its ubiquitous and versatile nature, glycosylation
hold promise for novel personalized-theranostics (therapeutics
and diagnostics), assuming one can differentiate between the glyco-patterns of the normal versus the disease state.
3. Aberrant cell-surface glycosylation in cancer
Glycosylation is remarkably dynamic and commonly modified
in cancer leading to the expression of cancer-associated antigens
[19], sometimes referred to as ‘‘onco-fetal antigens’’ that recapitulate expression normally limited to embryonic tissues [19,20]. The
two basic principles that phenotypically guide these changes are
incomplete synthesis and neo-synthesis of cancer-associated cell surface glycans [21–23]. These changes commonly apply to earlystage cancers and to advanced-stage cancers, respectively [24]. In
general, a shift from the normal glycosylation pathway leads to altered glycan expression due to one or more of the following
changes: (1) under- or overexpression of glycosyltransferases
deregulated at the level of epigenetics [25,26], transcription [27–
31], post-transcription [32] and/or chaperone [33]; (2) altered glycosidase activity [34–36,36,37]; (3) altered expression of glycoconjugate acceptor together with availability and abundance of the
sugar nucleotide donors [38]; (4) altered sugar nucleotide transporter activity [39]; and (5) improper function of the Golgi structure [40] where many of the glycosyltransferases are harbored
[41]. Evidence is accumulating that aberrant glycosylation contributes to various aspects of cancer development and progression,
including proliferation, invasion, angiogenesis, metastasis and
immunity [12,16,42]. Yet, oncogenic glycosylation is not random
but rather is limited to a distinct subset of glycans that become
modified, enriched or decreased on the tumor cell surface and
mediate either promotion or inhibition of tumor progression
[12,43,44].
4. Altered sialic acids expression
As early as 1960s there was considerable evidence that the surface properties of cancer cells are different than those of normal
cells [45] and that sialic acids largely contribute to that phenotypic
change [46–48]. Sialic acids (Sia) are nine-carbon backbone a-keto
acidic sugars with their carboxylate group normally negatively
charged at physiological pH. They are capping vertebrate glycans
and found a-linked to underlying sugars via their second carbon
to either galactose (a2 3Gal or a2 6Gal), N-acetylgalactosamine
(a2 6GalNAc) or to another Sia (a2 8Sia) [49] (but in some cases
also to N-acetylglucosamine (a2 6GlcNAc) [50]). Changes in Sia
level, linkage and distribution are associated with various aspects
of malignant transformation [44,51–54].
General increase in cell surface Sia was shown to promote metastatic potential [19,51,52,55–57] and result from various routes
[44]. Changes to the core structures of N-glycans are some of the
most common aberrant glycosylation in cancer. Increased activity
of the b1-6GlcNAc branching enzyme, N-acetylglucosaminyltransferase V (GlcNAc-TV or MGAT5), lead to larger and more branched
N-glycans thus providing additional acceptors for terminal sialylation (Fig. 2) [38,44,58]. Together with increased expression of sialyltransferases [59,60], these changes contribute to increased cell
surface sialylation and metastatic potential [38,38]. Similarly,
aberrant O-linked glycosylation can lead to increased sialylation.
Carcinomas (tumors of epithelial origin) overproduce mucins that
are heavily glycosylated high molecular weight glycoproteins
(e.g., MUC1, MUC4, MAC6, MAC5AC) characterized by dense clusters of O-glycans, although N-glycans can also be present [61–
64]. Cancer-associated mucin-type O-glycans tend to be truncated
due to a shift in the normal enzymatic machinery and are usually
highly sialylated and less sulphated (Fig. 2) [61–63,65,66].
Increased sialylation is also evident by expression of polysialic
acid (polySia or PSA) that is an oncofetal antigen associated with
various types of cancers (e.g., neuroblastoma, non-small cell lung
carcinoma, breast cancer) [12,67], as well as other diseases [67].
PSA is synthesized in the Golgi by the polysialyltransferases
ST8SiaII and ST8SiaIV, generating N-glycans with a linear homopol-
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Fig. 2. Common cancer-associated glycans. Some of the most common aberrant glycan motifs in cancer are described. Sialylated glycoconjugates are shaded in grey.
ymer of a2 8-linked Sia (n = about 8 to over 100; Fig. 2). These are
mainly conjugated to the neural cell adhesion molecule (NCAM)
[68–70], but can also be conjugated to other acceptors [69,71]. It
is more frequently expressed in high-grade tumors than in lowgrade tumors [72], and progression is associated with higher
expression level of PSA and its biosynthesizing enzymes (mainly
ST8SiaII) [73,74]. In addition, PSA has an anti-adhesive effect on
cell-cell interactions thus facilitating the detachment of cells from
the primary tumor, and thereby promoting cancer invasion and
metastasis [67,70,75,76].
Changes in expression of a2 3/6-linked Sia have also been described in cancer. Mass spectrometry analysis of human serum sialo-glycoproteins revealed that many sialylated glycans featured
increased levels of a2 6 sialylation in breast cancer [77] and lung
cancer samples [78], while a2 3 sialylation was decreased in lung
cancer [78] but increased in prostate cancer samples [79], in malignant brain tumors [80] and in ovarian serous carcinoma [81]. In
addition, tumor cells often express increased levels of a2 6Sia,
mainly due to upregulation of the ST6Gal-I [82–86] or ST6GalNAc
sialyltransferases [87–89] that respectively conjugate terminal
Sia to N-glycans or O-glycans and glycolipids [59,60,90]. There is
compelling evidence that a2 6Sia (but not a2 3Sia) negatively
regulate binding of the pro-apoptotic galectins, that typically bind
b-galactose-containing glycoconjugates, thereby promoting tumor
cell survival [56,91–93].
In addition to changes in the general expression of Sia, increased activity of sialyltransferases also leads to the overexpression of certain terminal glycan structures in various malignant
tissues. Rather than a single change in glycosylation, each type of
malignant tissue is characterized by a set of changes in glycan
expression that can be broadly expressed on various cancers [94–
96]. Fig. 2 summarizes some of the most common aberrant glycan
motifs in cancer including the aforementioned b1-6GlcNAc
branching in N-linked structures (competing with b1-4GlcNAc)
and polysialic acid (PSA) in N-linked structures; as well as Thomsen–Friedrich (T or TF), Thomsen–Nouvelle (Tn) and sialyl-Tn
(STn or CD175s) in O-linked structures; sialyl Lewis x (sialyl-Lex,
SLex or CD15s), sialyl-Lea (SLea or CA19-9) and Ley in either Nlinked, O-linked, or lipid-linked structures; and Globo H and the
gangliosides GM2 and GD3 in lipid-linked structures
[12,43,57,59]. Notably, many of these cancer-associated glycans
carry sialic acids at their tips, and those sialoglycans can be conjugated to either protein- or lipid-carriers (Fig. 2). Following is an
overview of the expression of specific sialoglycans in malignancy
and their biological effects that are commonly facilitated by interactions with lectins.
4.1. Expression of siayl-Lewis structures
The histoblood group Lewis (Le) antigens are found in most human epithelial tissues at the tips of various glycolipids and glycoproteins [97]. The biosynthetic acceptors for Le antigens are two
unique disaccharides to which activated fucose and sialic acid residues are added by specific fucosyltransferases and sialyltransferases, respectively. Type-1 disaccharide (Galb1-3GlcNAc) serves
as the precursor for Lea, Leb and SLea, while the Type-2 (Galb14GlcNAc) serves as the precursor for Lex, Ley and SLex (Fig. 3). Normally, Lea and Leb (Type-1 based) are widely expressed, while Lex
and Ley (Type-2 based) are found at relatively low levels. However,
the expression of the sialylated antigens, SLea and SLex, is significantly enhanced in cancer (Fig. 2) [24,57]. The expression of these
cancer-associated antigens results mainly from incomplete synthesis (e.g., increased expression of ST3Gal IV [57] and impairment
of subsequent a2–6 GlcNAc sialylation lead to expression of SLea
instead of the normal Disialyl-Lea [50]; Fig. 3), but in some cases
represent neo-synthesis (e.g., induction of SLex expression on adult
T-cell leukemia) [24,98]. Abnormal expression of such cancer-associated carbohydrate antigens can be intimately associated with genetic transformation within the cells [99]. For example, it has been
shown that in adult T-cell leukemia, the strong and constitutive
expression of SLex is due to the transcriptional activation of the
fucosyltransferase, Fuc-T VII (encoded by FUT7), which is the
rate-limiting enzyme in SLex synthesis in these cells [99]. On the
other hand, in advanced stage cancers of epithelial origin (carcino-
V. Padler-Karavani / Cancer Letters 352 (2014) 102–112
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Fig. 3. Biosynthesis of Lewis antigens. Lewis antigens derive from the substitution of Type-1 (Galb1-3GlcNAc) and Type-2 (Galb1-4GlcNAc) disaccharide sequences by
subsequent addition of fucose and sialic acid residues [24,57,112]. Main enzymes involved are indicated (red or green). Cancer associated carbohydrate antigens (Ley, SLea,
SLex) are in bold-dashed boxes, with the sialylated antigens in gray shading. ST6GalNAc VI and FucT III (green) compete for their common substrate Lec and can lead to
expression of SLea instead of the normal Disialyl-Lea through incomplete synthesis [50]. Similarly, SLex is expressed in malignancy instead of the normal 6S-SLex (L-selectin
ligand; the suggested normal biochemical pathway in green [183,184]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
mas) other fucosyltransferase isoenzymes are involved, mainly
Fuc-T III and VI (which are not expressed by leukemic cells)
[100]. Epigenetic silencing of some glycogenes (e.g., by DNA methylation or histone modification) that are involved in synthesis of
complex glycans have also been implicated in the expression of
SLea/SLex in the early stage cancers (representing incomplete synthesis), while in advanced stages transcription of other glycogenes
are accelerated under hypoxic conditions [25]. In addition, increased expression of the genes for some sugar transporters (i.e.,
GLUT-1 or UDP-galactose transporters) was implicated in the enhanced expression of SLea/SLex [101].
Both SLea and SLex contribute to hematogenous metastasis, in
which blood-invading cancer cells adhere to blood vessels endothelial cells in a process that requires the presence of carbohydrate
ligands on cancer cells and at the same time E-selectin receptors on
endothelial cells [24,102]. SLea mediates adhesion of cancer cells
derived from the lower digestive organs (colon and rectum), pancreas and biliary tract, while the SLex mediates adhesion of breast,
ovarian and pulmonary cancer cells [102,103].
E-Selectin is not constitutively expressed on endothelial cells,
but once induced by an inflammatory stimulus (i.e., TNF, IL-1a,
IL-1b) can be vigorously shed from the endothelial cell surface. Elevated levels of serum E-selectin in patients bearing SLea/SLex-positive tumors predicts a high risk for developing metastasis
[102,104,105]. In addition, under hypoxic tumor micoenvironment
there is a significant induction of SLea and SLex expression with
concomitant increase in E-selectin binding activity, together promoting tumor vascularization and growth [106]. Two other members of the selectin family include P- and L-selectin; The P-selectin
expressed within endothelial cells or platelets is stored in specific
granules but translocated to the surface upon stimulation, while Lselectin is known to be constitutively expressed on leukocytes. It
was recently demonstrated that ST3Gal-IV and ST3Gal-VI are the
major sialyltransferases involved in the generation of functional
selectin ligands in vivo [107]. Each selectin member has a distinct
ligand specificity: E-selectin binds SLex, while L-selectin requires
SLex carrying 6-O-sulfation at the GlcNAc moiety (Neu5Aca23Galb1-4(Fuca1-3)(SO3 6)GlcNAcb-R; 6S-SLex), and P-selectin reacts only when the carbohydrate determinant is carried by the specific core protein PSGL-1, which has sulfated tyrosine residues at
its N-terminal region (in some cases replaced by other core proteins e.g., CD24) [24,108,109]. Importantly, all selectins cross-react
with 6S-SLex making it a ‘universal selectin ligand’ and potential
potent selectin inhibitor [24]. However, 6S-SLex is preferentially
expressed on non-malignant epithelial cells, and tends to decrease
upon malignant transformation. Finally, because most cancer cells
express the non-sulfated version of SLex they are likely more
responsive to the E-selectins [110].
4.2. Expression of Siayl-Tn
Incomplete synthesis of O-glycans lead to generation of the low
molecular weight sugar antigens Tn, T and STn that become enriched in cancer, especially on mucins (Figs. 2 and 4) [111]. STn
(Neu5Aca2-6GalNAca-O-R) can be detected in almost all carcinomas (e.g., pancreas, ovarian, colorectal, stomach, liver, and breast)
at variable frequencies (5–100%) but correlating with invasive and
aggressive potential [112–115]. Conversely, it is rare in normal
healthy tissues mostly limited to the upper digestive tract
[114,116,117], or covered with O-acetylation [115]. The STnexpressing glycoproteins in human carcinoma include MUC1,
CD44 and MUC2 [87,116,118,119]. Two major alterations in the
normal O-glycan synthesis pathway lead to overexpression of
STn: (i) mutations in the T-synthase chaperone COSMC that blocks
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Fig. 4. Biosynthesis of mucin O-glycans. Most common mucin type O-glycans are depicted, main enzymes involved are indicated (red) [111]. Cancer associated carbohydrate
antigens (T, Tn, STn) are in bold-dashed boxes, with the sialylated antigens in gray shading. Mutations in the T-synthase chaperone COSMC [120,121] and/or overexpression of
ST6GalNAc-I [88,89] allow accumulation of STn in cancer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
O-glycan elongation and shift the pathway towards generation of
GalNAc (Tn) [120,121,121], and/or (ii) overexpression of ST6GalNAc-I that combines sialic acid onto GalNAc to make the STn
[88,89] (Fig. 4). Likewise, transfection of ST6GalNAc-I cDNA can induce STn expression in various cancer cell lines expressing Core-1
and Core-2 glycans likely competing with active T-synthase (Core1 b1-3-galactosyltransferase) [57,88,122]. Expression of STn leads
to decreased adhesion and increased tumor growth, mobility,
migration and invasion [87,123,124].
Altogether, these characteristics prompted the design of the STn
cancer vaccine ‘Theratope’, in which STn is conjugated to the
immunogenic protein carrier keyhole limpet haemocyanin (KLH)
[125]. However, despite initial encouraging results [126–128] a
large scale Phase III trial on women with metastatic breast cancer
failed to demonstrate that Theratope improved median time to disease progression or overall patient survival [129,130]. However, it
is worth noting that the vaccine was given to all enrolled patients
without prior screening for the expression of STn, thus it is possible
that if the treatment was ‘personalized’ for the potential responsive
patients the outcome of the trial may have been different [57]. In
addition, it was demonstrated in mice that Theratope-induced
tumor protection was dependent on the quantity of anti-STn
antibodies raised by immunization [131].
4.3. Altered gangliosides expression
Gangliosides are sialic acid-containing glycosphingolipids. Biosynthesis of gangliosides (Fig. 5) involves the sequential addition
of sialic acids to the precursor lactosylceramide (LacCer) by ST3Gal
V (GM3 synthase), ST8Sia I (GD3 synthase) and ST8Sia V (GT3 synthase) that leads to the biosynthesis of the precursor of a-series, bseries and c-series gangliosides, respectively representing the
mono-, di- and tri-sialylated gangliosides (Fig. 5) [57]. These are
normally arranged in glycosynapses, with their hydrophobic cera-
mide moiety anchored in the outer leaflet of the plasma membrane
and the hydrophilic sialoglycans extending outward from the cell
surface [132,133]. Gangliosides function as regulatory elements
either as receptors in cell–cell trans recognition or interact laterally
in cis to regulate downstream signaling of various proteins (e.g.,
insulin, epidermal growth factor, and vascular endothelial growth
factor receptors) [134,135].
Some gangliosides are frequently overexpressed in cancer
(Fig. 2; [94]) and play a key role in the induction of invasion and
metastasis [136,137]: GD2 in neuroblastoma [138] and small cell
lung cancer (SCLC) [139], GD3 and its modified version 9-O-acetyl-GD3 in melanoma [94,140–142], and GM2 in SCLC [143], (all
showing some expression in normal tissues). Fucosylated GM1
(Fucosyl-GM1) is more tumor-restricted and expressed in SCLC
with limited expression in sensory nerves [94,144–146]. Cancerassociated gangliosides are expressed either on the cell surface or
shed by tumors, exerting an immunosuppressive effect by sensitizing T lymphocytes to apoptosis [147]. Consequently, there have
been several efforts to generate cancer vaccines to these gangliosides [148]. Recent studies in human breast cancer stem cells
(CSC) indicated a functional role for the overexpressed GD2 and
GD3. Reduction in their expression caused a phenotypic change
from CSC to a non-CSC suggesting a possible novel approach in targeting human breast CSCs to interfere with cancer recurrence
[149]. Another novel approach to improve on cancer specificity
concerns the type of terminal sialic acid in the sialylated-cancer
associated antigen.
5. Expression of dietary immunogenic Neu5Gc in cancer and
related anti-Neu5Gc xeno-autoantibodies
N-glycolylneuraminic acid (Neu5Gc) is a non-human Sia that
accumulates on human carcinomas [150,151]. Neu5Gc is an excellent candidate for personalized-theranostics due to its unique
V. Padler-Karavani / Cancer Letters 352 (2014) 102–112
107
Fig. 5. Biosynthesis of gangliosides. Lactosylceramide (LacCer) serves as the precursor for the sequential addition of sialic acids to generate the mono-, di- and tri-sialylated
gangliosides (top row). These are the precursors for biosynthesis of a-series, b-series and c-series gangliosides that can further be sialylated [57,185]. Some of the depicted
gangliosides are more frequent in malignancies (dashed line) as well as the non-depicted 9-O-acetyl-GD3 and Fucosyl-GM1, as described in the text.
characteristic properties: it originates from the diet, it combines
with various glycans generating neo-sialylated-antigens, and it is
immunogenic in humans.
The two major Sias in mammals are N-acetylneuraminic acid
(Neu5Ac) and Neu5Gc that differ by a single oxygen atom. Hydroxylation of nucleotide-activated Neu5Ac by CMP-Neu5Ac hydroxylase (CMAH) leads to generation of Neu5Gc. However, this
enzyme is specifically inactivated only in humans, but not in all
other mammals studied to date [152], with no alternative
Neu5Gc-synthesis pathway [153]. Despite that, Neu5Gc is compatible with human cells’ biochemical pathways and is metabolically
incorporated from dietary mammalian foods [154–157] (Neu5Gc is
enriched in red meat and milk products, but low or undetected in
chicken and fish) [154,158]. In vitro studies showed that Neu5Gc is
incorporated into tumor cells mainly by macropinocytosis then
delivered into the cytosol by a lysosomal transporter [155] that
can be up-regulated under hypoxic conditions [159]. It was also
shown that Neu5Gc metabolism is enhanced by high cell growth
rates [160]. Once in the cytosol Neu5Gc is activated and incorporated into cell surface glycans replacing the native Neu5Ac [155].
Consequently, Neu5Gc appears at low levels on the cell surface of
human epithelia and endothelia but is especially enriched on carcinomas [150,154,161]. Conversely, Neu5Gc is recognized as foreign by the human immune system that promotes a polyclonal
highly
diverse
antibody
response
in
all
humans
[154,160,162,163], recognizing multiple Neu5Gc-containing antigens [162]. Anti-Neu5Gc IgM/IgG arise in infants at 6 month old
coinciding with dietary-Neu5Gc, and are likely induced via dietary-Neu5Gc uptake by commensal bacteria [164]. Thus, Neu5Gcglycans are termed ‘xeno-autoantigens’ and the antibodies against
it are termed ‘xeno-autoantibodies’, because Neu5Gc is presented
as self (auto) on the cell surface but at the same time recognized
as foreign (xeno) [162]. Consequently, such antibodies are potential barrier for xenotransplantation [165], and it was shown that
burn patients transiently exposed to xeno-pig-skin had high serum
anti-Neu5Gc IgG even 10 years (and up to 30 years) after the actual
procedure [166]. Furthermore, human anti-Neu5Gc antibodies
could be affinity-purified from human sera and were shown to
bind human carcinomas [162,167].
5.1. Role of anti-Neu5Gc IgG in cancer promotion and diagnostics
The effect of anti-Neu5Gc antibodies on tumor growth was
tested in a relevant mouse model. Neu5Gc-deficient mice
(Cmah / ) were injected with Neu5Gc-positive tumors, and once
established were treated with low dose anti-Neu5Gc antibodies.
This resulted in chronic inflammation-mediated tumor growth
stimulation that was suppressed by an anti-inflammatory treatment [168]. These results supported the notion that the combination of Neu5Gc with circulating anti-Neu5Gc antibodies might
contribute to the high frequency of diet-related human carcinomas
[169–173]. These findings established a novel paradigm of chronic
inflammation-mediated disease induced by metabolized dietarysugar that also spurs an immune response [168]. It was also later
supported by follow up studies in humans [174]; Subsequent
development of a specialized sialoglycan-microarray with multiple
Neu5Gc-/Neu5Ac-glycans allowed testing anti-Neu5Gc IgG responses in sera samples of cancer versus non-cancer patients. This
led to discovery of the novel antibody carcinoma biomarker, antiNeu5Gc-Sialyl-Tn IgG [174]. Neu5Gc-Sialyl-Tn resembles the
well-known cancer-associated carbohydrate antigen STn (Fig. 2),
only that Neu5Gc replaces Neu5Ac [174]. To further establish
anti-Neu5Gc antibodies as potential cancer biomarkers for early
detection of cancer, larger and longitudenal population studies
are needed. Recently, a novel method for detection of the total
polyclonal anti-Neu5Gc antibodies response was developed that
may facilitate high-throughput screening of human sera samples
for such further studies [175].
Similar to Neu5Gc-Sialyl-Tn, other sialylated-cancer-associated
antigens (Fig. 2) can occupy the sialic acid Neu5Gc instead of
Neu5Ac and thus may result in enhaced theranostic potential. It
was shown that expression of GM2 ganglioside is induced in can-
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cers, but in addition its Neu5Gc content increases by hypoxia-induced transcription of a sialic acid transporter gene resulting in
(Neu5Gc)GM2 that has high cancer specificity [25]. Similarly,
(Neu5Gc)GM3 was described as a cancer-associated antigen and
potential therapeutic target [176,177].
5.2. Role of anti-Neu5Gc IgG in immunotherapy
Since anti-Neu5Gc antibodies recognize a unique cell surface
marker on cancer cells they are also potential immunotherapeutics. Early evidence suggested that human sera with high antiNeu5Gc humoral response could promote complement-dependent
killing of leukemic cells [160]. Likewise, human serum or affinitypurified antibodies rich with high anti-Neu5Gc-Sialyl-Tn IgG reactivity could promote killing of human cancer cells expressing this
unique Neu5Gc-antigen by both complement- or antibody-dependent cellular cytotoxicity (CDC or ADCC) [175]. Further studies in
the human-like Neu5Gc-deficient mice model (Cmah / ) suggested
that the impact of the anti-Neu5Gc immune response may be dosedependent: while a low dose of affinity-purified human IgG supported tumor growth [168], a higher dose inhibited tumor growth
[174]. Anti-Neu5Gc antibodies can bind to Neu5Gc-positive tumors, but they can also recognize acellular circulating Neu5Gcantigens such as Neu5Gc-sialoglycoproteins.
5.3. Effects of Neu5Gc-antigens on glycosylated therapeutic antibodies
Varying amounts of Neu5Gc are found on many clinically-used
biotherapeutic glycoproteins (e.g., antibodies, inhibitors, cytokines
etc.), likely originating from the production process within non-human mammalian cell lines and/or the addition of animal-derived
tissue culture supplements [178,179]. Circulating anti-Neu5Gc
antibodies can capture such Neu5Gc-containing biotherapeutics
(e.g., anti-cancer drug Erbitux (Cetuximab)) and generate immune
complexes that can mediate rapid drug clearance. This leads to decreased effective concentration of the administered drug [178] that
eventually can reduce its efficacy. Thus, individuals with high antiNeu5Gc antibodies response that are treated with such Neu5Gcglycosylated biotherapeutics would likely have a diminished response to the therapy. Such detrimental outcome may be avoided
if patients take initial personalized-screening for presence of circulating anti-Neu5Gc antibodies.
6. Conclusions and perspectives
It is now well accepted that nutrition is likely the most important environmental factor that modulates expression of genes involved in metabolic pathways leading to various diseases [180].
It was also suggested that consumption of micronutrients can even
have long-lasting effects on the health of adult descendants [181].
Thus, identification of the mechanisms that lead to glycosylation
changes in cancer is crucial for designing better therapeutic interventions. A unique cancer-associated-glycan generally reflects several genetic defects that modify the normal glycosylation
pathways.
Aberrant sialylation in cancer provides a plethora of targets,
that when combined with metabolic incorporation of dietaryNeu5Gc provides further cancer specificity and unique opportunities for novel personalized ‘glyco-nutrigenomics’ that reflects the
mounted immune response. Neu5Gc-containing tumor-associated
carbohydrate antigens (Neu5Gc-TACA) are potential biomarkers
and targets for drug discovery and vaccine development. They provide a unique opportunity for personalized-theranostics based on
both the expression of the Neu5Gc-target and the level of antiNeu5Gc antibodies combined with individual patients’ analysis of
Neu5Gc-TACA that should be pre-tested and monitored throughout the therapy. In addition, there is now convincing evidence that
aberrant sialylation can be limited to individual sialo-glycoproteins rather than to the whole cellular expressed glycoproteins,
and that these can influence tumor cell behavior at multiple levels
(e.g., regulating conformation, surface retention or clustering,
modulating receptors interactions) [56,182]. Further mechanistic
knowledge of such unique sialo-glycoprotein effects may expand
the potential clinical targets, that when combined with Neu5Gc
metabolic engineering would likely provide increased cancer specificity and theranostic potential.
Conflict of Interest
None declared.
Acknowledgements
I would like to thank Prof. Ajit Varki at The University of
California San Diego for introducing me to the Sialic acid research
and for his excellent mentorship over the years. This work was
supported in part by grants from the Israeli National Nanotechnology
Initiative and Helmsley Charitable Trust for a focal technology
area on Nanomedicines for Personalized Theranostics and by the
European Commission FP7 Marie-Curie grant (PIIF-GA-2012-327726)
to V.P-K.
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