Trends in Cancer

Special Issue: Celebrating 5 Years

Opinion

Targeting Glycosylation: A New Road for

Cancer Drug Discovery
Ana Filipa Costa,1,2,5 Diana Campos,1,2,5 Celso A. Reis,1,2,3,4,* and Catarina Gomes ,1,2,*

Highlights
Cancer is a deadly disease that encompasses numerous cellular modifications. Cancer is a leading cause of death,
Among them, alterations in glycosylation are a proven reliable hallmark of cancer, mainly due to the lack of efficacy of
existing cancer treatments, such as
with most biomarkers used in the clinic detecting cancer-associated glycans. chemotherapy and radiotherapy.
Despite their clear potential as therapy targets, glycans have been overlooked in
drug discovery strategies. The complexity associated with the glycosylation pro- Aberrant protein glycosylation is a hall-
cess, and lack of specific methodologies to study it, have long hampered progress. mark of cancer and specific glycans
actively drive tumor development and
However, recent advances in new methodologies, such as glycoengineering of cells progression.
and high-throughput screening (HTS), have opened new avenues of discovery. We
envision that glycan-based targeting has the potential to start a new era of cancer The process of drug discovery is com-
therapy. In this article, we discuss the promise of cancer-associated glycosylation plex and time-consuming; the develop-
ment of new technologies and more
for the discovery of effective cancer drugs. advanced methodologies, such as high-
throughput screening (HTS) approaches,
can overcome these limitations.
What Are We Still Missing in Cancer Treatment?
Cancer is one of the leading causes of global mortality. According to the World Health Organization, can- New targets in the field of glycobiology,
such as particular glycosyltransferases
cer was responsible for an estimated 9.6 million deaths in 2018. The traditional methods of cancer man-
and cancer-associated glycans, have
agement are chemotherapy, radiation therapy, and surgery. The success of such strategies relies on huge potential in cancer therapy that
several factors, such as the type of tumor and stage of the disease. Unfortunately, the majority of tumors needs to be explored.
are detected in an advanced stage, leading to treatment failure. This therapeutic failure often results in
A combination of advanced cancer drug
tumor recurrence and metastasis, which accounts for approximately 90% of cancer deaths [1].
discovery methodologies and targeting
of cancer-associated glycans has the
To overcome the problems of traditional cancer therapies, new approaches have focused on the potential to remodel the field of cancer
search for specific molecular targets, target therapy (see Glossary), for the selective elimination therapy.

of cancer cells. Despite the advent of target therapy, many of these therapies are only efficient in a
small percentage of patients [2]. In addition, some of the patients who initially respond to target
therapy, later develop resistance. Recent research has illuminated several underlying reasons
1
i3S - Instituto de Investigação e
for treatment failure and resistance, with tumor heterogeneity and cancer stem cells being the
Inovação em Saúde, Universidade do
best known. Porto, 4200-135 Porto, Portugal
2
IPATIMUP - Institute of Molecular
Pathology and Immunology, University
In light of this, the identification of reliable targets and the development of new-targeted therapies
of Porto, 4200-135 Porto, Portugal
for cancer are crucial. In fact, much effort has been expended towards that end. Understanding 3
Institute of Biomedical Sciences of Abel
the changes that occur in neoplastic cells and their interactions may be the key to rational drug Salazar - ICBAS, University of Porto,
4050-313 Porto, Portugal
design. Among the major changes, alterations in the glycosylation process are a well- 4
Medical Faculty, University of Porto,
established hallmark of cancer [3]. Glycosylation is the enzymatic process responsible for the 4200-319 Porto, Portugal
5
attachment of glycans (carbohydrates) to proteins, lipids, or other saccharides (Figure 1). This These authors contributed equally to
this work
major post-translational modification (PTM) occurs in the endoplasmic reticulum/Golgi compart-
ment of essentially all cells and it is mediated by the coordinated action of a portfolio of different
glycosyltransferase and glycosidase enzymes that, in a series of steps, form carbohydrate
*Correspondence:
structures [3,4]. It is comparable with phosphorylation in terms of the range of modified sites and celsor@ipatimup.pt (C.A. Reis) and
certainly exceeds it in terms of complexity and structural diversity. cgomes@ipatimup.pt (C. Gomes).

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© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Trends in Cancer

Given that glycosylation is fundamental to various biological processes, it is not surprising that Glossary
minor alterations in glycan structures severely impact the biology of a cell [4], with the develop- Fluorescence polarization:
ment of several pathologies, such as cancer [5]. Malignant cancer transformation is, in fact, fluorescence method that allows rapid
associated with aberrant glycosylation and the ability to detect some of these aberrant glycans and quantitative analysis of diverse
molecular interactions and enzyme
makes them useful serological cancer biomarkers that are currently used in the clinic, such as
activities.
the CA19-9 (SLea) (Figure 1, light blue boxes) and CA 72-4 (STn) (Figure 1, light green box). N-Glycans: glycans covalently linked to
an Asp residue of a protein via a nitrogen
Considering this, new targets in the glycobiology field, such as cancer-associated glycoforms atom at the consensus sequence
Asn-X-Ser/Thr.
and glycotransferases associated with the synthesis of such glycans, have potential to be used O-Glycans: glycans covalently linked to
for drug discovery. Despite their clear potential as therapy targets, the complexity associated a polypeptide via an oxygen atom
with the glycosylation process, and lack of specific methodologies to study it, have long through serine (Ser) or threonine (Thr)
been hampering further progress in this area. Recent advances in new methodologies have residues.
Glycolipids: lipids with a carbohydrate
yielded new possibilities. We envision that the combination of new cancer drug discovery attached by a glycosidic (covalent) bond.
methodologies and cancer-associated glycan targeting could remodel the field of cancer Glycome: the entire repertoire of glycan
therapy (Figure 2, Key Figure). structures in an organism.
Glycoproteins: proteins that carry one
or more glycans covalently attached to
In this opinion article, we will discuss the advantages of using glycan-specific targets for the the polypeptide backbone.
discovery of effective cancer drugs. The advances in new methodologies will be addressed, in Glycosidase: enzyme responsible for
particular, studies involving high-throughput screening (HTS) assays. the hydrolysis of glycosidic linkages,
thus degrading glycoconjugates.
Glycosylation: post-translational
Monitoring Glycosylation in Cancer as a Solution for Cancer Therapy modification consisting of an enzymatic
Improvement process that produces glycosidic
linkages of saccharides to other
Glycosylation, the most common form of protein PTM, has a huge impact on every cellular pro- saccharides, proteins, or lipids.
cess. It is essential for recognition, signaling, and interaction events inside the cell and between Glycosyltransferase: enzyme
cells and proteins and can play an important structural role in folding and defining the glycoprotein responsible for the initiation and
conformation, trafficking, and degradation [4,5]. Glycosylation is also essential in adhesion, cell- elongation of glycan chains.
High-throughput screening (HTS):
matrix interaction, protection against proteases and immune recognition, and membrane automatized methodology vastly used in
organization, among many other features [4]. So, it is not surprising that minor alterations in drug discovery due to capacity to test a
carbohydrate structure can significantly impact the biology of a cell, leading to malignant cancer large number of synthetic compounds in
miniaturized in vitro assays to identify
transformation. Not only do alterations in glycosylation accompany the changes in neoplastic
those capable of modulating the
cell behavior, they are also critical in the development and progression of the disease. For biological target of interest.
instance, it has been described that expression of immature truncated O-glycans [6] or expres- Lewis antigen: a human blood group
sion of SLea [7] can directly promote oncogenic transformation. In addition, impairment of normal system. Fucosylated carbohydrates
linked to lipids or to proteins. Lewis
glycosylation, for instance, core 3 glycans [8], also leads to increased susceptibility of developing
antigens comprise type 1 (Lewis a,
tumors. Lewis b) and type 2 (Lewis X, Lewis Y)
carbohydrates.
This aberrant glycosylation simply reflects the deregulation of the glycan biosynthetic pathway Scintillation proximity assay (SPA): a
technology utilized in HTS to measure
that can occur at different levels: (i) epigenetics, (ii) transcription, (iii) post-transcription, (iv) expres-
the activity and binding of drug targets
sion of important chaperones, (v) altered glycosidase activity, (vi) availability and abundance of the applying the phenomenon of
sugar nucleotide donors, (vii) altered sugar nucleotide transporter activity, and/or (viii) improper scintillation.
function of the Golgi apparatus where many of the glycosyltransferases are located [5,9]. Sialylation: covalent addition of sialic
acid to the terminal end of glycoproteins,
catalyzed by a sialyltransferase.
The cancer-specific glycoprofile has been associated with many different features of cancer cells. Target therapy: a set of therapeutic
For instance, it has been described as a glycoprofile leading to alteration in the location of cell sur- methods that allow the targeting of
face receptors and their sensitivity to key ligands required for cell growth and invasion [4,10,11]. specific molecules (e.g., mAbs and small
molecules).
Also, it is known to affect the ability of neoplastic cells to engage and differentially impact the ac-
tivity of infiltrating immune cells normally responsible for immunosurveillance [3,5,10]. Regarding
the altered glycans, increased sialylation, and polysialic acid synthesis, increased N-glycan
branching and the appearance of sialylated Lewis antigens in glycolipids and glycoproteins
are typical terminal modifications implicated in cancer [3,9,12,13] and they impact a wide

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Trends in Cancer

Figure 1. Schematic Representation of Glycosylation Diversity. The glycosylation process is a well-orchestrated enzymatic process of building sugar structures:
glycans. Glycans can be attached to a variety of molecules such as proteins (glycoproteins) or lipids (glycolipids) or exist as free structures, such as hyaluronic acid.

spectrum of key biological processes, including neoplastic cell survival, growth migration, metas-
tasis, and host antitumor immunity (reviewed in [3,10]). For instance, several lectin families of the
immune system were proven to play a key role in cancer, namely selectins, Siglecs, and galectins,
along with macrophage galactose-type lectin (MGL) and dendritic cell (DC)-specific ICAM-3-
grabbing non-integrin 1 (DC-SIGN) [14]. Glycan–lectin interactions are central axes of multiple as-
pects of cancer progression, such as immune evasion, cell proliferation, invasion, and extravasa-
tion. Interference in these interactions with specific inhibitors is currently being explored in clinical
trials as a promising novel strategy for unique and combination therapies [15–17]. For example,
multiple myeloma (MM) is initiated through the interaction between ligands present on MM cells
and adhesion molecules E- and P-selectin expressed on the vascular endothelium, which induce
rolling, adhesion, and extravasation. Most MM cancer therapies rely on direct malignant cell
targeting; however, recently a completed Phase I/II clinical trial in acute myeloid leukemia patients
using GMI-1271 (E-selectin antagonist) showed high remission rates and improved overall sur-
vival with favorable safety, and a Phase I clinical trial is being performed on MM patients
(NCT02811822). Currently, the safety of GMI-1359 (E-selectin and CXCR4 dual antagonist) is
being evaluated in a Phase I clinical trial (NCT02931214).

It is of no surprise that altered glycosylation presents huge potential for cancer research. How-
ever, the glycosylation process has been difficult to study in normal and aberrant circumstances.
The immense structural complexity and the fact that glycan biosynthesis is not under direct
genetic control have long hampered further progress in this area. Since specific glycan structures
are uniquely expressed on tumor cells, cancer-related glycans serve not only as biomarkers but

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Key Figure
Glycosylation Alterations as Drivers to Improve Cancer Therapy

Trends in Cancer

Figure 2. The diagram shows the actual status of cancer therapy. The treatment options rely on conventional therapy,
immunotherapy, and small-molecule inhibitors. The main drawbacks of each therapy regimen are indicated with red marks.
Considering that altered glycans play a role in cancer development, progression, and failure of these cancer treatment
options, a question is raised: Could targeting glycosylation be a solution for the development of effective cancer treatment?

also as promising targets for therapeutic strategies. Recent advances in new methodologies,
such as glycoengineering of cells allied to the great progress on analytical techniques, might un-
lock novel targets, overcoming the existing bottlenecks to examine new drugs [18,19]. This way,
using combinations of new glycan-based inhibitors, a coordinated attack on the cancer glycome
can be envisioned, which is expected to severely cripple the ability of tumor cells to grow and
metastasize [20,21].

The New Era of Cancer Drug Discovery

Although the process of drug discovery is known to be complex and time-consuming, the devel-
opment of new methodologies and more advanced technologies can help overcome these limi-
tations. Nowadays, the specific molecular targeting approaches available for use in clinical
practice are small-molecule agents, such as specific inhibitors [22], and monoclonal antibodies
(mAbs) [23] (Box 1). Although beneficial for treatment of cancer patients, both methodologies
have encountered some limitations, such as being restricted to a subset of cancer patients, or
have encountered therapy resistance.

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Box 1. Selective Therapies: Specific Inhibitors and mAbs

Antibodies are now an established option in cancer treatment, with more than 30 antibodies or antibody derivatives currently
approved by the FDA and European Medicines Agency and hundreds more in clinical trials [48]. Anticancer mAbs target a
broad range of antigens, including soluble proteins, cancer cell surfaces, and effector cell receptors. Such versatility allows
mAbs to exploit several mechanisms of action, including the direct blocking of growth factors, the interruption or induction
of signaling pathways, and the modulation of the immune system [48,49]. MAbs as therapeutic drugs offer some advan-
tages, such as relatively high specificity, low toxicity, and long half-life (i.e., weeks) in the human circulation. They have also
disadvantages, such as the laborious production process undergoing the relatively high heterogeneity due to, for example,
post-translational modifications (PTMs). However, these modifications offer opportunities to include additional functionalities
and thereby provide possibilities to further fine-tune the efficacy of some therapeutic antibodies [50,51].

Small-molecule inhibitors are generally thought to be less specific than therapeutic mAbs. However, this lower specificity is
potentially advantageous, albeit with some risk of increased toxicity, in that it confers the ability to inhibit several signaling
pathways at reduced plasma concentrations. Unlike mAbs, small-molecule agents can translocate through plasma mem-
branes and interact with the cytoplasmic domain of cell-surface receptors and intracellular signaling molecules [52].

The search for new therapeutic agents is a challenge and a main problem is the lack of infrastruc-
tures to test all new potential drugs. The emergence of HTS automatized methodology and its
capacity to test a large number of compounds had an important repercussion in the search for
new therapeutic drugs, particularly small-molecule agents. In fact, HTS approaches have had a
high impact on scientific research and biological testing, allowing for greater efficiency and
lower costs. HTS success rates are reasonable; roughly 50% successful obtention of a hit, con-
firmed by more than one assay in vitro and, where possible, in vivo [24,25]. However, the truth is
that only a few drugs on the market today are derived from these screenings, since they simply do
not reach the clinical trials phase or, when they do, they fail them. Common miscalculations with
these strategies include: (i) poorly validated targets; (ii) the use of small screening libraries; (iii) an
inadequate and incomplete follow-up capacity; (iv) highly artificial and nonphysiological assay
systems; (v) a severe lack of appropriate animal models for specific diseases, and (vi) unpredict-
able compound toxicity [25].

Several HTS assays have been developed in the past years, aiming for a cancer target therapy.
The majority of the approaches used in the cancer field are based on HTS of chemical and natural
compound libraries, with different protein kinases being the most common targets so far. The
FDA successfully approved several therapeutics found in small-molecule screens. Currently,
the FDA has approved more than 30 kinase inhibitors for cancer therapy. A few relevant ones
are gefitinib (2003), erlotinib (2004), sorafenib (2005), and lapatinib (2007) [25,26]. Unfortunately,
most of the hit candidates failed the validation phase. This drug family has some challenges, due
to drug resistance, toxicity, compromised effectiveness, and an inadequate knowledge of the se-
lectivity of these kinase inhibitors. Furthermore, clinical evaluation of these inhibitors has shown
that therapeutic responses vary amongst patients and between patient populations [27–29].

The investment in searching for new target therapies for cancer and different HTS assay optimi-
zation is expected to improve the rate of new drug approval in clinical trials and achieve more
effective therapy options for patients. However, there are still certain targets with therapy potential
that are poorly explored. A great example is the glycobiology field.

Glycosylation in Cancer and Drug Discovery: A Road to Be Explored

In addition to the earlier mentioned role in several aspects of tumor development and progression,
recent findings also suggest that glycans can impair or modulate cancer therapies. For instance,
resistance to the chemotherapeutic agent doxorubicin (DXR), a front-line treatment for some type
of cancers, is due to glycosylation at cell surface proteins, which may affect epitope accessibility
and drug binding to receptor proteins [30]. Additionally, cancer-associated glycans present at

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cancer cells can structurally impair the accessibility of the protein targets by therapeutic antibod-
ies [30–32]. At the forefront of immunotherapy, and with an unprecedented success, is the use of
antibodies to block immune checkpoints, such as the programmed cell death ligand 1 (PD-L1). In
2019, it was demonstrated that N-glycans inhibit PD-L1 antibody recognition at the tumor cell
surface, showing that therapeutic antibodies fail to bind to certain PD-L1 glycoforms. This led
to inefficient biomarker detection and the consequential premature exclusion of patients for treat-
ment [33]. These findings could have significant implications and may explain the failure of the
clinical trials targeting some of these markers. Also, the affinity of the therapeutic antibody
AR20.5, which targets MUC1, was shown to be glycan-dependent [31]. Other studies have
also highlighted the role of glycans in therapy resistance of currently used targeted therapies,
such as the mAbs against human epidermal growth factor receptor-type 2 (HER2), stressing
that this subject cannot be neglected. For instance, the interaction between herceptin (mAb
drug) and HER2 revealed an increase in the accessibility of the antibody following deglycosylation
of cell membrane proteins [30]. Also, Xiao and coworkers proposed an ingenious approach,
combining trastuzumab, an anti HER2 mAb, with a sialidase, an enzyme that removes sialic
acids from glycan structures. This antibody–enzyme conjugate selectively changed the tumor
cell glycocalyx, resulting in an increased activation of natural killer cells with consequent increased
cytotoxicity against tumor cells [34].

Novel strategies have been developed that focus on the glyco-specific issue. Various interesting
models and solutions have emerged, which aim at specific targeting and development of more
general approaches that can be applied to various glycosylation targets. For instance, recent
studies have used modified sugars, such as fluorinated analogs, to disturb glycosylation path-
ways [35–38]. However, this research has still been difficult to translate to clinical practice due
to the low specificity for cancer cells. More recently, several studies using genetically modified
T cells expressing chimeric antigen receptors (CAR-T) have also taken into consideration this
problem. For instance, Posey and coworkers successfully induced cancer cell cytotoxicity in
xenograft models of T cell leukemia and pancreatic cancer using a specific CAR that recognized
the cancer-associated Tn glycoform of MUC1 [39] (NCT03633773). However, the major
challenge in the CAR-T field is still the lack of efficient targeting for solid tumors [40]. Having
said that, discovery of new cancer drugs based on HTS still holds a great potential.

Different methodologies have been used for glyco-specific HTS assays (Table 1 and Box 2). Most
of these assays are either performed directly on purified protein targets, the so-called biochemical
based assays, or on cell models specifically displaying the features being tested, the cell-based
assays. The former is more laborious, involving the synthesis and testing of related compounds
and requiring further validation in cells and/or in vivo models, but generally achieving higher affinity
of the hits. The latter can directly achieve a biologically effective hit, yet in many cases, the actual
target is either unknown or needs work to be verified.

Donovan and colleagues were at the forefront of establishing a solid-phase glycosyltransferase

assay for HTS, aiming for drug discovery. Although other HTS methods for glycosyltransferases
were being developed at the time, this was the first screening of a natural compound library [41].
Thus, different assays have been developed targeting different glycosyltransferases, for instance,
GlcNAc-T5 and Core 2 GlcNAc-T [41]; ST3Gal3, ST6Gal1, ST3Gal1, FUT6, and FUT7, [42];
ppGalNAc-T2 and ppGalNAc-T3 [43]; and FUT 6 [44]. All these studies show very different and
interesting approaches to targeting aberrant glycosylation, especially in cancer. However, HTS
assays focused at glycan targets are mostly biochemical assays and the main concern is that
these approaches do not consider the effect of compounds on the cell itself and in the whole
‘system’ where these therapeutic targets are inserted. In fact, these enzymes can interact with

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Table 1. Different HTS Approaches Targeting Several Glycosyltransferases

HTS assay Target(s) Type of assay Refs
(biochemical vs. cell based)
Radiolabeled ligands
Solid-phase glycosyltransferase GlcNAc-T V and Core 2 GlcNAc-T Biochemical [41]
assay
Scintillation proximity assay FucTVII Biochemical [53]
Scintillation proximity assay FucTVII Biochemical [54]
Fluorescent methods
Fluorescence polarization O-GlcNAc transferase (OGT) Biochemical [58]
FRET O-GlcNAc transferase (OGT) Biochemical [60]
Fluorescence polarization ST3Gal III, ST6Gal I, ST3Gal I ST, Biochemical [42]
FUT6, and FUT7
Fluorescence detection of UDP, Galactosyltransferase B4GALT1 Biochemical [61]
GDP, and CMP
Cell-based fluorescence sensor ppGalNAc-T2 ppGalNAc-T3 Cell based [43]
Fluorogenically labeled FUT6 (adapted to all FUT) Biochemical [44]
oligosaccharide
Fluorescence polarization β-Kdo glycosyltransferases Biochemical [59]
(bacteria)
Other methods
Enzyme-linked lectin assay ppGalNAcT-1 Biochemical [62]
(ELLA)
Noncovalent carbohydrate FucT VI Biochemical [63]
microarray

Box 2. Most Relevant HTS Assays Targeting Glycosyltransferases

The adjustment of HTS to multiple approaches results in a group of interesting methodologies, adaptable to different
targets. Unfortunately, the variety of glyco-specific HTS assays is relatively poor in comparison. Even so, several different
and interesting approaches have been developed (see Table 1 in main text). The methods that use radioisotope have been
slowly eliminated, due to environmental and public health reasons, although the contribution of these systems for drug dis-
covery in general cannot be discarded and is worth mentioning. The first HTS approaches adapted to a screening of a
compound library consisted of a solid-phase glycosyltransferase assay for HTS [41]. The establishment of the scintilla-
tion proximity assay (SPA) technique resulted in two interesting studies focused on a particular glycosyltransferase,
FUT7 [53,54]. SPA is a common tool used in HTS and it is based on the phenomenon of scintillation, where the scintillant
is incorporated into fluomicrospheres (SPA beads); if a radioactive molecule binds to the fluomicrosphere, this stimulates
the scintillant to emit light [55].

Despite the potential of these methodologies, the establishment of fluorescence in HTS assay has opened a world of pos-
sibility for drug discovery in general and made possible the avoidance of radioactivity. Thus, the HTS fluorescence
methods for various glycobiological targets are currently more common, as they are highly sensitive [44]. Multiple assays
have been developed using fluorescence methods, the most persistent being fluorescence polarization, widely utilized
in HTS to investigate a variety of biological processes involving molecular interactions and enzymatic activity [56,57]. This
methodology was used to explore different glycotargets, such as O-GlcNAc transferase (OGT) [58], several sialyl- and
fucosyl-transferases [42], and even bacteria β-Kdo glycosyltransferases [59]. The fluorescence approaches are many
and are open to various possibilities and strategies. For instance, Gross et al. also proposed a fluorescence resonance
energy transfer (FRET)-based HTS for OGT [60]. In 2013, a highly sensitive universal assay based on enzyme-coupled fluo-
rescence detection of UDP, GDP, and CMP was reported and it is based on the use of ADP for conversion of resazurin to
highly fluorescent resorufin, which is determined by fluorescence measurement [61]. In 2019, another interesting study
was published by Zhang and colleagues, where a glycosidase-coupled enzyme approach was established, with
fluorogenically labeled oligosaccharides for fucosyltransferases [44]. Other methods were also utilized to find glycosylation
inhibitors/modulators, highlighting two research papers targeting ppGalNAcT-1 [62] and FuT-VI [63].

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several molecules, leading to the formation of abnormal glycans and disrupting the correct func- Outstanding Questions
tioning of cells. Thus, we envision that HTS using cellular systems for these targets will benefit the How can we get around the
search for new therapies. This way, we can evaluate compounds and molecules not only in terms inefficiency of currently used cancer
drug treatments?
of therapeutic action, but also obtain some information on the toxicity and specificity of the can-
didate, something that was not possible in biochemical assays. Are glycosylation alterations a cause or
a consequence of cancer?
A recent report in 2017 finally makes use of a cellular model to find an inhibitor of a specific
To what extent can altered glycans
ppGalNAc transferase, ppGalNAc-T3, that has been involved in cancer metastasis. Thus, a contribute to tumor development and
drug that blocks ppGalNAc-T3 could potentially prevent cancer cells from spreading. In this progression?
study, out of the over 20 000 compounds screened, one compound, T3Inh-1 selectively blocked
What are the reasons for the low drug
ppGalNAc-T3, with downstream effects on cells grown but without causing any toxic side effects
approval rates to the clinic?
[43]. A ppGalNAc-T2 sensor was used to assess the specificity of the tested drugs. This report
opens the door to new cancer glycan target studies using cellular systems for drug discovery What are the best approaches to
HTS. Moreover, a library of gRNAs that target all the glyco-related enzymes has been published, target glycan-specific antigens?
opening a window of opportunity for the fine tuning of different glycosylation pathways [45]. Alto- Should targeted delivery to cancer
gether, new possibilities have emerged in this area and we may envision that the development of cells be considered as a means of
even more accurate systems for HTS, such as 3D models, will contribute to solving the problem. improving the specificity towards
glycosylation in cancer versus
glycosylation in normal cells?
The remaining concern on targeting glycosylation in cancer is still the risky side effects. The up-
take of promising new drugs by nonmalignant cells may damage their homeostatic glycan biosyn-
thesis, counting for undesired effects. We may envision that a targeted-delivery system comes as
a smart solution to such limitations. In recent years, the use of nanoparticles (NPs) for the targeted
drug delivery has gained enormous application [46]. Additionally, novel strategies to functionalize
NPs, such as their surface decoration with antibodies, have been improving the efficiency of drug
delivery systems [47]. We envision that, in the near future, new approaches for cancer-specific
delivery of such promising new therapeutics will emerge.

Concluding Remarks
Cancer is a leading cause of death worldwide and most cancer-related deaths are due to the lack
of effective therapies. Cancer treatment regimens rely mostly on conventional chemotherapy and
on a few targeted therapies that can only be applied to a subset of cancer patients. Nevertheless,
there is increasing evidence that altered glycans are active players throughout cancer develop-
ment and progression. In addition, altered glycosylation in cancer also plays a role in the failure
of these cancer treatment options. In this direction, we have explored the specific case of glyco-
sylation in cancer as a promising target for drug discovery strategies (Figure 2). It is true that the
complexity of glycosylation is a challenge; still, efforts have been initiated to target altered glycans
in cancer (see Outstanding Questions). We mentioned a few of the diverse and interesting ap-
proaches targeting cancer-associated glycans, such as the use of carbohydrate analogs and
the development of glycan-specific CAR-T and we focused on HTS for the discovery of small
molecules with potential clinical application. We finished by underlining that, apart from the
need for development of new cancer drug strategies targeting glycosylation, it is worth exploring
effective cancer delivery systems to avoid side effects. We believe that exploring specific glycosyl-
ation targets may be the start of a new era of cancer therapy.

Acknowledgments
This work was supported by: (i) Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020—
Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020 (POCI-01-0145-FEDER-016585;
POCI-01-0145-FEDER-007274). (ii) Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020
Partnership Agreement, through the European Regional Development Fund (ERDF) project NORTE-01-0145-FEDER-
000029. (iii) Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e
Inovação through the research projects PTDC/MED-QUI/29780/2017 - POCI-01-0145-FEDER-029780.

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