Glycans: potential therapeutic targets for cholangiocarcinoma and their therapeutic and diagnostic implications

KEYWORDS:

• Bile ducts
• biomarker
• cancer
• glycan
• glycosylation
• liver
• targeted therapy

1. Introduction

Glycosylation is an important post-translational modification of cellular glycoprotein by the stepwise addition of sugar residues to form a complex glycan structure. Glycans play essential roles in many biological processes; such as cell-cell communication, cell adhesion, ligand–receptor interaction, self and nonself recognition, etc. Alteration of glycosylation has been considered a hallmark of cancer as it plays important role in tumor development and progression [1]. The aberrant glycosylation is possibly triggered by the overproduction of nucleotide-sugar donors and/or altered expression of glycosyltransferase and glycosidase enzymes [2]. Not only aberrant glycosylation, but the over-expression of carrier-proteins is also an important factor to promote tumor growth and metastasis. These aberrations are possibly the targets for cancer immunotherapy or chemo-sensitization. Also, the cancer-associated glycans and glycoconjugates can possibly be the biomarkers for diagnosis, monitoring, and prognostic prediction of the disease. Collective data in cholangiocarcinoma (CCA) revealed that glycosylation is altered during tumorigenesis and progression of CCA. The CCA-associated glycans were aberrantly expressed and play many essential roles in tumor metastasis and drug resistance, which were possibly used as the targets for CCA treatment. Moreover, the CCA-associated glycans that are elevated in patients’ sera are beneficial as biomarkers for diagnosis and prognosis of CCA.

2. Glycans and CCA

2.1. Roles of glycans in CCA metastasis and chemoresistance

Glycomics and glycoproteomics approach on CCA tissues revealed the aberrant glycosylation and consequent elevation of CCA-associated glycans in cancer cells compared with hepatocytes and normal biliary cells. A high level of expression of these glycans was associated with poor prognosis and shorter survival of CCA patients. This information agrees with the results from functional analyses of CCA-associated glycans which play important roles in the progression of CCA (Table 1).

Table 1. Applications of CCA-associated glycans and specific lectins in CCA

			Applications
Glycans	Applicable lectins	Functions	Marker	Targeted therapy	References
O-GalNAc	SBA WFA SJA VVL	Metastasis	Diagnosis Prognosis	Anti-metastasis	[3–5,7]
O-GlcNAc	sWGA	Metastasis	Prognosis	Anti-metastasis	[6]
Fucose	EUA-I	Metastasis	Prognosis	Anti-metastasis	[9]
Sialic acid	MAL-II	Chemoresistance	Prognosis	Chemo-sensitization	[10]

2.1.1. GalNAc glycans

N-acetyl galactosamine (GalNAc)-containing glycans were drastically increased in CCA compared with the normal bile ducts in adjacent tissues [3–5]. The O-linked GalNAc glycan was found to regulate CCA metastasis via Akt/Erk signaling pathway. Modulation of GalNAc modification by knockdown or overexpression of GalNAc-transferase (GalNT)-5 was shown to affect the metastatic ability of CCA cells [5].

2.1.2. GlcNAc glycans

O-GlcNAcylation, the reversible modification of nucleocytoplasmic proteins by N-acetyl glucosamine (GlcNAc), is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and was found to promote metastasis of CCA. O-GlcNAc regulated N-glycosylation through Erk/Akt signaling pathway and phosphorylation of Forkhead box-O3⁶. Blockade of surface N-glycan by a mannose-binding lectin from Pisum sativum resulted in significant suppression of the metastatic abilities of CCA cells [6].

2.1.3. Fucosylated glycans

Peripheral glycans, including fucosylated- and sialylated-glycans, were also demonstrated to promote the progression of CCA. Fucosylated-glycans, such as a(1,2)-fucose-modified glycan and Lewis-associated glycans were found to facilitate the migration and invasion of CCA cells [7–9]. Fucosyltransferase (FUT)-1, an enzyme response for a(1,2)-fucose modification, was found to regulate the EGF/EGFR activation in CCA cells [9]. Metastatic abilities of CCA cells were significantly suppressed by specific siRNA against FUT1 and FUT3. Neutralization of surface Lewis-associated antigens using a specific monoclonal antibodies against CA-S27 or sialyl-Lewis A (sLeA) could significantly reduce the metastatic abilities of CCA cells. The metastatic suppression effect was also observed after treatment with Ulex europaeus agglutinin-I (UEA-I, a(1,2)-fucose binding lectin).

2.1.4. Sialylated glycans

Different from other glycosylation processes, the terminal sialic acid was found to be involved in the chemoresistance of CCA. Suppression of sialic acid modification by a sialyltransferase inhibitor, 3Fax-peracetyl-Neu5Ac, could enhance the sensitivity of CCA cells to 5-fluorouracil (5-FU), a general chemotherapeutic drug used for cancer treatment [10].

These collective pieces of evidence provide the imperative information about the glycans in promoting CCA metastasis and chemoresistance, suggesting CCA-associated glycans as the potential targets for CCA treatment.

2.2. Glycan as a marker for diagnosis and prognostic prediction of CCA

Several CCA-associated glycans are highly expressed in patients’ tissues and sera, which can be used as the potential markers for diagnosis and prognostic prediction of CCA.

The information obtained through glycomic analysis using lectin-based approaches showed that many GalNAc-binding lectins; including Wisteria floribunda agglutinin (WFA) [4], Sophora japonica agglutinin (SJA) [3], and Vicia villosa lectin (VVL) [5]; exhibited a strong reactivity with preneoplastic bile ducts and CCA, but not normal bile ducts, suggesting their specificity to bile duct pathology. The glycans comprised in mucin glycoproteins are possibly been shredded and secreted into serum and bile of CCA patients. The CCA-associated glycoforms of mucin glycoproteins, WFA-bound MUC1 [4], and soybean agglutinin (SBA)-bound MUC5AC [11], were highly detected in serum and/or bile of CCA patients and applicable for the diagnosis of CCA. Lewis-associated glycans, including carbohydrate antigen 19–9 (CA19-9, sialyl-Lewis A) and carbohydrate antigen-S27 (CA-S27, Lewis-A-associated glycan), were highly detected in the sera of CCA patients compared with non-CCA controls [7,12]. A high level of these serum glycans was found to associate with poor prognosis and the short survival of CCA patients, suggesting their ability to be a poor prognostic marker for CCA.

Moreover, a high level of O-GlcNAc-modified proteins, MAL-II binding a(2,3)-sialylated glycan, and a(1,2)-fucose modified glycan in tumor tissues was associated with the unfavorable fate of CCA patients, suggesting their application as a potential poor prognostic indicator for CCA.

3. Expert opinion

Glycans are the potential targets for the treatment of CCA as well as biomarkers for diagnosis and prognostic prediction of the disease (Figure 1). As mentioned as above, most of the CCA-associated glycans; N-glycans, O-GlcNAc, O-GalNAc, and the terminal fucose and Lewis antigens; were found to play important roles in tumor metastasis. Thus, these CCA-associated glycans are probably promising targets for the development of anti-metastatic agents. Furthermore, sialic acid-modified glycan was found to be involved in the chemoresistance of CCA. Therefore, it is possible to target the sialic acid modification for chemo-sensitization in CCA treatment. The combination of glycosylation inhibitors and the standard chemotherapeutic drugs, such as 5-FU, gemcitabine, and cisplatin, is a fascinating approach to be considered to improve the chemotherapy of CCA.

Figure 1. Schematic diagram for biosynthesis of CCA-associated glycans

Alteration of glycosylation in CCA leads to the expression of CCA-associated glycans that are potentially used as the targets for CCA treatment and as the biomarkers for diagnosis and prognostic prediction of CCA. After the protein is translated from mRNA, several nucleocytoplasmic proteins, such as transcription factors, are possibly modified by N-acetylglucosamine (GlcNAc) under the control of O-GlcNAc transferase (OGT) then translocated into the nucleus. The synthesis of mucin-type O-glycans, serine, or threonine residue was initially modified with N-acetylgalactosamine (GalNAc) by GalNAc-transferases (GalNTs), followed by the stepwise glycan extension. Terminal glycosylation with fucose or sialic acids was regulated by fucosyltransferases (FUTs) and sialyltransferases (STs). After complete glycosylation, the glycoproteins were transferred to the cell membrane or secreted out of the cells. Several CCA-associated glycans were found to be the potential targets for CCA treatment. O-GalNAc, O-GlcNAc, and fucosylated glycans were found to participate in metastasis, while sialylated glycan was identified to involve in chemoresistance of CCA. The secreted CCA-associated glycans were applicable as the glycobiomarker for diagnosis and prognostic prediction of CCA.

Several approaches can possibly be used to target the CCA-associated glycans for CCA treatment [13]. Inhibition of glycan biosynthesis by suppression of glycosyltransferases such as OGT, GalNT, FUTs, and sialyltransferases using specific inhibitors or RNAi is one of the promising strategies for CCA treatment. Several glycosylation inhibitors have been synthesized and studied on many types of cancer (Table 2) [14,15]. Further, comprehensive in vivo and clinical studies are still needed on the efficiency of these inhibitors for CCA treatments. The application of RNAi-based treatment for cancer patients are recently facing with many obstacles regarding the efficiency and safety of RNAi delivery. The success of the development of RNAi-based treatment for CCA may lead to the success of the targeted-therapy of CCA in the future.

Table 2. Examples of glycosylation inhibitors

Types of inhibitor		Mechanism of inhibition	Effects
Metabolic inhibitors
	6-diazo-5-oxo-L-norleucine (DON)	Interfere metabolism of glycosylation precursors	Decrease UDP-GlcNAc
Tunicamycin		Blocking the formation of dolichol-PP-GlcNAc	Decrease N-linked glycans
Plant alkaloids
	Australine	Inhibit α-glucosidases I	Incomplete N-linked glycans synthesis
	Castanospermine	Inhibit α-glucosidases I and II	Incomplete N-linked glycans synthesis
	Deoxynojirimycin	Inhibit α-glucosidase I and II	Incomplete N-linked glycans synthesis
	Deoxynojirimycin	Inhibit α-glucosidases II	Incomplete N-linked glycans synthesis
	Kifunensin	Inhibit α-mannosidase I	Incomplete N-linked glycans synthesis
	Mannostatin A	Inhibit α-mannosidase II	Incomplete N-linked glycans synthesis
	Swainsonine	Inhibit α-mannosidase II	Incomplete N-linked glycans synthesis
Substrate and Nucleotide-sugar analogs
	Uridine analogs	Inhibit polypeptide GalNAc transferases (GalNTs)	Decrease O-linked glycans
	Alloxan	Inhibit O-GlcNAc transferase (OGT)	Decrease O-GlcNAc glycans
	Streptozotocin	Inhibit O-GlcNAc transferase (OGT)	Decrease O-GlcNAc glycans
	Peracetylated GlcNAc (Ac-GlcNAc)	Inhibit O-GlcNAc transferase (OGT)	Decrease O-GlcNAc glycans
	O- [2-acetamido-2-deoxy-D-glucopyranosylidene]amino-N-phenylcarbamate (PUGNAc)	Inhibit O-GlcNAcase (OGA)	Increase O-GlcNAc glycans
	Thiamet-G	Inhibit O-GlcNAcase (OGA)	Increase O-GlcNAc glycans
	GlcNAcstatin	Inhibit O-GlcNAcase (OGA)	Increase O-GlcNAc glycans
	Glycan substrate analogs	Inhibit specific glycosyltransferases	Decrease the target glycan structures

Targeted immunotherapy is also a great challenge for CCA treatment. The use of specific monoclonal antibodies or the development of the chimeric antigen receptor (CAR)-T cells that recognizes CCA-associated glycans are the anticipative strategies to improve the immunotherapy of CCA.

Using lectins that exhibit high affinity and specificity to bind the target CCA-associated glycans are one of the key strategies to be considered. Many lectins were demonstrated to exhibit strong anti-cancer activity and potentially be used for cancer treatment [16]. The clinical trial of a lectin from mistletoe has been performed on many types of cancer and it seems to be a promising anti-cancer drug without serious side effects to the patients [16]. Based on the in vitro studies, Pisum sativum agglutinin (PSA) and UEA1 could significantly suppress the metastatic ability of CCA cells and are considered potential anti-metastatic agents for CCA. However, to assure the clinical effectiveness of these lectins as an anti-metastatic agent for CCA, further in vivo and clinical studies are needed. The chimeric antibodies or lectins against CCA-associated glycans are also possible potential anti-cancer agents as they were presumed to provide the high therapeutic efficiency with lower side effects, comparing with the full-length molecules.

Although CCA-associated glycan seems to be a promising target for CCA treatment, it may not applicable for all CCA cases as a particular glycan is expressed only in some, but not all CCA cases. To achieve the most effective way of targeting the CCA-associated glycans for CCA treatment, their expressions in the resected- or biopsied-tissues or serum samples should be investigated prior to the treatment. Inclusion of the determination of CCA-associated glycans as one of the criteria of CCA management may facilitate the clinicians to select the most effective-targeted-therapy.

Besides the target for CCA treatment, glycans are also applicable as the biomarkers for diagnosis and prognostic prediction of the disease. CCA-associated glycans, especially GalNAc-containing glycans, were highly detected in tissue and/or serum of the CCA patients. The expression pattern of these glycans are variable among CCA patients [12]. Combined analysis of these CCA-associated glycans can improve the diagnostic and prognostic power of glycans [12]. To improve the diagnosis and prognostic prediction of CCA, the development of the promising multi-glycobiomarker panel is an important and urgent task to be considered.

As glycoproteins are composed of peptide and glycans, the lectin-based approaches are commonly used for the analysis of CCA-associated glycans. Development of diagnostic kit using lectin-lectin or lectin-antibody sandwich approaches may improve their efficiency to detect CCA-associated glycobiomarkers as it combines the preferential ability of monoclonal antibodies to proteins and of lectins to glycans.

4. Conclusion

The cumulative information showed that CCA cells exhibited the altered glycosylation, which leads to the aberrant expression of CCA-associated glycans. These tumor-associated glycans can be a potential biomarker for diagnosis and prognostic prediction of CCA as well as the targets for treatment.

Declaration of interests

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Acknowledgments

The author would like to thank Prof. Yukifumi Nawa for the English editing via KKU Publication Clinic.

Additional information

Funding

The works of the author were supported by the Faculty of Medicine, Khon Kaen University (IN63273); Thailand Research Fund (MRG6180107); Program Management Unit for Human Resources & Institutional Development, Research, and Innovation (630000050061).