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International Journal of
Molecular Sciences
Review
Glycans as Biomarkers in Prostate Cancer
Emma Scott and Jennifer Munkley *
Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK;
Emma.Scott@newcastle.ac.uk
* Correspondence: Jennifer.munkley@ncl.ac.uk
Received: 21 February 2019; Accepted: 17 March 2019; Published: 19 March 2019
Abstract: Prostate cancer is the most commonly diagnosed malignancy in men, claiming over
350,000 lives worldwide annually. Current diagnosis relies on prostate-specific antigen (PSA) testing,
but this misses some aggressive tumours, and leads to the overtreatment of non-harmful disease.
Hence, there is an urgent unmet clinical need to identify new diagnostic and prognostic biomarkers.
As prostate cancer is a heterogeneous and multifocal disease, it is likely that multiple biomarkers
will be needed to guide clinical decisions. Fluid-based biomarkers would be ideal, and attention is
now turning to minimally invasive liquid biopsies, which enable the analysis of tumour components
in patient blood or urine. Effective diagnostics using liquid biopsies will require a multifaceted
approach, and a recent high-profile review discussed combining multiple analytes, including changes
to the tumour transcriptome, epigenome, proteome, and metabolome. However, the concentration on
genomics-based paramaters for analysing liquid biopsies is potentially missing a goldmine. Glycans
have shown huge promise as disease biomarkers, and data suggests that integrating biomarkers
across multi-omic platforms (including changes to the glycome) can improve the stratification of
patients with prostate cancer. A wide range of alterations to glycans have been observed in prostate
cancer, including changes to PSA glycosylation, increased sialylation and core fucosylation, increased
O-GlcNacylation, the emergence of cryptic and branched N-glyans, and changes to galectins and
proteoglycans. In this review, we discuss the huge potential to exploit glycans as diagnostic and
prognostic biomarkers for prostate cancer, and argue that the inclusion of glycans in a multi-analyte
liquid biopsy test for prostate cancer will help maximise clinical utility.
Keywords: prostate cancer; biomarkers; liquid biopsy; glycans; glycosylation
1. Introduction
Prostate cancer is the most common cancer in men, and is a major clinical burden [1]. In the last
decade, research into prostate cancer has advanced rapidly, and large advances have been made in both
finding new treatments and understanding the underlying biology. However, several areas of urgent
unmet clinical need remain, including the identification of: (i) validated biomarkers to complement
prostate-specific antigen (PSA) for screening; (ii) prognostic biomarkers with the clinical utility to
distinguish indolent and aggressive disease; (iii) molecular stratification methods and predictive
biomarkers; and (iv) surrogate end point biomarkers that are valid measures of therapeutic response
and survival [2]. As prostate cancer is a heterogeneous and multifocal disease, it is likely that multiple
biomarkers will be needed to guide clinical decisions. Fluid-based biomarkers would be ideal, and
attention is now turning to minimally invasive liquid biopsies, which enable the analysis of tumour
components in patient biological fluids such as blood and urine. Urine is an amenable bodily fluid
for prostate cancer biomarker discovery. It is easily obtained in a non-invasive way, and due to the
proximity of the prostate to the bladder, urine may carry markers that reflect the development and
progression of prostate cancer [3]. Proximal fluids of the prostate such as expressed prostatic secretions
Int. J. Mol. Sci. 2019, 20, 1389; doi:10.3390/ijms20061389 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019, 20, 1389 2 of 20
(fluid secreted by the prostate after a digital rectal prostate massage) can also be collected in urine and
used as a source of biomarkers [4]. Whereas traditional tissue biopsies only provide limited snapshots
of the
of tumour andsuch
the prostate might fail to reflect
as expressed heterogeneity,
prostatic secretions (fluidliquidsecreted
biopsies by can provideafter
the prostate a comprehensive
a digital
of the prostate such as expressed prostatic secretions (fluid secreted by the prostate after a digital
viewrectal
of allprostate
cancerous massage)
lesions can also be collected
(primary in urine as
and metastases) and
wellusedasas a source
offering theofopportunity
biomarkers [4]. to track
rectal prostate massage) can also be collected in urine and used as a source of biomarkers [4].
Whereas
tumour traditional
evolution [5]. tissue biopsies only provide limited snapshots of the tumour and might fail to
Whereas traditional tissue biopsies only provide limited snapshots of the tumour and might fail to
reflect
A 2018heterogeneity,
high-profile review liquid biopsies
in Nature canGenetics
provide a comprehensive view of all cancerous usinglesions
reflect heterogeneity, liquid biopsies can providehighlighted
a comprehensive that effective
view of all diagnostics
cancerous lesionsliquid
(primary and metastases) as well as offering the opportunity to track tumour evolution [5].
biopsies
(primarywilland
require the multiparametric
metastases) as well as offeringanalysis of several
the opportunity analytes
to track tumour(including
evolution [5]. changes to the
A 2018 high-profile review in Nature Genetics highlighted that effective diagnostics using liquid
transcriptome,
A 2018epigenome,
high-profile proteome,
review in Nature and the metabolome)
Genetics highlighted within the same
that effective blood or using
diagnostics urine liquid
sample [6].
biopsies will require the multiparametric analysis of several analytes (including changes to the
biopsies will
The detection of require the
changes to multiparametric
the glycome, and analysis
more of several cancer-associated
specifically analytes (includingglycan changes to the
sugar groups,
transcriptome, epigenome, proteome, and the metabolome) within the same blood or urine sample
transcriptome,
was [6].
not Theincluded epigenome,
in this proteome,
review. Glycans and have
the metabolome)
shown hugewithin the same
promise as blood or urine
diagnostic sample for
biomarkers
detection of changes to the glycome, and more specifically cancer-associated glycan sugar
[6].[7–9],
cancer The detection
and recent of changes to the glycome,
data suggests and more specificallyacross
cancer-associated glycan sugar
groups, was not included in this that integrating
review. Glycansbiomarkers
have shown hugemulti-omic
promise asplatformsdiagnostic (which
groups, was not included in this review. Glycans have shown huge promise as diagnostic
includes changes
biomarkers forto the glycome)
cancer [7–9], andcan improve
recent the stratification
data suggests of patients
that integrating withacross
biomarkers prostate cancer [10].
multi-omic
biomarkers for cancer [7–9], and recent data suggests that integrating biomarkers across multi-omic
platforms
Glycans are (which includes changes
saccharides that cantobethe glycome)
attached tocan improve
proteins, the stratification
lipids, of patients
and other glycans with the
through
platforms (which includes changes to the glycome) can improve the stratification of patients with
prostate
enzymatic cancer [10].
prostateprocess known as glycosylation. Glycosylation is the most common posttranslational
cancer [10].
Glycans
modification, and areis saccharides
now known thatto can be attached
be essential to to proteins,every
virtually lipids, and other process
glycans through the [11].
Glycans are saccharides that can be attached to proteins, lipids,biological
and other glycans through in the bodythe
enzymatic process known as glycosylation. Glycosylation is the most common posttranslational
Glycans can either
enzymatic processbe added
knownsequentially
as glycosylation. to theGlycosylation
hydroxyl oxygen is the ofmostserine/threonine residues on the
common posttranslational
modification, and is now known to be essential to virtually every biological process in the body
target protein (O-linked
modification, and is now glycosylation),
known to beor as pre-assembled
essential to virtually blocks of 14 sugars
every biological thatinare
process thetransferred
body
[11]. Glycans can either be added sequentially to the hydroxyl oxygen of serine/threonine residues
[11]. Glycans can
co-translationally either be added sequentially to the hydroxyl oxygen ofthe
serine/threonine residues
on the target protein (O-linked glycosylation), or as pre-assembled blocks of 14 sugars that(N-linked
via the amide group of an asparagine residue on target protein are
on the target
glycosylation). How protein
much (O-linked
a protein glycosylation),
or amide
lipid isgroup or as pre-assembled
glycosylated blocks
on theof
dependsresidue 14 sugars
number that are
of glycosylation
transferred co-translationally via the of an asparagine on the target protein
transferred
sites(N-linked
present, and co-translationally
the expression viaofthe amide glycosylation
specific group of an asparagine
enzymesresidue within on
theonthe
celltarget protein
glycosylation). How much a protein or lipid is glycosylated depends the[9]. Glycans
number of are
(N-linked glycosylation). How much a protein or lipid is glycosylated depends on the number of
major building blocks
glycosylation of life [12],
sites present, and but have beenof
the expression hugely
specific understudied
glycosylation (Figure
enzymes1). This the
within is partly
cell [9].due to
glycosylation sites present, and the expression of specific glycosylation enzymes within the cell [9].
theirGlycans
complexity,are major building blocks
the difficulties of life [12],
studying them,butandhavethatbeen hugely
there is nounderstudied (Figure 1).glycans
clear link between This is and
Glycans are major building blocks of life [12], but have been hugely understudied (Figure 1). This is
DNA. partly due to their
However, this complexity,
is now the difficulties
changing, and studying
the importancethem, and
of that there
glycans is no
can be clear link between as an
conceptualised
partly due to their complexity, the difficulties studying them, and that there is no clear link between
glycans
extended and ofDNA.
model However,
the central dogma this(Figure
is now changing, and theto importance of is
glycans
rapidlycan be
glycans and DNA. However, this is now2).changing,
The technology study glycans
and the importance of glycans advancing,
can be
conceptualised as an extended model of the central dogma (Figure 2). The technology to study
and there is expected
conceptualised as to
anbe an explosion
extended modelof ofinterest
the centralin this area (Figure
dogma in the next 10 to
2). The 15 years, which
technology to study will be
glycans is rapidly advancing, and there is expected to be an explosion of interest in this area in the
helped by projects
glycans is rapidly such as the human
advancing, and there glycome project
is expected [13].
to be an explosion of interest in this area in the
next 10 to 15 years, which will be helped by projects such as the human glycome project [13].
next 10 to 15 years, which will be helped by projects such as the human glycome project [13].
Figure
Figure 1. Glycans,
1. Glycans, the understudied
the understudied major
major building
building blocks
blocks ofof life.
life. TheThe glycoproteomecombines
glycoproteome combines three
Figure 1. Glycans, the understudied major building blocks of life. The glycoproteome combines
three
highly highly adaptable
adaptable interdependent
interdependent biological
biological alphabets.
alphabets. Created
Created with with BioRender.
BioRender.
three highly adaptable interdependent biological alphabets. Created with BioRender.
Figure 2. The importance of glycans can be conceptualised as an extended model of the central
Figure
Figure 2. importance
2. The The importance of glycans
of glycans can can be conceptualised
be conceptualised as an
as an extended
extended model
model of of
thethe central
central dogma.
dogma. Created with BioRender.
dogma. Created with
Created with BioRender. BioRender.
2
2
Int. J. Mol. Sci. 2019, 20, 1389 3 of 20
Changes to glycans in cancer cells were first described 50 years ago [14], and confirmed by the
development of antibodies against tumour-specific antigens [15]. Aberrant glycosylation is a universal
feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer
type [16,17]. Cancer cells have numerous changes to glycans compared to normal tissue, including
changes to sialylation, fucosylation, the truncation of O-glycans, and N-glycan branching (see [18] for
more details). The importance of glycans in cancer is emphasised by the majority of the Food and
Drug Administration (FDA)-approved tumour markers being glycan antigens or glycoproteins [19–21].
Glycans have key roles in fundamental molecular and cell biology processes in cancer biology,
including cell signalling, tumour cell invasion, immune modulation, angiogenesis, interactions with
the cell matrix, and metastasis, which are linked to all of the recognised cancer hallmarks [17]. Glycans
secreted by cancer cells likely represent the altered glycosylation status of cancer cells, and are a largely
untapped resource of cancer biomarkers. There is a huge potential to exploit glycans to improve
early diagnosis, as biomarkers for prognosis and stratification, and as markers of specific therapeutic
targets [7].
The physiological function of the prostate is to act as a secretory gland, which generates and
secretes glycoproteins such as PSA into seminal fluid. As such, the prostate is a major secretor of
glycans, and there is an unrealised opportunity to detect aberrant glycosylation patterns in serum
and urine, and link this to prostate cancer status [22]. As prostate cancer progresses, the epithelial
glands become smaller and more rounded, potentially having a drastic effect on the normal secretory
pathways where proteins and lipids are glycosylated. Glycans can influence cell survival, proliferation,
and metastasis, and likely play a key role in these processes in prostate cancer [22]. A wide range of
alterations to glycans have been observed in prostate cancer, including changes to PSA glycosylation,
increased sialylation and fucosylation, increased O-GlcNacylation, the emergence of cryptic and
branched N-glyans, and changes to galectins and proteoglycans [22–24] (see Tables 1–3 for more
details). In this review, we discuss the huge potential to exploit glycans as diagnostic and prognostic
biomarkers for prostate cancer, and argue that it will be essential to include glycans as part of a
multi-analyte liquid biopsy test.
2. PSA Glycosylation
Prostate-specific antigen (PSA) is a glycoprotein enzyme that has been used widely as a biomarker
for prostate cancer, but has the disadvantage of low specificity and no prognostic value at diagnosis [25].
PSA screening for prostate cancer has led to the overdiagnosis and overtreatment of indolent disease,
resulting in unnecessary biopsies and treatments for non-aggressive cancers [26,27]. Refinements on
the PSA test, such as the Prostate Health Index (PHI) and 4Kscore, can improve diagnostic accuracy,
but have limited prognostic utility, and may miss some high-grade cancers [28]. Thus, there is
an urgent clinical need to shift the focus to identifying aggressive disease that needs immediate
treatment [29–31]. Advances in mass spectrometry have led to an increased interest in glycan
structures on cancer-associated proteins, and numerous studies have investigated whether a glycan
signature on PSA can be used to improve its clinical utility [21,24]. A recent study also linked a
single nucleotide polymorphism that affects PSA glycosylation to prostate cancer risk [32]. PSA has
a single N-glycosylation site at asparagine-69 (Asn-69) [24]. Approximately 50 glycoforms of PSA
have been described, but only some of these are found in aggressive prostate cancer. In particular,
complex biantennary glycoforms with α2,3-sialic acid have been closely linked to aggressive disease in
multiple studies [33–35] (Table 1). When combined with the PHI, the detection of α2,3-sialic acid PSA
glycoforms in a cohort of 79 patients showed 100% sensitivity and 94.7% specificity to differentiate
high-risk prostate cancer from low-risk and benign disease [34]. The robust prediction power of
α2,3-sialylated PSA to diagnose aggressive prostate cancer has been confirmed by other studies using
independent cohorts and different technologies [35]. Together, these studies provide strong evidence in
support of the clinical utility of PSA glycoforms to distinguish indolent and aggressive prostate cancer.
Int. J. Mol. Sci. 2019, 20, 1389 4 of 20
3. Sialyled Glycans
Sialylation is the process by which sialic acid residues are added to glycans as the terminal
monosaccharide. Abnormal sialylation is a universal feature of cancer cells that is linked to poor
prognosis and metastasis [9,36–40]. In prostate cancer, sialic acid levels have been investigated as
an adjunct to PSA in predicting prostate malignancy [41,42]. Increased levels of α2-3-linked sialic
acid have been detected on serum glycoproteins in patients with prostate cancer compared to benign
prostatic hyperplasia (BPH), and can be used to predict Gleason score with a higher specificity and
sensitivity than PSA [43,44]. Serum sialic acid levels have also been linked to pathological grade, and
elevated sialic acid may predict bone metastasis [45]. Together, these studies suggest serum sialic acid
as a promising diagnostic biomarker for prostate cancer that should be further investigated for use in
predicting disease progression.
An emerging tool for glycoproteomic
Sialylation is the processanalysis
by which is theacid
sialic use of azide
residues sugartoanalogs
are added glycans asas themimics
terminal of sialic
monosaccharide.
acid. This strategy utilises Abnormal sialylation
metabolically labelledisglycans a universal feature
with of cancer reporters
chemical cells that is linked
that to arepoor
ligated to
prognosis and metastasis [9,36–40]. In prostate cancer, sialic acid levels have been investigated as an
fluorescent probes, and offers a new avenue for probing changes to the glycome
adjunct to PSA in predicting prostate malignancy [41,42]. Increased levels of α2-3-linked sialic acid
by both imaging
and glycoproteomic have
analyses [24,46].
been detected on In prostate
serum cancer,
glycoproteins sialic acid
in patients analogs
with prostate have
cancer been used
compared to benignto identify
prostatic hyperplasia (BPH), and can be used to predict Gleason score with a higher specificity and
sialoglycoproteins linked to metastatic potential in cell lines derived from PC3 cells [47]. More recently,
sensitivity than PSA [43,44]. Serum sialic acid levels have also been linked to pathological grade,
sugar azide analogs have been sialic
and elevated utilised in prostate
acid may predict bone cancer tissue
metastasis slices
[45]. to identify
Together, these studiesglycoproteins
suggest serum that are
elevated or uniquely found in prostate cancer cells [48].
sialic acid as a promising diagnostic biomarker for prostate cancer that should be further
investigated for use in predicting disease progression.
As well as changes to serum sialic acid levels and sialoglycoproteins, tumour-associated sialylated
An emerging tool for glycoproteomic analysis is the use of azide sugar analogs as mimics of
glycans also changesialic
in prostate cancer.utilises
acid. This strategy Numerous
metabolically studies
labelledhave glycansreported
with chemical thereporters
upregulationthat are of the
sialylated blood groupligated to fluorescent
antigen Sialyl probes,
Lewis and offers
X (SLe X ) avenue
a new in prostatefor probing changes
cancer, andto the glycomethis
linked by both
with poor
imaging and glycoproteomic analyses [24,46]. In prostate cancer, sialic acid analogs have been used
prognosis in patients [49–52] (Table 1). SLe X , and its isomer SLe A , are the minimal recognition
to identify sialoglycoproteins linked to metastatic potential in cell lines derived from PC3 cells [47].
motif for ligands ofMoreselectins,
recently, which
sugar azideis aanalogs
family haveof beenlectins
utilisedwith rolescancer
in prostate in leukocyte
tissue slices to trafficking
identify and
glycoproteins thatXare elevated or uniquely found in prostate cancer cells [48].
tumour extravasation [53]. SLe could influence prostate cancer progression
As well as changes to serum sialic acid levels and sialoglycoproteins, tumour-associated
through numerous
mechanisms, including binding
sialylated glycansto also
E-selectin, the evasion
change in prostate of Natural
cancer. Numerous Killer
studies have(NK)
reported celltheimmunity,
upregulation and the
of the sialylated
promotion of bone metastasis blood group
[52,54–56]. Theantigen Sialyl Lewis Xof
upregulation (SLeSLe X prostate
X) in
has beencancer, and linked
detected onthis withMucin 1
PSA,
poor prognosis in patients [49–52] (Table 1). SLeX, and its isomer SLeA, are the minimal recognition
(MUC1), and prostatic acid
motif phosphatase
for ligands (PAP)
of selectins, whichproteins
is a family in of a panel
lectins with ofroles
10 malignant tissues (relative
in leukocyte trafficking and to
matched normal tissue), opening up a new avenue for the development of prostate cancer-specific
tumour extravasation [53]. SLe X could influence prostate cancer progression through numerous
mechanisms, including binding to E-selectin, the evasion of Natural Killer (NK) cell immunity, and
glycoprotein biomarkers [57]. The cancer-associated sialyl-Tn glycan (known as sTn) has also been
the promotion of bone metastasis [52,54–56]. The upregulation of SLeX has been detected on PSA,
linked to prostate cancer.
Mucin 1sTn is a and
(MUC1), truncated O-glycan
prostatic acid phosphatase containing
(PAP) proteins a sialic acidofα-2,6
in a panel linked
10 malignant to GalNAc
tissues
(relative to matched normal tissue), opening up a new
α-O-Serine/Threonine (Ser/Thr) (Table 1). The sTn glycan is upregulated in several cancer types andavenue for the development of prostate
cancer-specific glycoprotein biomarkers [57]. The cancer-associated sialyl-Tn glycan (known as sTn)
associated with metastasis
has also and poor toprognosis
been linked [37].sTn
prostate cancer. Inisprostate
a truncatedcancer,
O-glycansTn is expressed
containing a sialic acidinα-2,6
high-grade
prostate tumours [58,59], and can reduce prostate cancer cell adhesion [60,61]. The sTn antigen
linked to GalNAc α-O-Serine/Threonine (Ser/Thr) (Table 1). The sTn glycan is upregulated in is
several cancer types and associated with metastasis and poor prognosis [37]. In prostate cancer, sTn
being widely investigated as a circulating biomarker for other cancers [62,63], and it is likely that
is expressed in high-grade prostate tumours [58,59], and can reduce prostate cancer cell adhesion
innovative tools that[60,61].
wereThe developed
sTn antigen to detect
is being sTninvestigated
widely [63–65] will also have
as a circulating clinical
biomarker forutility for prostate
other cancers
[62,63],
cancer precision oncology. and it is likely that innovative tools that were developed to detect sTn [63–65] will also have
clinical utility for prostate cancer precision oncology.
Table 1. Summary of glycan
Table alterations
1. Summary of glycan in prostate
alterations cancer.
in prostate PSA:
cancer. prostate-specific
PSA: antigen.
prostate-specific antigen.
Structure Link to Prostate Cancer References
Glycan Glycan Structure Link to Prostate Cancer References
Example of N- Complex biantennary glycoforms with α2,3-sialic [33–35].
glycan on PSA acid have been closely linked to aggressive disease in
multiple
Complexstudies.biantennary
glycoforms with
Example of N-glycan on α2,3-sialic acid have
[33–35].
PSA been closely linked to
aggressive disease in
multiple studies.
α2-3 sialylated N-glycan
α2-3 sialylated N-glycan
4
Sialyl Tn (sTn) Expressed in high-grade prostate tumours. Can [58–61]
Int. J. Mol. Sci. 2019, 20, 1389 reduce prostate cancer cell adhesion. 5 of 20
Sialyl Lewis X Upregulated and linked to poor prognosis in
Table 1. Cont. [49–52,57].
(SLeX) Glycan Structure patients.
Link toDetected onCancer
Prostate PSA, MUC1 and PAP in
References
Sialyl Lewis X Upregulated and linked to poor prognosis in [49–52,57].
malignant tissue
(SLeX) Upregulated
patients. Detectedand linked
on PSA, MUC1 and PAP in
to poor prognosis in
Sialyl Lewis X (SLeX ) patients.tissue
malignant Detected on [49–52,57].
Core Fucosylation Sialyl Lewis X Increased
PSA,
Upregulated and linked
MUC1 ininpatient
to poor prognosis serum.
and [49–52,57].
PAP in Linked to aggressive [43,66–68]
Sialyl Tn (sTn) Expressed in high-grade prostate tumours. Can [58–61]
(SLeX)
malignant tissue
patients. Detected on PSA, MUC1 and PAP in
α3 α3malignant tissue disease.
reduce prostate cancer cell adhesion.
Sialyl Tn (sTn) Expressed in high-grade prostate tumours. Can [58–61]
Sialyl Tn (sTn) β4 β4
Expressed in high-grade prostate tumours. Can [58–61]
Sialyl Lewis X
Expressed
reduce incancer
Levelsprostate high-grade
Upregulated and linked to poor prognosis in
correlate
reduce prostate cancer cell adhesion.
cell
[49–52,57].
with adhesion.
Fucosyltransferase 8 (FUT8). [69]
(SLeX)
β2 β2 patients. Detected on PSA, MUC1 and PAP in
prostate tumours. Can
Sialyl Tn (sTn) malignant tissue [58–61]
reduce prostate cancer
α3 α6 cell adhesion.
Sialyl Tn (sTn) Expressed in high-grade prostate tumours. Can [58–61]
reduce prostate cancer cell adhesion.
β4
Core Fucosylation Increased in patient
Increased in patient serum. Linked to aggressive
[43,66–68]
α3
α6
α3 disease. serum. Linked to [43,66–68]
β4 R β4 aggressive disease.
Levels correlate with Fucosyltransferase 8 (FUT8). [69]
Core Fucosylation β2 β2 Increased in patient serum. Linked to aggressive [43,66–68]
Core Fucosylation
O-GlcNAcylation Core Fucosylation
Upregulated and[43,66–68]
linked to poor prognosis in
Increased in patient serum. Linked to aggressive
[70,71]
α3 α6
Core Fucosylation
α3 disease.
Levels correlate
Increased with Linked to aggressive
in patient serum. [43,66–68]
α3
β4
α3 disease.
Fucosyltransferase 8 [69]
α3 β4 β4 primary prostate cancer.
α3Levels correlate withdisease.
α6
α3 β2 R β2 (FUT8).
Fucosyltransferase 8 (FUT8). [69]
O-GlcNAcylation β4 α3 α6
β4 Upregulated and linked to poor prognosis in
Downregulated [70,71]
in castrate resistant prostate [72]
β4 β4 Upregulated and linked
β4 Levels
primary prostate cancer. correlate with Fucosyltransferase 8 (FUT8). [69]
β2 α6 to
β2 Downregulated inLevelspoor prognosis
correlate
castrate resistant prostate
in
with Fucosyltransferase
[72]
8[70,71]
(FUT8). [69]
β2 R β2 cancer(CRPC).
primary prostate[70,71]cancer.
O-GlcNAcylation O-GlcNAcylation Upregulated and linked to poor prognosis in
cancer(CRPC).
α3 α3 α6α6 Downregulated in
primary prostate cancer.
Branched N- castrate resistant[72]prostate
Downregulated in castrate resistant prostate
Linked to metastasis and disease-free survival.
[73] [72]
glycans β4β4 cancer(CRPC).
cancer(CRPC).
Branched N- Linked to metastasis and disease-free survival. [73]
Predictive biomarker for CRPC. [74]
Branched N- α6α6 Linked to metastasis and
Linked to metastasis and disease-free survival.
[73]
[73]
glycans glycans
Cryptic N-glycans RR disease-free survival.
N-linked oligomannose 9 (Man9) is increased in [75]
Branched N-glycans Predictive biomarker for CRPC. [74]
O-GlcNAcylation
O-GlcNAcylation Upregulated and
Upregulated and linked
linked totopoor
poor prognosis
prognosis in in [70,71]
[70,71]
Predictive
high-gradebiomarker
tumours for CRPC.
and linked to clinical outcome. [74]
Man9 Predictive biomarker for
primary prostate cancer.
[74]
Cryptic N-glycans N-linkedCRPC.
oligomannose
primary 9 (Man9)
prostate is increased in
cancer. [75]
high-grade tumours and linked
Downregulated in to clinical outcome.
castrate resistant prostate [72]
Man9
Downregulated in castrate resistant prostate [72]
Man5 N-linked oligomannose 5
9
cancer(CRPC).
(Man9) is increasedin
Cryptic N-glycans cancer(CRPC).
high-grade tumours 5 and [75]
R
Man5
linked to clinical
outcome.
Branched N- Linked to metastasis and disease-free survival. [73]
R
Branched N- Linked to metastasis and disease-free survival. [73]
glycans
glycans Predictive biomarker for CRPC. [74]
4. Fucosylation
4. Fucosylation Predictive biomarker for CRPC. [74]
Fucosylation describes the attachment of a fucose residue to a glycan, and consists of terminal
4. Fucosylation
Fucosylation describes the attachment
fucosylation and core fucosylation.of a fucose Increased residue to a glycan,
core fucosylation has beenand
Fucosylation describes the attachment of a fucose residue to a glycan, and consists of terminal
detectedconsists of terminal
in the serum of
5
patients with prostate cancer, is associated with disease progression [43,67,68] (Table 1), and may
fucosylation and core fucosylation.
fucosylation
influence
and core
prostate
Increased
fucosylation.
cancer cell
core fucosylation
Increased
trafficking
core fucosylation
through an
has has been
been
E-selectin
detected detected
in
dependent
the serum of
mechanism
in the
[56].
serum
The
of
patients with prostate cancer, is associated with disease progression [43,67,68] (Table 1), and may
patients with prostate cancer,
influence prostateiscancer
fucosyltransferase associated
FUT8 with
is the only
cell trafficking enzyme
through disease
anresponsibleprogression
E-selectin [43,67,68]
for the α-1,6-linked
dependent mechanism core The
[56]. (Table that
fucosylation 1), and
adds fucose toFUT8
may influence prostate cancer cellthetrafficking
fucosyltransferase inner GlcNAc
is the only enzymeon N-glycans
responsible[76].
through an FUT8
for the expression
α-1,6-linked
E-selectin coreisfucosylation
increased in
dependent high-grade and
that
mechanism [56].
addsmetastatic
fucose to theprostate
inner GlcNAccancer on [73,77].
N-glycansThe [76]. increased fucosylation
FUT8 expression of inglycoproteins
is increased high-grade andin aggressive
The fucosyltransferasemetastatic
FUT8
prostateis the only
prostate
cancer cancer enzyme
[73,77].
correlates withThe responsible
FUT8increased
[69], and forhasthe
fucosylation
this of α-1,6-linked
been glycoproteins core
in aggressive
recently linked fucosylation
to castrate resistance, that
prostate
cell cancer
survival, correlates with FUT8
andN-glycans
lower PSA [69], and this [78]
production has been
(Tablerecently linkedtogether,
1). Taken to castratethese
resistance,
findings implicate and
adds fucose to the inner GlcNAc
cell dysregulated
on
survival, and lower PSA production
[76]. FUT8
[78] (Table
expression
1). Taken together, and
is increased
these the
findings
in high-grade
implicate of castrate
fucosylation in prostate cancer progression development
metastatic prostate cancer [73,77].
dysregulated
resistance. Thewith
fucosylation
In line increased
in prostate cancer
this, several fucosylation
progression of andglycoproteins
fucosylated glycoproteins
the development ofincastrate
are being investigatedaggressive prostate
as potential
resistance. In line with this, several fucosylated glycoproteins are being investigated as potential
cancer correlates with non-invasive
FUT8 [69],
non-invasive and this
predictive
predictive has been
biomarkers.
biomarkers. recently
These
These include linked
include
decreased to castrate
decreased
fucosylation on PSA resistance,
fucosylation on PSA
[33,79] and cell survival,
[33,79] and
integrins
integrins [80], [80],elevated
and and elevated fucosylated haptoglobin [68].
and lower PSA production [78] (Tablefucosylated
1). Taken haptoglobin [68].
together, these findings implicate dysregulated 5
fucosylation in prostate cancer progression and the development of castrate resistance. In line with
5. 5. O-GlcNAcylation
O-GlcNAcylation
this, several fucosylatedThe glycoproteins
The addition
addition of O-GlcNAc areto being
of O-GlcNAc proteins investigated
to proteins
(known (known
as as potential
as O-GlcNAcylation)
O-GlcNAcylation) non-invasive
plays a fundamental
plays a fundamental role predictive
role
in cellular processes,
in cellular including
processes, transcription,
including epigenetics,
transcription, cell signalling,
epigenetics, proteostasis, proteostasis,
cell signalling, and and 5
biomarkers. These include decreased
bioenergetics
bioenergetics[81,82][81,82] fucosylation
(Table (Table
1). In 1).cancer on
In cells,
PSA
cancerelevated
[33,79]
cells, pools
elevated
and
of Uridine integrins
pools ofdiphosphate
[80],
N-
Uridine diphosphate
and elevated
N-
fucosylated haptoglobinacetylglucosamine
[68].
acetylglucosamine (UDP-GlcNAc)
(UDP-GlcNAc) drive the O-GlcNAcylation
drive of key targets
the O-GlcNAcylation in the
of key cytoplasm,
targets in the cytoplasm,
nucleus, and mitochondrion, and can alter key hallmarks of cancer [83,84]. O-GlcNAcylation is
nucleus, and mitochondrion, and can alter key hallmarks of cancer [83,84]. O-GlcNAcylation is
catalysed by O-GlcNAc transferase (OGT) (for more details, see [85]). OGT uses UDP-GlcNAc as a
catalysed by O-GlcNAc transferase (OGT) (for more details, see [85]). OGT uses UDP-GlcNAc as a
substrate, which is produced by the hexosamine biosynthetic pathway (HBP). HBP acts as a sensor
substrate,
for the which
nutritional stateisofproduced
the cell, andbyhas
theahexosamine biosynthetic
key role in the pathway
metabolic rewiring (HBP).inHBP
observed acts as a sensor
cancer
[86].for the nutritional
In primary prostate state ofglobal
cancer, the cell, and has a keyis role
O-GlcNAcylation in the
elevated metabolic
relative rewiring
to benign diseaseobserved
[70], in cancer
and [86]. In primary
O-GlcNAc levels prostate
correlate cancer, global Gleason
with a higher O-GlcNAcylation is elevated
score and reduced relative
patient to benign
prognosis [71]. disease [70],
BothandOGTO-GlcNAc
and enzymeslevels correlate
in the with a change
HBP pathway higher inGleason
prostatescore and
cancer. OGTreduced patient in
is upregulated prognosis [71].
primary
Bothprostate
OGT andcancer, and thisinis the
enzymes linked
HBP to apathway
higher Gleason
changescore, reduced time
in prostate to biochemical
cancer. OGT is upregulated in
recurrence, and increased
primary prostate c-Myc
cancer, and this stability
is linked [87]. Similarly,
to a higher Gleason UDP-N-Acetylglucosamine
score, reduced time to biochemicalInt. J. Mol. Sci. 2019, 20, 1389 6 of 20
5. O-GlcNAcylation
The addition of O-GlcNAc to proteins (known as O-GlcNAcylation) plays a fundamental
role in cellular processes, including transcription, epigenetics, cell signalling, proteostasis,
and bioenergetics [81,82] (Table 1). In cancer cells, elevated pools of Uridine diphosphate
N-acetylglucosamine (UDP-GlcNAc) drive the O-GlcNAcylation of key targets in the cytoplasm,
nucleus, and mitochondrion, and can alter key hallmarks of cancer [83,84]. O-GlcNAcylation is
catalysed by O-GlcNAc transferase (OGT) (for more details, see [85]). OGT uses UDP-GlcNAc as a
substrate, which is produced by the hexosamine biosynthetic pathway (HBP). HBP acts as a sensor for
the nutritional state of the cell, and has a key role in the metabolic rewiring observed in cancer [86].
In primary prostate cancer, global O-GlcNAcylation is elevated relative to benign disease [70], and
O-GlcNAc levels correlate with a higher Gleason score and reduced patient prognosis [71]. Both
OGT and enzymes in the HBP pathway change in prostate cancer. OGT is upregulated in primary
prostate cancer, and this is linked to a higher Gleason score, reduced time to biochemical recurrence,
and increased c-Myc stability [87]. Similarly, UDP-N-Acetylglucosamine Pyrophosphorylase 1
(UAP1,the last enzyme in the HBP) is also overexpressed in prostate cancer tissue [88]. Although
HBP is upregulated in localised prostate cancer where it promotes disease progression, in advanced
castrate-resistant disease, the inhibition of HBP enhances tumour growth [72]. Metabolic rewiring
during disease progression is believed to promote the downregulation of HBP in castrate-resistant
disease, and in particular, loss of the Glucosamine-Phosphate N-Acetyltransferase 1 (GNPNAT1)
enzyme may serve as a marker of progression to castrate resistance [72].
6. Branched and Cryptic N-Glycans
Changes to N-glycans are common in cancer, and have been linked to metastasis in multiple
tumour types [89,90]. Common changes include the branching of complex biantennary glycans to
triantennary and tetraantennary structures, and the emergence of cryptic N-glycans [9] (Table 1).
In prostate cancer, β-1,6-GlcNAc tri-branched and tetra-branched N-glycans are linked to both
metastasis in xenograft models and disease-free survival in patients [73]. Serum N-glycan signatures
have shown promise as diagnostic and predictive biomarkers in prostate cancer [91]. Changes to
branched N-glycans can help distinguish BPH and prostate cancer [43,92], and increased serum
triantennary and tetraantennary N-glycans have clinical utility to predict castrate-resistant prostate
cancer [74]. In recent work, a muti-omic study identified tetraantennary and tetrasialylated N-glycans
using mass specrometery as part of a biomarker panel to improve the stratification of patients with
indolent and aggressive prostate cancer, and predict patient survival [10].
Cryptic N-glycans are the precursors, cores, and internal sequences of N-glycans that are usually
masked by other sugar moieties, but occur in cancer due to alterations in glycan synthesis and
processing [22,93]. As illustrated in Table 1, they contain the same mannose cores, but differ in the
terminal sugar moieties. Cryptic N-glycans expose internal sequences that are normally ‘cryptic’ to
the immune system [94]. Several studies have investigated how cryptic N-glycans change among
different Gleason grades of prostate cancer and in metastatic disease [73,94–96]. Of particular interest,
the cryptic N-glycan Man9 has been detected in the serum of men with prostate cancer, and serum
Man9 autoantibodies may help differentiate high-grade tumours and predict clinical outcome [75].
The further development and validation of assays to detect anti-Man9 antibodies in patient serum is
expected to be useful clinically.
7. The F77 Antigen
An antibody against the F77 antigen was initially isolated from mice injected with PC3 cells, and
has provoked great interest as a prostate cancer-specific diagnostic and therapeutic tool. F77 was
first identified as a glycolipid with α1,2-fucose linkages, and linked to the glycosylation enzymes
Fucosyltransferase 1 (FUT1) and Core 2 Branching Enzymes 1-3 (GCNT1, GCNT2, and GCNT3) [97–99].Int. J. Mol. Sci. 2019, 20, 1389 7 of 20
However, F77 has since been detected on O-glycan proteins, including a spliced isoform of CD44
found in the sera of prostate cancer patients [100]. The antibody against F77 can inhibit the growth of
PC3 and DU145 tumour xenografts in nude mice, and can be used to differentiate primary and
metastatic prostate cancer from non-malignant prostate tissue, [99]. Immunohistochemistry staining
tissue sections detected F77 in 112 of 116 primary and 29 of 34 metastatic prostate cancer tissues, while
no signal was detected in normal prostate tissue [99].
8. Glycolipids
Many of the glycans found attached to proteins are also found on glycolipids (glycolipids are
lipids modified by one or more glycans). The aberrant expression of glycolipids in cancer has potential
roles in disease progression and anti-tumour immunity (see [101] for more details). In prostate cancer,
the F77 antigen (see Section 7 above) is known to contain a glycolipid with α1,2-fucose linkages, and
preliminary studies have linked increased ganglioside GD1a to castrate-resistant disease [102,103].
9. Proteoglycans
Proteoglycans are proteins that are heavily glycosylated. The core protein has one or more
glycosaminoglycan (GAG) chains attached (such as chondroitin sulphate, heparin sulphate, or keratin
sulphate). Proteoglycans are a major component of the extracellular matrix and interact with growth
factors, chemokines, and other extracellular matrix proteins. They have roles in cell signalling,
adhesion, cell growth, and apoptosis, and have an established role in cancer progression [104].
Several proteoglycans have been found to be important in prostate cancer, including versican, decorin,
biglycan, lumican, and syndecan-1 (summarised in Table 2), and data from a range of studies implicate
proteoglycan alterations to prostate cancer cell survival and metastasis [105]. Proteoglycan expression
patterns might be useful as predictive and prognostic biomarkers in patients with prostate tumours [22].
Of particular interest, increased levels of versican [106], biglycan [107], and syndecan-1 [108,109] are
linked to poor prognosis.
Table 2. Proteoglycans with roles in prostate cancer.
Proteoglycan Link to Prostate Cancer References
Modulates binding to the extracellular matrix (ECM) and
Versican enhances motility. [106,110–112]
Associated with poor prognosis.
Suppresses tumour growth by inhibiting both androgen
Decorin receptor (AR) and epidermal growth factor (EGF). [106,113,114]
Decreased in prostate cancer.
Biglycan Associated with poor prognosis and PTEN deletion. [107]
Lumican in stroma tissue suppresses cancer progression.
Lumican [115]
Potential marker in prostate cancer staging.
Linked to disease progression. Upregulates sonic
Perlecan [116,117]
hedgehog signalling.
Role in the epithelial-to-mesenchymal transition (EMT).
[118,119]
Syndecan-1 Maintains stability of prostate tumour initiating cells.
Poor prognosis. [108,109]
10. Galectins
Cancer cells may also display altered expression of proteins that interact with glycans. A key
example of this is the galectins, which are a group of glycan-binding proteins with an established
role in tumour biology [120]. Several galectins have been implicated in prostate cancer biology
(summarised in Table 3). A unique galectin signature has been identified in prostate cancer tissue, withInt. J. Mol. Sci. 2019, 20, 1389 8 of 20
the upregulation of galectin-1 and downregulation of galectins 3, 4, 9, and 12 observed during disease
progression [121]. Of particular interest, galectin-3 is linked to tumour progression [122] and has a role
in the prostate cancer bone metastasis niche [123]. Immunohistochemistry staining of galectin-3 in
tissues from 83 patients had 91.7% sensitivity, 64% specificity, and 73% accuracy in predicting PSA
biochemical recurrence [124].
Table 3. Galectins with roles in prostate cancer.
Galectin Link to Prostate Cancer References
Upregulated during disease progression. Linked to angiogenesis. [121]
Galectin-1
Potential therapeutic target in CRPC. [125,126]
Promotes prostate tumour growth and invasion.
[127]
Potential diagnostic marker.
Galectin-3 High in early stages, lost in advanced disease.
[123,124]
May predict biochemical recurrence.
Role in bone metastasis niche. [122]
Linked to metastasis and reduced survival. [128]
Galectin-4 Part of O-glycosylation-mediated signalling circuit drives
[129]
metastatic CRPC.
Galectin-8 Linked to metastasis. Proposed as prognostic biomarker. [130]
11. Upregulation of Glycosylation Enzymes
One of the underlying causes of aberrant glycosylation in cancer is the dysregulated expression of
glycosylation enzymes within the cancer cell [16]. Growing evidence links the differential expression of
glycosylation enzymes to the progression of prostate cancer [57,78,88,131–136] (summarised in Table 4).
Androgens are known to drive the development and progression of prostate cancer, and the first
line of treatment for men with advanced disease is androgen deprivation therapy (ADT) [137].
Recently, we identified a set of glycosylation enzymes that were regulated by androgens both in
seven men undergoing ADT, and in prostate cancer cell lines. Importantly, these enzymes are linked to
prostate cancer cell survival, and are upregulated in primary prostate cancer relative to normal and
benign tissue [131,132]. The androgen-regulated glycosylation enzymes in prostate cancer include:
(i) ST6GalNac1, which synthesises the cancer-associated sTn antigen, (ii) GCNT1, which is linked to
the synthesis of sLeX , (iii) GalNAc transferase 7 (GALNT7), which is part of a gene signature associated
with androgen receptor splice variant-7 (AR-V7), and (iv) the HBP enzyme UAP1, which is highly
overexpressed in prostate cancer [57,60,88,132,138]. Several cancer-associated glycans, including sTn,
Tn, and sLeX are also androgen regulated in prostate cancer cell lines [132], but this is yet to be studied
in primary patient tissue. Preliminary studies have found that GCNT1 can be detected in post-digital
rectal examination (post-DRE) urine from 35 prostate cancer patients by immunoblotting, and used
to predict the extracapsular extension of prostate cancer (the receiver operating characteristic (ROC)
curve analysis for GCNT1 was 0.7614, compared to 0.7455 for PSA) [139]. The UAP1 gene can also
be detected in plasma and urine, and can be used as part of a gene panel to predict prostate biopsy
results and prognosis [140,141]. Altered expression of the fucosyltransferases FUT6 and FUT8 are
important in advanced prostate cancer. FUT6 is upregulated in distant metastases, and may play a role
in metastasis to bone [136]. FUT8 expression increases in aggressive and castrate-resistant prostate
cancer, and is linked to poor prognosis in patients (these studies were based on cell lines, and a small
number of tissue samples and will need repeating in additional larger cohorts) [77,78]. Taken together,
these studies suggest that glycosylation enzymes are a promising but yet unexploited resource of
diagnostic and prognostic biomarkers for prostate cancer, and may also provide new insights into
candidate glycoprotein alterations.Int. J. Mol. Sci. 2019, 20, 1389 9 of 20
Table 4. Summary of glycosylation enzymes with roles in prostate cancer. HBP: hexosamine
biosynthetic pathway.
Glycosylation
Role in Glycosylation Link to Prostate Cancer References
Enzyme
Regulated by androgens. Upregulated in
Transfers α-2,6 sialic acid
ST6GALNAC1 tumour tissue. Linked to the synthesis of [60,61,142]
to O-linked GalNAc
sTn. Reduced cell adhesion
Increased in aggressive disease. Closely
related to extraprostatic extension and
lymph node metastasis. Increases tumour [133,134]
growth on orthotopic inoculation into the
mouse prostate.
Forms core-2-branched Resistance to NK cell immunity. [52]
GCNT1
O-linked glycans Regulated by androgens [132]
Associated with higher levels of core 2 O
[57]
sLex in PSA, PAP, and MUC1
Linked to F77 antigen. [97]
Detected in post-DRE urine. Indicator of
[139]
extracapsular extension
Forms core-2-branched Linked to invasion. Potential role in
GCNT2 [143]
O-linked glycans integrin signalling.
Upregulated in malignant PCa as part of a
[144]
glycosylation gene signature.
Initiation of
GALNT7 Androgen regulated and linked to prostate
O-glycosylation
[132]
cancer cell viability.
Correlates with AR-V7 in CRPC. [138]
Part of O-glycosylation mediated signalling
Generates the common
C1GALT1 circuit that drives CRPC and is linked to [129]
core 1 O-glycan structure
poor survival.
Addition of sialic acid to Upregulated. Linked to reduced survival
[135]
ST6Gal1 galactose-containing and metastasis.
N-glycan Regulated by androgens. [132]
Upregulated in malignant prostate cancer
[144]
Mannose trimming of as part of a glycosylation gene signature.
EDEM3
N-glycans
Androgen regulated and linked to prostate
[132]
cancer cell viability.
Biosynthesis of β1,6
MGAT5 GlcNAc-branched Link to metastasis in mouse models. [145]
N-glycans
Highly overexpressed (correlates negatively
Last enzyme in HBP with Gleason score). Linked to increased
UAP1 pathway. Produces UDP-GlcNAc. Protects prostate cancer cells [88]
UDP-GlcNAc from endoplasmic reticulum (ER) stress.
Regulated by androgens.
HBP pathway. Produces
GNPNAT1 GNPNAT1 is decreased in CRPC. [72]
UDP-GlcNAc
Upregulated in distant metastases. Role in
FUT6 Fucosylation [136]
prostate cancer metastasis to bone.
Transfers fucose to Increased in aggressive prostate cancer and
[77]
FUT8 core-GlcNAc of the linked to poor prognosis.
N-glycans Increased in CRPC. [78]Int. J. Mol. Sci. 2019, 20, 1389 10 of 20
12. Exosomes
Exosomes are small vesicles secreted from most cell types that are present in body fluids.
There is increasing evidence that exosomes are involved in carcinogenesis, and they are emerging as
a rich source of tumour-specific proteins and biomarkers for cancer detection and progression [146].
Exosomes secreted by the prostate are structurally unique, and can be isolated from seminal fluid, tissue,
blood, or urine for further analyses, providing a novel and easily isolatable source of tumour-specific
proteins [147]. Glycans are important in exosome function [148]. A recent review discussed the
potential to exploitexosomes as powerful tools to diagnose aggressive prostate cancer as early as
possible, as well as predict patient prognosis and response to treatment [149]. Exosomes are a rich
source of glycans [23,150], but are still largely unexplored in prostate cancer. The profiling of N-linked
glycans from prostate cancer exosomes indicates a global decrease in large branched triantennary and
tetraantennary glycans that reflects clinical status [151]. A better understanding of the presence of
glycoproteins and glycan profiles of exosomes could improve current approaches of diagnosis and
prognosis. As discussed by Tkac et al., glycan markers are likely to be enriched in exosomes, which
will aid in assay design [23].
13. Tissue Imaging of Glycans
Changes to glycans can be profiled in clinically relevant tissues using recently developed
innovative approaches. A sialyltransferase-based chemoenzymatic histology assay can detect
differences between unsialylated glycans in normal, cancerous, and metastatic prostate tissue
sections [152], and bioorthoganol labelling has been used to identify sialoglycoproteins in prostate
cancer tissue slices [48]. Advances in imaging mass spectrometry mean that glycans can now be
identified directly on patient tissue, allowing for analysis of the spatial distribution of glycans.
N-glycans can be profiled on formalin-fixed paraffin embedded (FFPE) tissue samples and tissue
microarrays (TMAs) using matrix-assisted laser desorption/ionization imaging mass spectrometry
(MALDI-IMS) [24,153–155]. In this recently developed approach, glycans are released from their
protein carrier and analysed directly on tissue. More than 40 individual glycan structures can be
detected, and tumour-specific glycans can be grouped relative to histopathology localisations [155].
A major advantage of MALDI-MS imaging is that the best starting material is FFPE tissue, which
is widely available in pathology labs. Ongoing work in this emerging area aims to identify
tumour-specific glycan biomarker panels indicative of aggressive prostate cancer, as well as identify
a second tier of biomarkers by linking glycans back to their original glycoprotein carriers [23]. It is
hoped that information on the various types of glycans within cancer tissue can be used in the future
to enable the development of lectin-based assays for use in the pathology lab [155].
14. A Multi-Omic Liquid Biopsy Test
The development and progression of prostate cancer from localised, organ-confined disease to
biochemically progressive and then to metastasis is prolonged. To capture this evolving landscape,
serial tissue biopsies are needed, which is difficult and costly to execute in the clinic. In addition,
prostate cancer preferentially metastasises to bone, which is a site that is very difficult to sample.
Tumour heterogeneity can also lead to aggressive tumours being missed, or underestimation of the
tumour landscape [156]. Liquid biopsies offer a potential solution to overcome the practical and
technical challenges of the traditional tissue biopsy [157]. The concept of a ‘liquid biopsy’ relies on the
principle that cancer cells are shed directly into blood, urine, and other body fluids. These cancer cells
can be captured and used to derive information to improve diagnosis, prognosis, or treatment. Liquid
biopsies may also capture a more complete representation of a cancer, which encompasses tumour
heterogeneity [158].Int. J. Mol. Sci. 2019, 20, 1389 11 of 20
Flow cytometery techniques were able to detect circulating prostate cancer cells in men
with metastatic disease as early as the 1970s [159]. Today, technologies can use bodily fluids to
profile genomic mutations, copy number alterations, and obtain information about the tumour
transcriptome, epigenome, proteome, metabalome, and glycome. Among prostate cancer liquid
biopsies, circulating tumours cells (CTCs) are the most extensively evaluated biomarkers [160], but
other clinically relevant phenotypes have also shown promise. These include detection of the androgen
receptor variant V7 (AR-V7) [161], circulating miRNAs [162–164], TMPRSS2:ERG, and prostate cancer
antigen 3 (PCA3) [165–167], circulating free DNA (cfDNA) (reviewed in [158]), detection of gene
methylation [168,169], and the analysis of tumour-derived exosomes [170].
Individual biomarkers in liquid biopsy can often not accurately predict disease state due to
heterogeneity in phenotype across individuals and over time. To address this challenge, effective
diagnostics using liquid biopsies will require multiparameter strategies to combine information from
multiple analytes. A recent study by Murphy et al. suggested that integrating biomarkers across
multi-omic platforms (including changes to the epigenome, transcriptome, proteome, and glycome)
can improve the stratification of patients with prostate cancer [10]. By the analysis of four types of
DNA methylation, four coding and nine non-coding RNAs, 27 peptides, and 13 glycans (in a cohort of
158 radical prostatectomy patients), combined with clinical parameters, it was possible to effectively
distinguish indolent and aggressive prostate cancer with area under the ROC curve (AUC) = 0.91 (age,
PSA level, Gleason score, and DRE gave an AUC of 0.67). This strongly suggests that multivariate
models (built from different -omics data) will lead to superior accuracy over individual markers for
prostate cancer diagnosis and disease stratification [10], and together with the data discussed in this
review, clearly demonstrate that the inclusion of glycans in a multi-analyte liquid biopsy test for
prostate cancer should help improve clinical utility.
15. Conclusions and Future Perspectives
Glycans contribute to many aspects of prostate cancer, and likely represent a huge and largely
untapped resource of biomarkers with clinical utility. The summary provided by this review suggests
that glycan-based biomarkers should be exploited as powerful tools to diagnose prostate cancer as
soon as possible, and to accurately determine tumour aggressiveness and patient prognosis. Liquid
biopsies hold great potential as non-invasive assays to monitor tumour heterogeneity and evolution,
and select personalised therapy. As new markers are identified, their clinical impact must be translated
through assay development, followed by clinical reproducibility and sensitivity testing [171]. These
efforts raise new biological and technical challenges [158,172], yet constitute critical steps towards
precision oncology. The ultimate goal in the field will be to clinically validate several types of molecules,
including glycans, within a multi-analyte liquid biopsy test. Furthermore, looking towards the future,
artificial intelligence-based tools may be useful to automatically discover and detect these signatures
and move the field towards multiparametric analyses [173]. Alongside this, the development of
novel glycan-targeting drugs (for examples, see [174–179]) will likely lead to new personalised
glycan-based therapies.
Funding: This work was supported by Prostate Cancer UK through a Research Innovation Award (RIA16-ST2-011).
Conflicts of Interest: The authors declare no conflict of interest.
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