Glycosylation: Rising Potential for Prostate Cancer Evaluation

Review Glycosylation: Rising Potential for Prostate Cancer Evaluation

Anna Kałuza˙ * , Justyna Szczykutowicz and Mirosława Ferens-Sieczkowska

Department of Chemistry and Immunochemistry, Wroclaw Medical University, Sklodowskiej-Curie 48/50, 50-369 Wroclaw, Poland; [email protected] (J.S.); [email protected] (M.F.-S.) * Correspondence: [email protected]; Tel.: +48-71-770-30-66

Simple Summary: Aberrant protein glycosylation is a well-known hallmark of cancer and is associated with differential expression of enzymes such as glycosyltransferases and glycosidases. The altered expression of the enzymes triggers cancer cells to produce glycoproteins with speciﬁc cancer-related aberrations in glycan structures. Increasing number of data indicate that glycosylation patterns of PSA and other prostate-originated proteins exert a potential to distinguish between benign prostate disease and cancer as well as among different stages of prostate cancer development and aggressiveness. This review summarizes the alterations in glycan sialylation, fucosylation, truncated O-glycans, and LacdiNAc groups outlining their potential applications in non-invasive diagnostic procedures of prostate diseases. Further research is desired to develop more general algorithms exploiting glycobiology data for the improvement of prostate diseases evaluation.

Abstract: Prostate cancer is the second most commonly diagnosed cancer among men. Alterations in protein glycosylation are conﬁrmed to be a reliable hallmark of cancer. Prostate-speciﬁc antigen is the

biomarker that is used most frequently for prostate cancer detection, although its lack of sensitivity and speciﬁcity results in many unnecessary biopsies. A wide range of glycosylation alterations in

Citation: Kałuza,˙ A.; Szczykutowicz, prostate cancer cells, including increased sialylation and fucosylation, can modify protein function J.; Ferens-Sieczkowska, M. and play a crucial role in many important biological processes in cancer, including cell signalling, Glycosylation: Rising Potential for adhesion, migration, and cellular metabolism. In this review, we summarize studies evaluating the Prostate Cancer Evaluation. Cancers prostate cancer associated glycosylation related alterations in sialylation, mainly α2,3-sialylation, core 2021, 13, 3726. https://doi.org/ fucosylation, branched N-glycans, LacdiNAc group and presence of truncated O-glycans (sTn, sT 10.3390/cancers13153726 antigen). Finally, we discuss the great potential to make use of glycans as diagnostic and prognostic biomarkers for prostate cancer. Academic Editor: Sandra J. van Vliet Keywords: glycosylation; glycans; prostate cancer; prostate specific antigen; biomarkers Received: 25 June 2021 Accepted: 21 July 2021 Published: 24 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Prostate cancer was the second most commonly occurring cancer and the fifth leading published maps and institutional affil- cause of cancer death among men in 2020 [1,2]. In 1986, prostate-specific antigen (PSA) iations. was approved by the United States Food and Drug Administration (FDA) to screen men for prostate cancer (PCa). PSA is present in small quantities in the serum of men with a healthy prostate (up to 2.5 ng/mL), whereas a concentration above 4 ng/mL is considered indicative of prostate cancer or benign prostatic hyperplasia (BPH) [3,4]. Frequently, PSA screening for prostate cancer has limited sensitivity and specificity, which can lead to Copyright: © 2021 by the authors. overdiagnosis and overtreatment of indolent disease, resulting in unnecessary, invasive Licensee MDPI, Basel, Switzerland. This article is an open access article biopsy and treatments for non-aggressive cancers [5,6]. Hence, in the following years distributed under the terms and serum PSA screening in association with digital rectal exam (DRE) and Gleason scoring conditions of the Creative Commons of prostate biopsy samples was approved by the FDA for the early detection of prostate Attribution (CC BY) license (https:// cancer [7]. Subsequent approaches for refining the specificity and sensitivity of the serum creativecommons.org/licenses/by/ PSA test involved calculating the proportion of free PSA (fPSA) to total PSA (tPSA), and 4.0/). PSA complexed with alpha-1-antichymotrypsin and α2-macroglobulin to total PSA [7], as

Cancers 2021, 13, 3726. https://doi.org/10.3390/cancers13153726 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3726 2 of 22

well as focusing on the individual molecular forms of PSA such as proPSA, benign PSA, and intact PSA. A multifactorial PSA test, such as the Prostate Health Index (PHI) and the four-kallikrein panel (4Kscore), can improve diagnostic accuracy but has limited prognostic usefulness, and may miss some aggressive tumors [8–10]. More recently, imaging techniques such as multiparametric magnetic resonance imaging (mpMRI) and prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/computed tomography (CT) have evaluated as a potential tool for staging prostate cancer in men before radical treatment. However, the limitations of both techniques were identified. Detection of clinically significant PCa in multiparametric MRI is changeable and depends on a few factors, including tumor location and volume, Gleason score, moreover the analysis of multiparametric prostate MRI is operator dependent [11]. Meanwhile, the sensitivity of PSMA PET/CT technique for the detection of metastases is reduced, presumably due to limitations in the spatial resolution of detecting small tumor deposits in primary and recurrent prostate cancer [12]. Researchers also compared the clinical utility of prostate cancer antigen 3 (PCA3) with PSA in a serum test [13] and widely examined PSA post- translational modification such as glycosylation [14–16]. Due to the high importance of glycan alterations observed in prostate cancer, including increased sialylation and core fucosylation, the emergence of truncated O-glycans and branched N-glycans, in this review we discuss the great potential to make use of glycans as diagnostic and prognostic biomarkers for prostate cancer. Our review focuses on PSA, the glycoprotein that has been studied in the most detail so far, but the variety of scientific approaches is remarkable, therefore unfractionated biological material such as whole serum were also considered. Not all glycoepitopes important for prostate cancer proliferation have been observed for PSA, thus analyzing only a selected glycoprotein, a lot of data relevant to the assessment of glycosylation changes in prostate disease may be lost. Therefore, presented work does not provide evident solutions for the selection of glycoepitopes that could effectively act as prognostic biomarkers, the studies available so far do not allow for such far-reaching conclusions.

2. Action of Androgens in the Prostate Gland The biomolecules secreted in prostatic fluid are engaged in the regulation of prostate epithelium homeostasis and the process of ejaculation. The agents that provide these functions are kallikreins, which include PSA and kallikrein-related peptidase 2 (hK2). The others are citrate, an intermediate product of the Krebs cycle, and Zn2+, a chemical element actively stored within the cytoplasm of the prostatic epithelial cells [17,18]. Physiological growth and development of prostatic epithelium, as well as the regulation of its secretory functions, depend on the appropriate action of androgen steroid hormones [19]. The action of androgens is mediated via the androgen receptor (AR), a ligand-activated transcription factor and member of the steroid hormone nuclear receptor family, which mediates androgen signaling by binding to androgen response elements (AREs) in both normal prostate tissue and prostate cancer [20–22]. Healthy prostate cells store the largest amount of zinc ions of all soft tissues in the human body. This remarkable property is based on the fact that prostatic epithelial cells retain Zn2+ through androgen-dependent Zn2+ cellular uptake and maintain the cycle in which specific zinc transporters are involved [17,18]. The accumulation of Zn2+ and citrate within the prostate, suppression of the Krebs cycle, and the release of fluid from the gland are regulated by androgens. In prostate tissue, a more potent derivative 5α- dihydrotestosterone (DHT), synthesized from testosterone by 5α-reductase enzyme, is the primary ligand for the AR [23]. Hundreds of genes have been identified that are regulated via DHT in prostate epithelial cells, many of which are essential genes involved in the maintenance of prostate homeostasis [24,25]. Circulating testosterone level and intraprostatic DHT concentration decrease gradually with ageing, causing the gland to malfunction by reducing its ability to sustain a healthy tissue level of intracellular Zn2+, KLK-secreted proteins and citrate within the prostate liquid. Cancers 2021, 13, 3726 3 of 22

Normal prostate epithelial cells display characteristic behaviour in relation to their metabolic pathway. They are programmed to produce citrate, unlike most cells which oxidize it [26]. The inhibition of the Krebs cycle in normal prostate cells results in the accumulation and subsequent secretion of citrate as a component of semen. This distinctive process of citrate production is supported by another speciﬁc feature of the prostate epithelial cells: the ability to store high concentrations of zinc, which have been shown to inhibit m-aconitase, the enzyme that catalyses the oxidation of citrate in the Krebs cycle [27,28]. Accumulation of zinc in prostate epithelium is driven by an increased amount of the zinc transporter ZIP1 in tissue [27,28]. In normal prostate epithelial cells, through the accumulation of zinc, the Krebs cycle is inhibited, so these cells are energetically inefﬁcient. This leads to an ATP production process different than present in most cells [29]. In contrast, prostate cancer cells reverse this process, leading to reactions that cause zinc loss and citrate oxidation, which is a major change in energy metabolism [18,28]. It was discovered long ego that prostate cancer cells do not exhibit the standard Warburg effect, observed in most cancer cells, whereby high glucose uptake and lactate release are considered hallmarks of most tumors [30]. As opposed to most cancer cells that utilize aerobic glycolysis, prostate cancer cells show a higher level of citric acid cycle activity compared to normal cells [26,28,31]. Apart from that, androgens are crucial for the development and metastatic progression of prostate cancer. Undoubtedly, androgen deprivation therapy (ADT) is the widely accepted initial treatment for symptomatic metastatic prostate cancer. In the early stages of diseases ADT is effective, but after 2–3 years the patients may develop castration-resistant prostate cancer (CRPC), which is ultimately lethal [32,33].

3. Prostatic Inflammation, Benign Prostatic Hyperplasia and Prostate Cancer Recent data support the role of chronic prostatic inflammation as a predisposing factor for development of BPH [34] and prostate cancer [35]. Several authors have highlighted the significance of prostatic inflammation in the origin of BPH, pointing out that the prostate is an immunocompetent gland in which a small number of inflammatory cells such as T and B lymphocytes, mast cells and macrophages are physiologically present [36]. In adults, chronic inflammatory prostate infiltrates vary greatly in BPH and healthy tissues. The most common cells of infiltrates from patients with BPH are CD19+ or CD20+ B lymphocytes, CD4+ T lymphocytes and macrophages [37]. The B and T cells as well as macrophages occurring in the adult prostate can contribute to the damage of both epithelial and stromal cells, induce cytokine production and increase the concentration of growth factors that can stimulate an anomalous remodelling process. Interestingly, IL-8 has been suggested as a link between chronic prostate inflammation and the development of BPH. Several studies have indicated significantly elevated IL-8 expression in epithelial prostate cells and emphasized that this can trigger the expression of fibroblast growth factor (FGF) and stromal-epithelial growth factor signaling and subsequently induce abnormal proliferation of prostatic cells [38]. In consequence, tissue damage can initiate a chronic wound healing process that can induce prostate enlargement, potentially resulting in BPH [36].

4. Biology of Prostate-Specific Antigen Prostate fluid is a rich source of prostate-derived proteins that can be used for biomarker discovery in a number of prevalent benign and malignant prostatic diseases including prostatitis, benign prostatic hyperplasia and prostate cancer. Several glycosylated proteins have been accepted as cancer biomarkers by the FDA, including prostate-specific antigen [39,40]. Prostate-specific antigen (PSA), also known as human kallikrein 3 (KLK3), is currently widely used as the gold standard biomarker for screening and diagnosis of prostate cancer [4]. PSA is an organ-specific glycoprotein, secreted by the epithelium and periurethral glands. PSA occurs as a 237-amino acid serine protease with chymotrypsin- like activity, and its transcription is regulated by androgens [41,42]. PSA is secreted as an inactive proenzyme (proPSA) into seminal fluid, which during the liquefaction process is Cancers 2021, 13, 3726 4 of 22

converted by kallikrein-related peptidase 2 to a 33 kDa mature active form [3,43,44]. In the early stage of prostate cancer development, disruption of the basal cell layer and the basement membrane of the prostate epithelium results in leakage of PSA into the peripheral circulation, and this circulating blood form of PSA is identified in the early detection tests for prostate cancer [3]. The serum PSA level increases with the clinical severity of disease and is proportional to tumor volume, whereas after radical prostatectomy its level in serum reaches undetectable values [41,45]. Schroeder et al. showed that PSA-based screening of prostate cancer reduced the rate of death by 20% but simultaneously was correlated with a high degree of overdiagnosis [46]. PSA has a single N-glycosylation site at asparagine (Asn-69), verified in X-ray crystal structures [47,48]. In 2013, the results of a comprehensive multi-laboratory ABRF study examining N-glycosylation of Asn-69 in healthy donors were announced, and they made it possible to describe the structure of glycans attached to PSA [3,48,49]. The most abundant glycans identified were four biantennary glycan structures, consisting of three mannose, two galactose subunits, and four β-N-acetylglucosamine residues, estimated to comprise approximately 80% of the total number of PSA-related glycans. These all have one or two terminal sialic acid residues and the presence or absence of a core fucose. Two dominant hybrid structures have also been reported [3,48,49].

5. Glycosylation Changes in Cancer Protein glycosylation is a post-translational process that enhances molecular heterogeneity as well as functional diversity within cell populations. Tumor cells show a wide range of glycosylation rearrangement in relation to their unchanged counterparts. Hako- mori and Kannagi were the ﬁrst to postulate the two main mechanisms underlying the cancer-associated changes of carbohydrate structures, called incomplete synthesis and neo-synthesis [50]. The incomplete biosynthesis pathway, typical for the early stages of a tumor, is the result of defective synthesis of typical glycans present in normal epithelial cells, leading to the synthesis of truncated structures such as short-chain O-GalNAc glycans (Tn, T, sialyl-Tn, and sialyl-T antigens). In contrast, neo-synthesis occurs extensively in advanced stages of cancer, and is associated with the induction of some genes involved in the glycosylation pathway, which results in de novo expression of antigens such as Lewis blood group related antigens (Le) and their sialylated counterparts: sialyl Lewis A (sLea) and sialyl Lewis X (sLex) antigens (Figure1)[ 50,51]. Altered expression of glycans may result from several biological factors: (1) overexpression or underexpression of the relevant glycosyltransferases in the Golgi apparatus, (2) changed glycosidase activity, (3) alterations in tertiary peptide backbone composition, and (4) the availability and sufﬁciency of the sugar nucleotide donors and cofactors [52–54]. The presence and molecular density of the glycans affect the half-life of many different types of membrane receptor proteins, including glucose transporters, cytokine receptors, transforming growth factor beta (TGF-β) and epidermal growth factor receptor (EGFR), which are involved in tumor formation and cell migration associated with cancer progression [54–56]. Cancers 2021, 13, 3726 5 of 22 Cancers 2021, 13, x 5 of 20

FigureFigure 1. OverexpressedOverexpressed glycan glycan structures structures typical typical for PCa. for PCa. (A) Highly (A) Highly branched branched (tetra-antennary) (tetra-antennary) N-glycan N with-glycan LacdiNAc with LacdiNAcmotif; (B) Biantennarymotif; (B) Biantennary N-glycan terminated N-glycan terminated with α2,3-linked with α2,3 SA;- (linkedC) Biantennary SA; (C) Biantennary N-glycan with N- coreglycan fucose with and core bisecting fucose andGlcNAc; bisecting (D) sTnGlcNAc; truncated (D) sTn O-glycan; truncated (E) O sT-glycan; O-glycan (E) antigen; sT O-glycan (F) core-2-O-glycan antigen; (F) core with-2-O sLe-glycanX antigen. with BluesLeX boxesantigen. indicate Blue boxesglycosyltransferases indicate glycosyltransferases involved in the involved synthesis in ofthe particular synthesis structures. of particular structures.

6.6. Sialylated Sialylated N N-Glycans-Glycans SialylatedSialylated glycans glycans are are the the ligands ligands of of various various pr proteinsoteins involved involved in in crucial crucial biological biological processes;processes; cell cell surface surface sialylated sialylated glycans glycans are are engaged engaged in in the the immune immune response, response, signal signal transductiontransduction and and embryonic embryonic development. development. The The sialic sialic acid acid residues residues operate operate as as receptors receptors forfor specific specific ligands, ligands, such such as as siglecs siglecs and and sele selectinsctins [57,58] [57,58].. Many Many reports reports have have also also stated stated thatthat sialylated sialylated glycans glycans are are involved involved in in oncogenesis oncogenesis and and malignant malignant progression progression [58] [58].. The The transformationtransformation of of healthy healthy cells cells into into heterogeneous heterogeneous cancer cancer cells cells is is accompanied accompanied by by the the appearanceappearance of of an abnormal abnormal sialylation sialylation pattern, pattern, which which is isreflected reflected in a in large a large group group of sialy- of lated glycoproteins secreted by tumor cells [58,59]. One of the decisive glycomic features sialylated glycoproteins secreted by tumor cells [58,59]. One of the decisive glycomic associated with malignant and metastatic progression is the presence of N-glycans termi- features associated with malignant and metastatic progression is the presence of N- nated with α2,6-N-acetylneuraminic acid (Neu5Ac) residues, controlled by the action of glycans terminated with α2,6-N-acetylneuraminic acid (Neu5Ac) residues, controlled by β-galactoside-α2,6-sialyltransferase I (ST6Gal-I). Expression of this enzyme is modified in the action of β-galactoside-α2,6-sialyltransferase I (ST6Gal-I). Expression of this enzyme various malignancies, including prostate, breast and ovarian cancer [60–62]. The presence is modified in various malignancies, including prostate, breast and ovarian cancer [60– of α2,6-linked sialic acids on tumor cells is crucial because α2,6-sialylation can execute 62]. The presence of α2,6-linked sialic acids on tumor cells is crucial because α2,6- alternative biological outcomes compared to α2,3-sialylation. One accurate example is sialylation can execute alternative biological outcomes compared to α2,3-sialylation. One the impact of α2,6-sialylation on galectin-dependent cell behaviours. Numerous studies accurate example is the impact of α2,6-sialylation on galectin-dependent cell behaviours. indicate that α2,6-sialylation of galactose acts as a general inhibitor of galectin binding, in Numerous studies indicate that α2,6-sialylation of galactose acts as a general inhibitor of contrast to α2,3-sialylation with various binding effects to some particular galectins. There- galectin binding, in contrast to α2,3-sialylation with various binding effects to some fore, glycans capped with α2,6-sialic acid globally act as a significant negative regulator of particularmany key galectins. galectin functions. Therefore, One glycans of the important capped with activities α2,6-sialic of cell acid surface globallyα2,6-sialyation act as a significantis to suppress negative the binding regulator of pro-apoptotic of many key galectins, galectin at functions. the same time One inducing of the important cancer cell activitiessurvival [ of63 ,64 cell]. surface α2,6-sialyation is to suppress the binding of pro-apoptotic galectins,Additionally, at the same elevated time inducing sialylation cancer in cancer cell survival can be related [63,64] to. the formation of polysialic acid,Additionally, which is associated elevated with sialylation several types in cancer of cancers can and be relatedis often presentto the formationin high-grade of polysialictumors [58 acid,,65]. which Other is main associated sialylated with antigens several associatedtypes of cancers with cancer and is are often sLe presenta and sLe inx . a highThese-grade have tumors been shown [58,65] to. Other be remarkably main sialylated expressed antigens in many associated malignant with tumors, cancer including are sLe andhepatic, sLex. These renal andhave breast been shown cancers to [ 66be]. remarkably Additionally, expressed sLex antigen in many expression malignant leveltumors, has includingbeen associated hepatic, with renal a diminished and breast cancers chance of[66] survival. Additionally, for prostate sLex cancerantigen patients expression [67]. levelSLex hasand been itsisomer associated sLe awithare cruciala diminished recognition chance determinants of survival for for prostate selectins, cancer vascular patients cell

Cancers 2021, 13, 3726 6 of 22

adhesion molecules belonging to a large family of C-type lectins. In the process of inflammation, selectins mediate the first stage of leucocytes’ adhesion to the endothelium during leucocyte extravasation [68]. In cancer cells, sLex interactions with selectins regulate the metastatic process by tethering platelet-tumor cell emboli and promoting their arrest on the endothelium, therefore contributing to malignant behaviour and the progression of metastasis [69]. Over the years, PSA glycosylation changes have been analysed by mass spectrometry, and the level of α2,3-linked sialic acid was reported to be remarkably different in prostate cancer patients compared to a control group, stressing the importance of PSA sialylation in distinguishing cancer patients from healthy men (Figure1B) [ 14,16,70]. In patients with prostate cancer, serum PSA contains increased levels of α2,3-linked sialic acid connected to the terminal galactose residue compared to healthy individuals [14,71]. Additionally, Ohyama et al. detected that binding of prostate cancer related PSA to Maackia amurensis lectin specific for α2,3-linked sialic acid was more intense than binding of PSA from healthy individuals [72]. Serum glycoproteins of prostate cancer patients showed increased levels of α2,3-linked sialic acid in relation to serum of patients with benign prostatic hyperplasia. The authors suggested that this feature can be used to predict the Gleason score with greater sensitivity and specificity than PSA concentration, used to date [73]. In metastatic prostate cancer two main forms of PSA were detected in the serum: a free form and alpha-1-antichymotrypsin (ACT)-complexed PSA. Both were characterized by mostly sialylated biantennary glycan structures, but the presence of several multi-antennary complex structures was also observed [14]. Yoneyama et al. developed a more sensitive diagnostic PSA test, in which they used a magnetic microbead-based immunoassay, that directly measured the amount of α2,3-linked sialic acid on the free serum PSA. The new assay showed a sensitivity of 95% and a specificity of 72% in a measurement carried out on a cohort of over 300 serum samples, from patients who underwent biopsy, including 138 PCa and 178 non-PCa patients with a PSA level less than 10.0 ng/mL [16]. This solution was more sensitive and accurate than the conventional PSA level and the free PSA-percent tests in the diagnosis of prostate cancer. Another study conducted in 13 BPH and 34 prostate cancer patients’ sera (including 17 Gleason grade 5 and 17 Gleason grade 7 samples), which considered N-glycans from the whole serum glycoproteins, indicated that tetra-antennary tetra-sialylated N-linked oligosaccharide levels were higher in the serum samples from patients with Gleason score 7 compared to Gleason score 5. Conversely, levels of tetra-antennary tetra-sialylated glycans with terminal fucose and tri-antennary tri-galactosylated glycans were significantly lower in serum from patients with a Gleason score of 7 compared to a Gleason score of 5 [73]. Moreover, the detection of α2,3-linked sialic acid PSA glycoforms combined with the PHI in a cohort of 79 patients showed 100% sensitivity and 94.7% specificity. The proposed analysis proved superior to PSA to distinguish aggressive prostate cancer from low-risk and benign disease (Table1)[74]. Cancers 2021, 13, x 7 of 20

Cancers 2021, 13, x 7 of 20

Cancers 2021, 13, 3726 Table 1. Summary of sialylated N-glycan alterations in prostate cancer. 7 of 22 Table 1. Summary of sialylated N-glycan alterations in prostate cancer. Type of Author Year Structure Sample Groups Main Results GTypelycans of AuthorReferences Year Structure TableSample 1. Summary Groups of sialylated N-glycanMain alterations Results in prostate cancer. Glycans Serum of PCa patients Serum α2,3-linked sialic acid of PSA in PCa References Yoneyama 2014 Serumvs. serum of PCa of non patients-PCa Serumgroup wasα2,3 significantly-linked sialic higheracid of than PSA inin nonPCa- Author Year Type of Glycans Structure Sample Groups Main ResultsYoneyama[16] 2014 vs. serumpatients of non -PCa group was significantlyPCa group higher than in non- References [16] patients Level of α2,3-linkedPCa sialicgroup acid of PSA from PCaSerum serum of PCa vs. patientscontrol vs. serum of Serum α2,3-linked sialic acid of PSAPihikova in PCa 2016 group was Yoneyama 2014 non-PCa patientsLevelprostate of α2,3 cancer-linked serum sialicsignificantly wasacid increasedof PSA higher from than in non-PCa group [16] PCaserum serum (healthy vs. control men) Pihikova[70] 2016 prostatecompared cancer to healthyserum was individuals increased serum (healthy men) Level of α2,3-linked sialic acid of PSA[70] from prostate PCa serum vs. controlSignificant serumcompared increase to healthy in α2,3 -individualslinked sialic acid Pihikova 2016 BPH serum vs. PCa cancer serum was increased compared to (healthy men)fromSignificant total serum increase glycoproteins in α2,3-linked in PCa sialic group acid Saldova 2011 [73] [70] BPH serumserum vs. PCa healthy individuals fromcomparing total serum with glycoproteins BPH group was in PCa detected group Saldova 2011 [73] serum Significant increase in α2,3-linked sialic acid from total BPH serum, low-risk comparing with BPH group was detected BPH serum vs. PCa serumThere was a significantserum glycoproteinsincrease of α2,3 in- PCa group comparing with Saldova 2011 [73] BPH PCaserum, serum, low -risk sialylatedThere was PSA a significantin the group increase of highBPH of-risk groupα2,3 PCa- was detected α2,3-sialylated intermediatePCa serum,-risk PCa Llop 2016 [75] sialylatedpatients comparedPSA inThere the with group was the aof significantintermediate high-risk PCa increase- of α2,3-sialylated PSA α2,3-sialylated intermediateserum,BPH serum, and high-risk low-risk- riskPCa PCa serum, Llop 2016 [75] α2,3-sialylated patientsrisk PCa, compared low-riskin the PCa,with group andthe intermediate ofBPH high-risk groups PCa- patients compared with serum,PCaintermediate-risk serum and highsamples-risk PCa serum, and Llop 2016 [75] risk PCa, low-risk PCa,the intermediate-riskand BPH groups PCa, low-risk PCa, and PCahigh-risk serum samples PCa serum samplesSerum % α2,3-sialic acid of PSA was BPH serum vs. PCa BPH groups significantlySerum % α2,3 higher-sialic in acidpatients of PSA with was PCa Ishikawa 2017 [76] BPH serumserum vs. PCa Serum % α2,3-sialic acid of PSA was significantly BPH serum vs. PCasignificantly serum compared higher to inBPH patients patients with PCa Ishikawa 2017 [76] Ishikawa 2017 [76] serum higher in patients with PCa compared to BPH patients The combinationcompared of to% BPHα2,3 -patientsSA PSA and PHI BPH serum vs. PCa The combination of % α2,3-SAFerrer- PSABatalle and 2017 PHI Thedifferentiates combination high of -%risk α2,3 PCa-SA patients PSA and from PHI Ferrer-Batalle 2017 BPHBPH serumserum serum vs. PCa vs. PCa serum differentiates high-risk PCa patientsFerrer-Batalle from[74] the 2017 low and thedifferentiates low and intermediate high-risk PCa-risk patients PCa patients from [74] serum intermediate-risk PCa patients[74] PCa urine samples the low and intermediate-risk PCa patients Highly sialylated urinary N-glycans were Yang 2017 withPCaPCa variedurine urine samples Gleason samples with varied Highly sialylated urinary N-glycans were upregulated Yang 2017 Gleason scoresHighlyupregulated sialylated in metastatic urinary Ncancer-inglycans metastatic patients were cancer patientsYang[77] 2017 [77] with variedscores Gleason upregulated in metastatic cancer patients [77] scores BPH serum vs. PCa Tetra-antennary tetra-sialylated glycans BPHserumBPH serum serum(17 Gleason vs. vs. PCa PCa serum (17 Tetra-antennary tetra-sialylated glycans were Tetra-antennary wereTetra increased-antennary in tetrathe serum-sialylated samples glycans from Saldova 2011 Saldova 2011 Tetra-antennary tetrasialylated Gleasonserumgradegrade (17 5 andGleason 5 and17 17 Gleason grade increased in the serum samples from patients with Tetratetrasialylated-antennary patientswere increased with Gleason in the serumscore 7 samples compared from to Saldova[73] 2011 [73] Gleasongrade 5 gradeand7 samples) 17 7 Gleason score 7 compared to Gleason score 5 tetrasialylated patients with Gleason score 57 compared to [73] Gleasonsamples) grade 7 Gleason score 5 samples) A decreased amount of tri-antennary, tri- Tri-antennary, Indolent, significant, Agalactosylated decreased amount tri-sialylated of tri-antennary, glycans with tri- triTri-galactosylated-antennary, Indolent,aggressive significant, PCa galactosylatedand without tri -corsialylatede fucose glycans residue with Gilgunn 2020 tritri--sialylatedgalactosylated with accordingaggressive Epstein’s PCa correspondingand without to the cor transitione fucose residue of PCa from Gilgunn[78] 2020 triand-sialylated without withcore accordingcriteria Epstein’s correspondingindolent state to throughthe transition significant of PCa and from [78] andfucose without residue core criteria indolent stateaggressive through disease significant and fucose residue aggressive disease

Sialylated Serum PCa vs. Serum PSA sialylated glycoforms bearing Gratacós-Mulleras glycoformsSialylated standardSerum PCaSP PSA vs. of SerumGalNAc PSA moieties sialylated were glycoforms heightened bearing in Gratacós2020-Mulleras bearingglycoforms GalNAc standardhealthy SP men PSA of GalNAcaggressive moieties werePCa patients heightened in 2020[79] bearingmoieties GalNAc healthy men aggressive PCa patients [79] moieties

The most recent study considering whole serum N-linked glycans in various prostate cancerThe stages most recent aimed study to investigate considering whether whole serum an altered N-linked glycosylation glycans in various pattern prostate could cancerdifferentiate stages distinct aimed forms to investigate of prostate whether cancer, an including altered glycosylation indolent, significant, pattern could and differentiateaggressive PCa. distinct N-glycan forms profiling of prostate was performed cancer, includingon 117 prostate indolent, cancer significant, serum samples and aggreusing ssive an PCa. automated, N-glycan high profiling-throughput was performed analytical on 117 platform, prostate which cancer exploitsserum samples ultra- usingperformance an automated, liquid chromatography high-throughput for high analytical resolution platform, separation which of N exploits-glycans. ultra The- performanceresults reveal liquided a decreased chromatography amount for of high tri-antennary, resolution tri separation-galactosylated of N -glycans. tri-sialylated The resultsglycans revealwith anded awithout decreased core amount fucose residues, of tri-antennary, corresponding tri-galactosylated to the transition tri-sialylated of PCa glycansfrom the with indolent and without stage through core fucose significant residues, and corresponding aggressive disease. to the transition Additionally, of PCa an from the indolent stage through significant and aggressive disease. Additionally, an

CancersCancers 2021 2021, ,13 13, ,x x 77 ofof 2020

TableTable 1. 1. Summary Summary of of sialylated sialylated N N--glycanglycan alterations alterations in in prostate prostate cancer. cancer.

TypeType of of AuthorAuthor Year Year StructureStructure SampleSample G Groupsroups MainMain Results Results GGlycanslycans ReferencesReferences SerumSerum of of PCa PCa patients patients SerumSerum α2,3 α2,3--linkedlinked sialic sialic acid acid of of PSA PSA in in PCa PCa YoneyamaYoneyama 2014 2014 vs.vs. serum serum of of non non--PCaPCa groupgroup was was significantly significantly higher higher than than in in non non-- [16][16] patientspatients PCaPCa group group LevelLevel of of α2,3 α2,3--linkedlinked sialic sialic acid acid of of PSA PSA from from PCaPCa serum serum vs. vs. control control PihikovaPihikova 2016 2016 prostateprostate cancer cancer serum serum was was increased increased serumserum (healthy (healthy men) men) [70][70] comparedcompared to to healthy healthy individuals individuals SignificantSignificant increase increase in in α2,3 α2,3--linkedlinked sialic sialic acid acid BPHBPH serum serum vs. vs. PCa PCa fromfrom total total serum serum glycoproteins glycoproteins in in PCa PCa group group SaldovaSaldova 2011 2011 [73] [73] serumserum comparingcomparing with with BPH BPH group group was was detected detected BPHBPH serum, serum, low low--riskrisk ThereThere was was a a significant significant increase increase of of α2,3 α2,3-- PCaPCa serum, serum, sialylatedsialylated PSA PSA in in the the group group of of high high--riskrisk PCa PCa α2,3α2,3--sialylatedsialylated intermediateintermediate--riskrisk PCa PCa LlopLlop 2016 2016 [75] [75] patientspatients compared compared with with the the intermediate intermediate-- serum,serum, and and high high--riskrisk riskrisk PCa, PCa, low low--riskrisk PCa, PCa, and and BPH BPH groups groups PCaPCa serum serum samples samples SerumSerum % % α2,3 α2,3--sialicsialic acid acid of of PSA PSA was was BPHBPH serum serum vs. vs. PCa PCa significantlysignificantly higher higher in in patients patients with with PCa PCa IshikawaIshikawa 2017 2017 [76] [76] serumserum comparedcompared to to BPH BPH patients patients TheThe combination combination of of % % α2,3 α2,3--SASA PSA PSA and and PHI PHI BPHBPH serum serum vs. vs. PCa PCa FerrerFerrer--BatalleBatalle 2017 2017 differentiatesdifferentiates high high--riskrisk PCa PCa patients patients from from serumserum [74][74] thethe low low and and intermediate intermediate--riskrisk PCa PCa patients patients PCaPCa urine urine samples samples HighlyHighly sialylated sialylated urinary urinary N N--glycansglycans were were YangYang 2017 2017 withwith varied varied Gleason Gleason upregulatedupregulated in in metastatic metastatic cancer cancer patients patients [77][77] Cancers 2021, 13, 3726 scoresscores 8 of 22

BPHBPH serum serum vs. vs. PCa PCa TetraTetra--antennaryantennary tetra tetra--sialylatedsialylated glycans glycans serumserum (17 (17 Gleason Gleason TetraTetra--antennaryantennary werewere increased increased in in the the serum serum samples samples from from SaldovaSaldova 2011 2011 gradegrade 5 5 and and 17 17 Table 1. Cont. tetrasialylatedtetrasialylated patientspatients with with Gleason Gleason score score 7 7 compared compared to to [73][73] GleasonGleason grade grade 7 7 GleasonGleason score score 5 5 Author Year Type of Glycans Structuresamples)samples) Sample Groups Main Results References

AA decreased decreased amount amount of of tri tri--antennary,antennary, tri tri-- TriTri--antennary,antennary, A decreased amount of tri-antennary, Indolent,Indolent, significant, significant, galactosylatedgalactosylated tri tri--sialylatedsialylated glycans glycans with with Tri-antennary,tri tri-galactosylatedtri--galactosylatedgalactosylated tri-galactosylated tri-sialylated glycans with and Indolent,aggressiveaggressive signiﬁcant, PCa PCa aggressiveandand PCawithout without cor coree fucose fucose residue residue GilgunnGilgunn 2020 2020 Gilgunn 2020 tri-sialylated withtritri- and-sialylatedsialylated without with with core without core fucose residue corresponding to the accordingaccordingaccording Epstein’s Epstein’s Epstein’s correspondingcorresponding criteria to to the the transition transition of of PCa PCa from from [78][78] [78] fucoseandand residue without without core core transition of PCa from indolent state through criteriacriteria indolentindolent state state through through significant significantsigniﬁcant and and and aggressive disease fucosefucose residue residue aggressiveaggressive disease disease

SialylatedSialylated SerumSerum PCa PCa vs. vs. SerumSerum PSA PSA sialylated sialylated glycoforms glycoforms bearing bearing GratacósGratacós--MullerasMulleras Sialylated glycoformsglycoformsglycoforms bearing Serum PCa vs. standard SP PSA of Serum PSA sialylated glycoforms bearing GalNAc Gratacós-Mulleras 2020 standardstandard SP SP PSA PSA of of GalNAcGalNAc moieties moieties were were heightened heightened in in 20202020 GalNAcbearingbearing moieties GalNAc GalNAc healthy men moieties were heightened in aggressive PCa patients [79] healthyhealthy men men aggressiveaggressive PCa PCa patients patients [79][79] moietiesmoieties

TheThe most most recent recent study study considering considering whole whole serum serum N N--linkedlinked glycans glycans in in various various prostate prostate cancercancer stages stages aimed aimed to to investigate investigate whether whether an an altered altered glycosylation glycosylation pattern pattern could could differentiatedifferentiate distinct distinct forms forms of of prostate prostate cancer, cancer, including including indolent, indolent, significant, significant, and and aggreaggressivessive PCa. PCa. N N--glycanglycan profiling profiling was was performed performed on on 117 117 prostate prostate cancer cancer serum serum samples samples usingusing an an automated, automated, high high--throughputthroughput analytical analytical platform, platform, which which exploits exploits ultra ultra-- performanceperformance liquid liquid chromatography chromatography for for high high resolution resolution separation separation of of N N--glycans.glycans. The The resultsresults reveal revealeded a a decreased decreased amount amount of of tri tri--antennary,antennary, tri tri--galactosylatedgalactosylated tri tri--sialylatedsialylated glycansglycans withwith andand withoutwithout corecore fucosefucose residues,residues, correspondingcorresponding toto thethe transitiontransition of of PCaPCa fromfrom the the indolent indolent stage stage through through significant significant and and aggressive aggressive disease. disease. Additionally, Additionally, an an

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The most recent study considering whole serum N-linked glycans in various prostate cancer stages aimed to investigate whether an altered glycosylation pattern could differentiate distinct forms of prostate cancer, including indolent, significant, and aggressive PCa. N-glycan profiling was performed on 117 prostate cancer serum samples using an automated, high-throughput analytical platform, which exploits ultra-performance liquid chromatography for high resolution separation of N-glycans. The results revealed a decreased amount of tri-antennary, tri-galactosylated tri-sialylated glycans with and without core fucose residues, corresponding to the transition of PCa from the indolent stage through significant and aggressive disease. Additionally, an increase in hybrid, oligomannose, bisecting GlcNAc and monoantennary glycans was observed (Table1)[ 78]. Another interesting investigation regarded the main sialylated PSA glycoforms from the serum of aggressive PCa patients in relation to standard PSA from seminal plasma of healthy men. Exploiting Sambucus nigra affinity chromatography, the α2,6-linked sialic acid glycoforms were separated from α2,3-linked glycoforms, then PSA N-glycans were analysed by hydrophilic interaction liquid chromatography. The results indicated that levels of serum PSA sialylated glycoforms bearing GalNAc moieties (LacdiNAc) were raised in aggressive PCa patients. Concomitantly, levels of disialylated core fucosylated biantennary structures with α2,6-linked sialic acid, which were previously indicated as major PSA glycoforms characteristic for standard PSA from healthy men, were significantly lowered in aggressive PCa (Table1)[79].

7. Fucosylated N-Glycans Fucosylated glycans are synthesized by a series of fucosyltransferases. The modification generally occurs as core fucosylation and terminal fucosylation, the latter including specific Lewis blood group antigens such as Lex, Ley, Lea, and Leb. The core fucosylation of protein relies on addition of a fucose residue to the innermost N-acetylglucosamine (GlcNAc) residue of N-glycans via an α1,6-linkage, and is catalysed by fucosyltransferase Fuc-TVIII (Figure1C), encoded by the FUT8 gene [80]. Altered expression of FUT8 and FUT6 is an important feature in several cancers such as high-grade prostate cancer and breast cancer [80,81]. The FUT6 gene encodes α1,3-fucosyltransferase and is upregulated in distant metastases. It was also reported that the product of this gene can participate in metastasis to bones [82]. Furthermore, expression of FUT6 might trigger prostate cancer cell trafficking through an E-selectin-dependent mechanism [83,84]. Overexpression of FUT8 has been recently linked with aggressive and castrate-resistant prostate cancer, as well as being associated with a poor prognosis for patients [85,86]. More recent data indicate that FUT8 is involved in controlling the function of cancer cell membrane receptors [87]. Core fucosylation transforms cell surface molecules as well as the tumor microenvironment, and thus the extracellular matrix and growth factors, supporting cancer progression. FUT8 promotes cancer cell invasiveness by remodelling the core fucosylation of the TGF-β receptor [88,89], as the presence of core fucose strongly affects the binding affinity of the TGF-β receptor and thus TGF-β induced epithelial-mesenchymal transformation (EMT) [90,91]. In the serum of prostate cancer patients increased core fucosylation of glycans has been found, compared to patients with BPH as well as men without known prostatic disease. These findings suggest that core fucosylation is associated with disease progression [73,92,93]. Saldova et al. established a high-throughput HPLC assay and used it for quantitative analysis of N-glycans from the whole serum glycoproteins of BPH and prostate cancer samples. Additionally, the samples were split into low and higher grade Gleason groups. A significant increase in core-fucosylated biantennary glycans in prostate cancer related to BPH was observed, but no changes in these glycans were associated with Gleason scores (Table2)[73]. Cancers 2021, 13, x 9 of 20 CancersCancers 20212021,, 1313,, xx 99 ofof 2020

Cancers 2021, 13, 3726 10 of 22 Table 2. Summary of fucosylated, bisected, tri- and tetra-antennary N-glycan alternations in prostate cancer. TableTable 2. 2. SummarySummary of of fucosylated, fucosylated, bisected, bisected, t triri--- and and tetra tetra---antennaryantennary N N---glycanglycan alternations alternations in in prostate prostate cancer. cancer. Author, Year Type of Glycans TableStructure 2. Summary of fucosylated,Sample Groups bisected, tri- and tetra-antennaryMain N-glycanResults alternations in prostateAuthor,Author, cancer. Year Year TypeType of of G Glycanslycanslycans StructureStructure SampleSample G Groupsroupsroups MainMain Results Results References ReferencesReferences In the serum of PCa patients increased Type of Glycans StructureHealthy individuals Sample Groupsserum vs. InInIn the thethe serum serumserum of ofof PCa PCaPCa patients patientspatients increased increasedincreased Main Results Kyselova 2007 Author, Year References HealthyHealthy individuals individuals serum serum vs. vs. fucosylation of glycans compared to healthy KyselovaKyselova 2007 2007 PCa patients serum fucosylationfucosylationfucosylation of ofof glycans glycansglycans compared comparedcompared to toto healthy healthyhealthy [91] HealthyPCaPCa individualspatients patients serum serum serum vs. PCa individualsIn the serum was offound PCa patients increased fucosylation[91][91][91] of Kyselova 2007 individualsindividualsindividuals was waswas found foundfound patients serum A significantglycans increase compared in core-fucosylation to healthy individuals was found [91] BPH serum vs. PCa serum (17 AA significant significant increase increase in in core core---fucosylationfucosylationfucosylation BPHBPH serum serum vs. vs. PCa PCa serum serum (17 (17 biantennary glycans in PCa serum relative to Saldova 2011 GleasonBPH grade serum 5 and vs. PCa17 Gleason serum (17biantennarybiantennaryA glycans glycans signiﬁcant in in PCa PCa increase serum serum relative relative in core-fucosylation to to SaldovaSaldova biantennary 2011 2011 GleasonGleason grade grade 5 5 and and 17 17 Gleason Gleason BPH was observed, no changes were associated [73] Saldova 2011 Gleasongrade grade 7 samples) 5 and 17 GleasonBPHBPH was was observed, observed,glycans no no in changes changes PCa serum were were associated relativeassociated to BPH was[73][73][73] observed, no gradegrade 7 7 samples) samples) with Gleason scores [73] grade 7 samples) withwith changesGleason Gleason scores scores were associated with Gleason scores Fucosylation of glycans was increased in the LNCaP-an androgen dependent FucosylationFucosylation of of glycans glycans was was increased increased in in the the LNCaPLNCaP-anLNCaP---anan androgen androgen androgen dependent dependent dependent rapidly cell proliferatingFucosylation and more of invasive glycans PC3 was cell increased Shah in 2015 the rapidly cell line and PC3- an androden rapidlyrapidly proliferating proliferating and and more more invasive invasive PC3 PC3 cell cell ShahShah 2015 2015 Shah 2015 cellcell line line and and and PC3 PC3 PC3---- an an anandroden androden androden line relativeproliferating to the slow and growing, more and invasive less PC3 cell line[85]relative to the Fucosylated or/and independent cell line linelineline relative relativerelative to toto the thethe slow slowslow growing, growing,growing, and andand less lessless [85][85][85] [85] FucosylatedFucosylated or/andor/andor/and independentindependentindependentindependent cell cellcell line lineline cell line invasiveslow LNCaP growing, cell line and less invasive LNCaP cell line Fucosylated invasiveinvasiveinvasive LNCaP LNCaPLNCaP cell cellcell line lineline FUT8 expression was elevated in metastatic PCa FUT8FUT8 expression expressionFUT8 was wasexpression elevated elevated in in was metastatic metastatic elevated PCa PCa in metastatic PCa tissues PC3PC3 vs. LNCaP vs. LNCaP tissues compared to normal prostate tissues PC3PC3 vs. vs. LNCaP LNCaP tissuestissuestissues compared comparedcompared to toto normal normalnormalcompared prostate prostateprostate tonormal tissues tissuestissues prostate tissues and and FUT8 overexpression in LNCaP Wang 2014 Wang 2014 andand FUT8FUT8 overexpression overexpressionFUT8 in in LNCaP overexpressionLNCaP in LNCaPWangWang 2014 2014 metastaticmetastatic PCa tissues tissues vs. vs. normalcells increased PCa cell migration, while loss of [80] [80] metastaticmetastatic PCa PCa tissues tissues vs. vs. cellscells increased increasedcells PCa PCa increased cell cell migration, migration, PCa cell while while migration, loss loss of of while loss[80][80][80] of FUT8 in PC3 normal prostateprostate tissues tissues FUT8 in PC3 normalnormal prostate prostate tissues tissues FUT8FUT8 in inin PC3 PC3PC3cells decreased cell motility cells decreased cell motility cellscells decreaseddecreased cellcell motilitymotility LCA- and AALLCA--immunoassays and AAL-immunoassays detected detected increased PCaPCa serum serum samplessamples with differentLCALCA--- and and AAL AAL---immunoassaysimmunoassaysimmunoassays detected detecteddetected Wang 2019 Wang 2019 PCaPCa serum serum samples samples with with increased fucosylatedfucosylated PSA, PSA, results results were were correlatedWangWang with2019 2019 higher differentGleason Gleason scores increasedincreasedincreased fucosylated fucosylatedfucosylated PSA, PSA,PSA, results resultsresults were werewere [94] [94] differentdifferent Gleason Gleason scores scores correlated with higher GleasonGleason scores scores [94][94][94] correlatedcorrelated with with higher higher Gleason Gleason scores scores

EPS-urine pools from aggressive The presence of bi-antennary structures with EPSEPSEPS-urine---urineurine pools pools pools from from from aggressive aggressive aggressive TheThe presence presence of of bi biThe---antennaryantennary presence structures structures of bi-antennary with with Nyalwidhe structures 2013 with Bisected PCa, indolent PCa and bisecting-GlcNAc residue was increased with NyalwidheNyalwidhe 2013 2013 Nyalwidhe 2013 Bisected BisectedBisected PCa,PCa,PCa, indolent indolent indolent PCa PCa and PCaand and bisectingbisecting---GlcNAcGlcNAcbisecting-GlcNAc residue residue was was increased increased residue with with was increased[95] with non-cancer urine samples disease severity [95][95][95] [95] nonnonnon-cancer---cancercancer urine urine urine samples samples samples diseasedisease severity severity disease severity

Serum samples from healthy Tri- and tetra-antennary glycans were SerumSerumSerum samples samples from from from healthy healthy healthy men,TriTri--- and and tetra tetratetra---antennaryantennary glycans glycans were were Ishibashi 2014 men, BPH, early stage-PCa, PCa significantlyTri- higher and in tetra-antennary CRPC patients compared glycans were Ishibashi signiﬁcantlyIshibashiIshibashi 2014 20142014 higher in Ishibashi 2014 men,men,BPH, BPH, BPH, early early early stage-PCa, stage stage---PCa,PCa, PCaPCaPCa withsignificantlysignificantly higher higher in in CRPC CRPC patients patients compared compared [96] with ADT and CRPC patients to theCRPC other patientsgroups compared to the other[96][96][96] groups [96] withwith ADT ADT and and CRPC CRPC CRPC patients patients patients tototo the thethe other otherother groups groupsgroups MGAT5B gene overexpression was observed Tri- and Tri- and MGAT5BMGAT5B gene gene overexpression overexpression was was observed observed TriTri--- and and in all prostate cancerMGAT5B cell linesgene compared overexpression to was observed tetra-antennarytetra-antennary ininin all allall prostate prostateprostate cancer cancercancer cell cellcell lines lineslines compared compared to to tetratetratetra---antennaryantennary ProstateProstate cancer cancer cells cells LNCaP, LNCaP, PC- PC-3,normal prostatein all prostate epithelium. cancer β1,6 cell-branched lines compared Lange to normal 2012 prostate Lange 2012 ProstateProstate cancer cancer cells cells LNCaP, LNCaP, PC PC--- normalnormal prostate prostate epithelium. epithelium. β1,6 β1,6---branchedbranched LangeLange 2012 2012 3, LuCaPLuCaP 23.1, 23.1, and and DU- DU-145145 oligosaccharidesepithelium. wereβ1,6-branched oligosaccharides[97] were [97] 3,3, LuCaP LuCaP 23.1, 23.1, and and DU DU---145145 oligosaccharidesoligosaccharides were were [97][97][97] restricted torestricted metastatic toprostate metastatic cancer prostate cancer xenografts restrictedrestricted to to metastatic metastatic prostate prostate cancer cancer xenografts xenograftsxenografts The amount of the LacdiNAc moieties was Normal seminal fluid and TheThe amount amount of of the the LacdiNAc LacdiNAc moieties moieties was was Peracaula 2003 NormalNormal seminal seminal fluid fluid and and increased in LNCaP glycans in relation to PeracaulaPeracaula 2003 2003 prostate cancer cells LNCaP increasedincreasedincreased in inin LNCaP LNCaPLNCaP glycans glycansglycans in inin relation relationrelation to toto [71] prostateprostate cancer cancer cells cells LNCaP LNCaP structures obtained from normal SP PSA [71][71][71] structuresstructures obtained obtained from from normal normal SP SP PSA PSA Abundance of multisialylated LacdiNAc Haga 2019 AbundanceAbundance of of multisialylated multisialylated LacdiNAc LacdiNAc HagaHaga 2019 2019 Sera from PCa vs. BPH sera structures was significantly upregulated in the [98] SeraSera from from PCa PCa vs. vs. BPH BPH sera sera structuresstructures was was significantly significantly upregulated upregulated in in the the [98][98][98] PCa patients compared to the BPH group PCaPCa patients patients compared compared to to the the BPH BPH group group In prostate cancer the upregulation of the Seminal fluid, serum of BPH InInIn prostate prostateprostate cancer cancercancer the thethe upregulation upregulationupregulation of ofof the thethe SeminalSeminal fluid, fluid, serum serum of of BPH BPH β4GalNAcT4 was observed, and were correlated Fukushima 2010 LacdiNAc motif and PCa patients, and LNCaP β4GalNAcT4β4GalNAcT4 was was observed, observed, and and were were correlated correlated FukushimaFukushima 2010 2010 LacdiNAcLacdiNAc motif motif andand PCa PCa patients, patients, and and LNCaP LNCaP with the overexpression of the LacdiNAc groups [99] cell line withwith the the overexpression overexpression of of the the LacdiNAc LacdiNAc groups groups [99][99][99] cellcell line line for PCa-derived PSA forforfor PCa PCa---derivedderived PSA PSA The LacdiNAc-PSA immunoassay allowed for TheThe LacdiNAc LacdiNAc---PSAPSA immunoassay immunoassay allowed allowed for for distinction between PCa and BPH within the distinctiondistinction between between PCa PCa and and BPH BPH within within the the PSA gray zone Kaya 2015 BPH serum vs. PCa serum PSAPSA gray gray zone zone KayaKaya 2015 2015 BPHBPH serum serum vs. vs. PCa PCa serum serum The LacdiNAc-PSA determination revealed that [100] TheThe LacdiNAc LacdiNAc---PSAPSA determination determination revealed revealed that that [100][100][100] levels of LacdiNAc-PSA were significantly levelslevelslevels of ofof LacdiNAc LacdiNAcLacdiNAc---PSAPSA were were significantly significantly higher in PCa sera than in BPH sera higherhigher in in PCa PCa sera sera than than in in BPH BPH sera sera Studies utilizing the two prostate cancer cell lines LNCaP and PC3 have shown that StudiesStudies utilizingutilizing thethe twotwo prostateprostate cancercancer cellcell lineslines LNCaPLNCaP andand PC3PC3 havehave shownshown thatthat fucosylation of glycans is increased in the rapidly proliferating and more invasive PC3 fucosylationfucosylation ofof glycansglycans isis increasedincreased inin thethe rapidlyrapidly proliferatingproliferating andand moremore invasiveinvasive PC3PC3 cell line compared to the slow growing, less invasive LNCaP cell line [85]. Wang et al. cellcell lineline comparedcompared toto thethe slowslow growing,growing, lessless invasiveinvasive LNCaPLNCaP cellcell lineline [85][85]... WangWangWang etetet aaal.l.l. detected expression of α1,6-fucosyltransferase solely in PC3, and not in LNCaP cells. detecteddetected expression expression of of α1,6 α1,6--fucosyltransferasefucosyltransferase solely solely in in PC3, PC3, and and not not in in LNCaP LNCaP cells. cells. Afterwards the authors found that FUT8 expression was elevated in metastatic PCa AfterwardsAfterwards the the authors authors found found that that FUT8FUT8 expressionexpression was was elevated elevated in in metastatic metastatic PCa PCa tissues compared to normal prostate tissues. Using PC3 and LNCaP cells as models, they tissuestissues comparedcompared toto normalnormal prostateprostate tissues.tissues. UsingUsing PC3PC3 andand LNCaPLNCaP cellscells asas models,models, theythey

Cancers 2021, 13, x 9 of 20

Table 2. Summary of fucosylated, bisected, tri- and tetra-antennary N-glycan alternations in prostate cancer.

Author, Year Type of Glycans Structure Sample Groups Main Results References In the serum of PCa patients increased Healthy individuals serum vs. Kyselova 2007 fucosylation of glycans compared to healthy PCa patients serum [91] individuals was found A significant increase in core-fucosylation BPH serum vs. PCa serum (17 biantennary glycans in PCa serum relative to Saldova 2011 Gleason grade 5 and 17 Gleason BPH was observed, no changes were associated [73] grade 7 samples) with Gleason scores Fucosylation of glycans was increased in the LNCaP-an androgen dependent rapidly proliferating and more invasive PC3 cell Shah 2015 cell line and PC3- an androden line relative to the slow growing, and less [85] Fucosylated or/and independent cell line invasive LNCaP cell line FUT8 expression was elevated in metastatic PCa PC3 vs. LNCaP tissues compared to normal prostate tissues and FUT8 overexpression in LNCaP Wang 2014 metastatic PCa tissues vs. cells increased PCa cell migration, while loss of [80] normal prostate tissues FUT8 in PC3 cells decreased cell motility LCA- and AAL-immunoassays detected PCa serum samples with Wang 2019 increased fucosylated PSA, results were different Gleason scores [94] correlated with higher Gleason scores

EPS-urine pools from aggressive The presence of bi-antennary structures with Nyalwidhe 2013 Bisected PCa, indolent PCa and bisecting-GlcNAc residue was increased with [95] non-cancer urine samples disease severity

Serum samples from healthy Tri- and tetra-antennary glycans were Ishibashi 2014 men, BPH, early stage-PCa, PCa significantly higher in CRPC patients compared [96] Cancers 2021, 13, 3726 with ADT and CRPC patients to the other groups 11 of 22 MGAT5B gene overexpression was observed Tri- and in all prostate cancer cell lines compared to tetra-antennary Prostate cancer cells LNCaP, PC- normal prostate epithelium. β1,6-branched Lange 2012 3, LuCaP 23.1, and DU-145 Table 2. Cont.oligosaccharides were [97] restricted to metastatic prostate cancer Type of Glycans Structure Sample Groupsxenografts Main Results Author, Year References The amount of the LacdiNAc moieties was Normal seminal fluid and The amount of the LacdiNAc moietiesPeracaula was increased 2003 in Normal seminal fluid and prostateincreased in LNCaP glycans in relation to Peracaula 2003 prostate cancer cells LNCaP LNCaP glycans in relation to structures[71] obtained from cancer cells LNCaP structures obtained from normal SP PSA [71] normal SP PSA Abundance of multisialylated LacdiNAc Haga 2019 Abundance of multisialylated LacdiNAc structures was Sera from PCa vs. BPH sera structures was significantly upregulated in the [98] Haga 2019 Sera from PCa vs. BPH sera significantly upregulated in the PCa patients compared to PCa patients compared to the BPH group [98] In prostate cancer the upregulationthe of BPH the group LacdiNAc motif Seminal fluid, serum of BPH β4GalNAcT4In was prostate observed, cancer and the were upregulation correlated ofFukushima the β4GalNAcT4 2010 was LacdiNAc motif andSeminal PCa patients, fluid,serum and LNCaP of BPH and Fukushima 2010 with the overexpressionobserved, andof the were LacdiNAc correlated groups with the overexpression[99] of PCa patients,cell line and LNCaP cell line [99] for PCathe-derived LacdiNAc PSA groups for PCa-derived PSA The LacdiNAc-PSA immunoassay allowed for distinctionThe between LacdiNAc-PSA PCa and BPH immunoassay within the allowed for distinction between PCa and BPH within the PSA gray zone PSA gray zone Kaya 2015 Kaya 2015 BPHBPH serum serum vs. PCa vs. serum PCa serum The LacdiNAc-PSA determination revealed that levels of The LacdiNAc-PSA determination revealed that [100] [100] levels ofLacdiNAc-PSA LacdiNAc-PSA were were significantly significantly higher in PCa sera than in higher in PCa sera than in BPH seraBPH sera Studies utilizing the two prostate cancer cell lines LNCaP and PC3 have shown that fucosylation of glycans is increased in the rapidly proliferating and more invasive PC3 cell line compared to the slow growing, less invasive LNCaP cell line [85]. Wang et al. detected expression of α1,6-fucosyltransferase solely in PC3, and not in LNCaP cells. Afterwards the authors found that FUT8 expression was elevated in metastatic PCa tissues compared to normal prostate tissues. Using PC3 and LNCaP cells as models, they

Cancers 2021, 13, 3726 12 of 22

Studies utilizing the two prostate cancer cell lines LNCaP and PC3 have shown that fucosylation of glycans is increased in the rapidly proliferating and more invasive PC3 cell line compared to the slow growing, less invasive LNCaP cell line [85]. Wang et al. detected expression of α1,6-fucosyltransferase solely in PC3, and not in LNCaP cells. Afterwards the authors found that FUT8 expression was elevated in metastatic PCa tissues compared to normal prostate tissues. Using PC3 and LNCaP cells as models, they also conﬁrmed that FUT8 overexpression in LNCaP cells increased PCa cell migration, while the silencing of FUT8 expression in PC3 cells reduced cell motility. These results suggest the association of FUT8 with aggressive prostate cancer (Table2)[91,101]. In a recent study Clark et al. evaluated the effect of altered α1,6-fucosyltransferase expression on extracellular vesicles (EVs) in a prostate cancer cell model. They found that increased cellular expression of FUT8 can reduce the number of vesicles secreted by prostate cancer cells and simultaneously enhance the protein abundance correlated with cell motility and prostate cancer metastasis. Overexpression of FUT8 can also cause changes in glycans presented on EV-derived glycoproteins [101]. Wang and co-workers developed a quantitative lectin immunoassay using Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) to evaluate the level of PSA fucosylated glycoforms in serum samples from prostate cancer patients with different Gleason scores. The results demonstrated that both LCA and AAL immunoassays identiﬁed an increased level of fucosylated serum PSA. These results were concomitantly correlated with higher Gleason scores. Finally, the authors concluded that the determined fucosylated PSA forms could be valuable biomarkers to differentiate between aggressive and non-aggressive prostate cancer (Table2)[94].

8. Branched N-Glycans During malignant transformation and cancer progression, a frequently occurring change in the glycosylation pattern is the increased expression of complex β1,6-branched N-glycans [102]. The raised level of GlcNAc-branching N-glycans is a result of increased activity of mannoside N-acetylglucosaminyltransferase 5 (GnT-V), which is encoded by the MGAT5 gene, often activated in cancer cells [59]. Activity of GnT-V leads to the formation of complexed tri- and tetra-antennary structures (Figure1A), which can affect the stability, functional activity and half-life of proteins, as well as membrane dynamics [61,103]. Furthermore, branched N-glycans can be modified via β1,4-galactosyltransferases (β1,4- GalTs), thus elongated with poly-N-acetyllactosamine repeats, and finally capped with sialic acid or fucose. Poly-N-acetyllactosamine moieties are ligands for galectins, a family of evolutionarily conserved carbohydrate-binding proteins. These lectins bind glycans with high avidity and affinity, forming multivalent galectin-glycan lattices that control glycoprotein clustering and endocytosis, to regulate receptor signaling and activation [104]. Galectins play an essential role in cancer, participating in neoplastic transformation, survival of cancer cell, angiogenesis and tumor metastasis [59,105]. In contrast to the function of GnT-V, GnT-III catalyses the attachment of bisecting GlcNAc N-glycans via a β1,4-linkage (Figure1C), inhibiting further conversion and extension of the glycan, as seen in β1,6-branched structures. GnT-III counteracts the role of GnT-V in the neoplastic process by participating in the inhibition of cancer metastasis [59]. Numerous studies have indicated that N-acetylglucosaminyltransferase V is an important tumorigenesis- and metastasis-associated enzyme in prostate cancer [106]. Lange et al. reported that β1,6-GlcNAc tri- and tetra-branched N-glycans were increased in cell line xenograft mouse models of prostate cancer (Table2)[ 97]. Additionally, it was found that patients with castration-resistant prostate cancer had both overexpressed transcription levels of N-glycan branching enzymes and increased tri- and tetra-antennary N-glycans [91,96]. Slightly different observations were reported for direct expressed-prostatic secretion (EPS) and EPS urine samples. A decline in the amount of tri- and tetra-antennary glycans in advanced prostate cancer samples with a Gleason score of 8 and 9 was detected, and at the same time many biantennary structures with a bisecting-GlcNAc residue were observed, suggesting antimetastatic activity of GnT-III [95]. In some recent research it has been empha- Cancers 2021, 13, 3726 13 of 22

sized that changes in branched N-glycans can help to distinguish between BPH and prostate cancer, and furthermore increased levels of serum tri- and tetra-antennary N-glycans can be clinically useful for predicting castrate-resistant prostate cancer (Table2)[ 96]. In a recent study, tetra-antennary N-glycans were identiﬁed as part of a biomarker panel to reﬁne the distinction of patients with indolent and aggressive prostate cancer and predict patient survival [107].

9. LacdiNAc Structures In general, LacdiNAc structures are rarely observed in normal mammalian cells, while their frequency is significantly increased in prostate, ovarian, and pancreatic cancers [98,108,109]. The terminal modification of N-glycans results from the β4-linkage of N-acetylgalactosamine to N-acetylglucosamine to form the LacdiNAc unit (GalNAcβ1→4GlcNAc). Two human β4- N-acetylgalactosaminyltransferases (β4GalNAcTs)—β4GalNAcT3 and β4GalNAcT4—are involved in biosynthesis of the LacdiNAc motif (Figure1A). Although the enzymes have high sequence homology and similar substrate specificities, they present different tissue distribution: the B4GALNT3 gene is mainly expressed in the human testis, stomach and colon, while the B4GALNT4 gene is expressed in the human brain and ovary [109]. LacdiNAc structures have been documented for PSA N-glycans purified from seminal plasma of healthy individuals and from the human prostate cell line LNCaP. The amounts of the LacdiNAc moieties were increased in PSA obtained from prostate cancer cells [71,109]. Furthermore, the occurrence of LacdiNAc groups on PSA has been observed in numerous studies using Wisteria floribunda agglutinin (WFA) [98–100,110]. Interestingly, in prostate cancer the upregulation of β4GalNAcT4 was observed, while no changes were noted for β4GalNAcT3 [99,109]. Additionally, a correlation between enzyme upregulation and overexpression of LacdiNAc epitopes in prostate cancer derived PSA was found (Table2)[ 99]. When prostate cancer and benign prostatic hyperplasia PSA were compared, BPH patients’ glycoproteins contained predominantly terminally sialylated, complex-type biantennary N-glycans, whereas PSA from patients with prostate cancer presented an increased amount of similar biantennary complex-type glycans, but with one LacdiNAc unit, only partially sialylated [109]. Haga and co-workers observed that abundance of multiple sialylated LacdiNAc structures was significantly upregulated in PCa patients compared to the BPH group, and further established a new, highly sensitive PCa-specific diagnostic model: the “PSA G-index”. It is based on the relative abundance of the two di- and tri-sialylated LacdiNAc glycoforms. Both above-mentioned PSA glycoforms showed a significant correlation with Gleason scores. In the same study, histochemical staining analysis with WFA lectin showed that PCa cells overexpressed glycoproteins containing LacdiNAc moieties (Table2)[ 98]. The low sensitivity of tests developed so far encourages the use of a combination of several markers in the assay, suggesting that such a combination could ultimately constitute a biomarker panel for prostate cancer detection, as proposed by Yoneyama et al. To identify clinically significant prostate cancer (CSPC), they evaluated the amount of LacdiNAc-glycosylated prostate-specific antigen (LDN-PSA) and LDN-PSA normalized by prostate volume (LDN-PSAD). During the experiment, they measured LDN-PSA, total PSA, and ratio of free PSA to total PSA values in 718 men who underwent a prostate biopsy and in 174 prostate cancer patients who underwent radical prostatectomy. In the cohort of prostate biopsy patients LDN-PSAD demonstrated significantly higher clinical performance to discriminate CSPC compared with LDN-PSA, PSAD, total PSA and free/total PSA ratio. The authors concluded that LacdiNAc-glycosylated PSA is significantly more efficient than the conventional PSA test in identifying patients with CSPC [111].

10. Truncated O-Glycans Biosynthesis of O-glycans is initiated by transfer of a single N-acetylgalactosamine residue to serine or threonine by polypeptide N-acetylgalactosaminyltransferases ppGalNAc- Ts (GALNTs), to form a simple Tn antigen [112]. In subsequent stages enzymatic extension Cancers 2021, 13, 3726 14 of 22 Cancers 2021, 13, x 12 of 20

Cancers 2021, 13, x 12 of 20

of Tn antigen with galactose builds core 1 O-glycans (T antigen), which can be further ex- tendedwhich with can β be1,6-N-acetylglucosamine further extended with to β1,6 produce-N-acetylglucosamine core 2 O-glycans (Figureto produce1E,F). core GCNT1 2 O- isglycans reported (Figure to be involved 1E,F). GCNT1 in the formation is reported of core to be 2 branched involved O-glycans in the formation and in synthesis of core 2 which can be further extended with β1,6-N-acetylglucosamine to produce x core 2 O- ofbranched the cancer-associated O-glycans and antigen in synthesis sLex [113 of, 114 the]. cancer Increased-associated GCNT1 antigen expression sLe has[113,114] been . glycans (Figure 1E,F). GCNT1 is reported to be involved in the formation of core 2 linkedIncreased to prostate GCNT1 cancer expression progression has been and is linked a predictor to prostate of recurrence cancer after progression radical prostatec- and is a branched O-glycans and in synthesis of the cancer-associated antigen sLex [113,114]. tomypredictor [114, 115of recurrence]. Expression after of bothradical GCNT1 prostatectomy and the sLe [114,115]x antigen. Expression is controlled of by both androgens GCNT1 Increased GCNT1x expression has been linked to prostate cancer progression and isx a inand prostate the sLe cancer antige cells.n is controlled Moreover, by sLe androgensx is the major in prostate sialylated cancer antigen cells. Moreover, related to sLe poor is predictor of recurrence after radical prostatectomy [114,115]. Expression of both GCNT1 prognosisthe majorand sialylated metastasis antigen in PCa related [48, 113to poor]. prognosis and metastasis in PCa [48,113]. and the sLex antigen is controlled by androgens in prostate cancer cells. Moreover, sLex is Alternatively,Alternatively, attachment attachment of of the the N-acetylneuraminic N-acetylneuraminic acid acid molecule molecule to to the the Tn Tn antigen antigen the major sialylated antigen related to poor prognosis and metastasis in PCa [48,113]. cancan generategenerate sialo-Tnsialo-Tn antigen (sTn) (sTn) (Figure (Figure 1D).1D). Formation Formation of of the the sTn sTn antigen antigen is regulated is regu- Alternatively, attachment of the N-acetylneuraminic acid molecule to the Tn antigen latedby expression by expression of of sialyltransferase sialyltransferase ST6GalNAc ST6GalNAc-I.-I. Such Such modifications modifications are are frequently frequently dysregulated,candysregulated, generate sialo oftenoften-Tn occur antigen during (sTn) the the (Figure neoplastic neoplastic 1D). transformationFormation transformation of the process processsTn antigen and and are is are associatedregulated associ- atedbywith expression with numerous numerous of malignancies, sialyltransferase malignancies, including including ST6GalNAc breast, breast, - colorectal,I. Such colorectal, modifications ovarian ovarian and and aregastric gastric frequently cancers can- cersdysregulated,[116,117] [116,117. Munkley]. Munkleyoften occuret al. et foundduring al. found that the that expressionneoplastic expression transformation of the of thesialyltransferase sialyltransferase process andST6GalNAc ST6GalNAc-Iare associated-I and andwiththe thecancer numerous cancer-associated-associated malignancies, sialyl sialyl-Tn-Tn includingantigen antigen is regulated breast, is regulated colorectal, by androgens by androgens ovarian in prostate and in prostate gastric cancer cancerscancer cells, cells,[116,117]and and is involved. is Munkley involved in etreducing in al. reducing found cell that cell adhesion, expression adhesion, leading leading of the tosialyltransferase to transformation transformation ST6GalNAc towards a a more-I more and mesenchymal-likethemesenchymal cancer-associated-likecell cell sialylphenotype phenotype-Tn antigenin ina a mouse mouseis regulated model model by of of androgens prostate prostate cancer, cancer, in prostate during during cancer a a process process cells, termedandtermed is epithelial-mesenchymal involved epithelial in- mesenchymal reducing cell transition adhesion, transition (EMT) leading (EMT) [118,119 to ].[118,119] transformation This fact. is This worth towards fact emphasizing, is a worth more asmesenchymalemp thehasizing, sTn antigen -aslike wasthe cell sTn detected phenotype antigen in half wasin a of mousedetected all high-grade model in half of prostate prostateof all high cancer cancer,-grade tissue during prostate samples a process cancer from patientstermedtissue samples with epithelial diagnosed from-mesenchymal patients prostate with disease transition diagnosed [118,120 (EMT) prostate]. Additionally, [118,119] disease ST6GalNAc-I . [118,120] This fact. A expressiondditionally is worth, wasempST6GalNAc foundhasizing, to-I be as expression increased the sTn antigen in was primary found wasprostate todetected be increased tumors in half and of in decreasedall primary high- grade prostate in metastatic prostate tumors tissuecancer and comparedtissuedecrea samplessed to in benign metastatic from prostate patients tissue tissue. withcomparedSeveral diagnosed to studies benign prostate have prostate shown disease tissue. that [118,120] increasedSeveral. Astudiesdditionally expression have, ofST6GalNAcshown sialylated that - TnincreasedI expression antigens expression inhibits was found formation of sialylated to be of increased a Tn solid antigens tumor in primary inhibits massand prostate formation promotes tumors of cell a solid andde- tachmentdecreatumor sed mass from in andmetastatic the tumor promotes tissue (Table cell 3compared)[ detachment118,121 ].to Thesebenign from results theprostate tumor indicate tissue. (Table that Several ST6GalNAc-I3) [118,121] studies. These mayhave playshownresu alts significant thatindicate increased that role ST6GalNAc inexpression tumor cell- Iof may invasion sialylated play anda significantTn migration antigens role [inhibits118 in,119 tumor ].formation In cell earlier invasion of research a solid and oftumormigration Arai massand [118,119]co-workers, and promotes. In earlier the cell presence research detachment of of sTnArai from in and the co the serum-workers, tumor or tumor(Table the presence tissue3) [118,121] biopsyof sTn. Thesein was the correlatedresuserumlts orindicate withtumor cancer that tissue ST6GalNAc progression biopsy was-I and maycorrelated worse play survivala with significant cancer outcomes progressionrole in prostatetumor and cell cancer worse invasion patients. survival and Additionally,migrationoutcomes [118,119]in theprostate expression. In cancer earlier of patients. research sialyl-Tn Additionally, of MUC-1, Arai and a protein co the-workers, expression that plays the presenceof a sialyl protective- Tnof sTnMUC role in- for1,the a theserumprotein mucosal or that tumor epithelial plays tissue a protective surface biopsy [ 122was role], correlatedfor was the negatively mucosal with correlatedcancerepithelial progression surface with survival [122] and, worsewas outcomes negatively survival and positivelyoutcomescorrelated correlatedin with prostate survival with cancer outcomes higher patients. serum and Additionally, positively PSA levels correlated (Table the expression3)[ 123with]. higher of sialyl serum-Tn PSAMUC levels-1, a protein(Table 3) that [123] plays. a protective role for the mucosal epithelial surface [122], was negatively Tablecorrelated 3. Summary with of survival truncated outcomes O-glycanalternations and positively in prostate correlated cancer. with higher serum PSA levels Table(Table 3. Summary 3) [123] .of truncated O-glycan alternations in prostate cancer. Author, Year Type of Glycans Structure Sample Groups Main Results ReferencesAuthor, Year Type of Glycans StructureTable 3. SummarySample of truncated Groups O-glycan alternationsMain in prostate Results cancer. The presence of sialyl-Tn MUC-1 was References The presence of sialyl-Tn MUC-1 was correlated Author, Year Type of Glycans Structure ProstateProstateSample cancer cancer Groups specimens correlated withMain cancer Results progression, with cancer progression, positively correlated AraiReferencesArai 2005 2005 specimensvs. normal vs. normal prostate positively correlated with higher serum withThe presencehigher serum of sialyl PSA-Tn level MUC in -PCa1 was patients, correlated and [123[123]] prostateProstate specimenscancer specimens PSA level in PCa patients, and negatively withnegatively cancer correlated progression, with positively survival correlated outcomes Arai 2005 vs. normal prostate correlated with survival outcomes withExpression higher serumof sTn PSAwas inducedlevel in PCa by androgens patients, and in [123] sialylsialyl-Tn-Tn (sTn) (sTn) antigen antigen specimens negativelyExpressionprostate correlated cancer of sTn cells with was and survival induced is mediated outcomes by by Munkley 2015 primary and metastatic androgensExpression of in sTn prostateST6GalNAc was induced cancer-I. by cells androgens and is in sialyl-Tn (sTn) antigen Munkley 2016 tumors ST6GalNAcprostatemediated -cancerI was by significantlycells ST6GalNAc-I. and is mediated up-regulated by in Munkley 2015 primary and Munkley[118,119] 2015 primary and metastatic primaryST6GalNAc-I prostate ST6GalNAccarcinoma was significantly but-I. relatively down- Munkley 2016 metastatic tumors Munkley 2016 tumors up-regulatedST6GalNAcregulated- inI in was primaryestablished significantly prostate metastatic up- carcinomaregulated tissue. in [118,119] but relatively down-regulated in [118,119] primarysialyl-T prostate antigen carcinomawas extensively but relatively elevated down in all - established metastatic tissue. sialyl-T (sT) VCaP, LNCaP, DU145, PC- prostateregulated cancer in cellestablished lines (VCaP, metastatic LNCaP, tissue. DU145, Bai 2020 antigen 3 vs. RWPE-1 cells sialyl-TsialylPC- antigen-T3) antigen compared was was to extensivelyextensively normal RWPE elevated elevated-1 cells, in inall [124] especially in PC-3 cells sialylsialyl-T-T (sT) (sT) VCaP, VCaP, LNCaP, LNCaP, DU145, DU145, PC-allprostate prostate cancer cancer cell celllines lines (VCaP, (VCaP, LNCaP, LNCaP, DU145, BaiBai 2020 2020 antigenantigen PC-3 vs.3 vs. RWPE-1 RWPE-1 cells cells DU145,PC- PC-3)3) compared compared to normal to normal RWPE RWPE-1-1 cells, [124[124]] especially in PC-3 cells cells, especially in PC-3 cells An increase in core-2-O-linked sLex antigens on core-2-O-linked sLexx Malignant vs. non- Chen 2014 PSA, MUC-1, and PAP in malignant relative to antigens malignant prostate tissues [113] Annon increase-malignant in core prostate-2-O-linked tissues sLe wasx antigens observed on core-2-O-linked sLexx Malignant vs. non- Chen 2014 PSA, MUC-1, and PAP in malignant relative to antigens malignant prostate tissues [113] non-malignant prostate tissues was observed

Cancers 2021, 13, x 12 of 20

which can be further extended with β1,6-N-acetylglucosamine to produce core 2 O- glycans (Figure 1E,F). GCNT1 is reported to be involved in the formation of core 2 branched O-glycans and in synthesis of the cancer-associated antigen sLex [113,114]. Increased GCNT1 expression has been linked to prostate cancer progression and is a predictor of recurrence after radical prostatectomy [114,115]. Expression of both GCNT1 and the sLex antigen is controlled by androgens in prostate cancer cells. Moreover, sLex is the major sialylated antigen related to poor prognosis and metastasis in PCa [48,113]. Alternatively, attachment of the N-acetylneuraminic acid molecule to the Tn antigen can generate sialo-Tn antigen (sTn) (Figure 1D). Formation of the sTn antigen is regulated by expression of sialyltransferase ST6GalNAc-I. Such modifications are frequently dysregulated, often occur during the neoplastic transformation process and are associated with numerous malignancies, including breast, colorectal, ovarian and gastric cancers [116,117]. Munkley et al. found that expression of the sialyltransferase ST6GalNAc-I and the cancer-associated sialyl-Tn antigen is regulated by androgens in prostate cancer cells, and is involved in reducing cell adhesion, leading to transformation towards a more mesenchymal-like cell phenotype in a mouse model of prostate cancer, during a process termed epithelial-mesenchymal transition (EMT) [118,119]. This fact is worth emphasizing, as the sTn antigen was detected in half of all high-grade prostate cancer tissue samples from patients with diagnosed prostate disease [118,120]. Additionally, ST6GalNAc-I expression was found to be increased in primary prostate tumors and decreased in metastatic tissue compared to benign prostate tissue. Several studies have shown that increased expression of sialylated Tn antigens inhibits formation of a solid tumor mass and promotes cell detachment from the tumor (Table 3) [118,121]. These results indicate that ST6GalNAc-I may play a significant role in tumor cell invasion and migration [118,119]. In earlier research of Arai and co-workers, the presence of sTn in the serum or tumor tissue biopsy was correlated with cancer progression and worse survival outcomes in prostate cancer patients. Additionally, the expression of sialyl-Tn MUC-1, a protein that plays a protective role for the mucosal epithelial surface [122], was negatively correlated with survival outcomes and positively correlated with higher serum PSA levels (Table 3) [123].

Table 3. Summary of truncated O-glycan alternations in prostate cancer.

Author, Year Type of Glycans Structure Sample Groups Main Results References The presence of sialyl-Tn MUC-1 was correlated Prostate cancer specimens with cancer progression, positively correlated Arai 2005 vs. normal prostate with higher serum PSA level in PCa patients, and [123] specimens negatively correlated with survival outcomes Expression of sTn was induced by androgens in sialyl-Tn (sTn) antigen prostate cancer cells and is mediated by Munkley 2015 primary and metastatic ST6GalNAc-I. Cancers 2021, 13, 3726 Munkley 201615 of 22 tumors ST6GalNAc-I was significantly up-regulated in [118,119] primary prostate carcinoma but relatively down- regulated in established metastatic tissue. Table 3. Cont.sialyl-T antigen was extensively elevated in all sialyl-T (sT) VCaP, LNCaP, DU145, PC- prostate cancer cell lines (VCaP, LNCaP, DU145, Bai 2020 antigen 3 vs. RWPE-1 cells PC-3) compared to normal RWPE-1 cells, Author,[124] Year Type of Glycans Structure Sample Groups Main Results especially in PC-3 cells References

An increase in core-2-O-linked sLex Malignant vs. An increase in core-2-O-linked sLex antigens on corecore-2-O-linked-2-O-linked sLexx Malignant vs. non- antigens on PSA, MUC-1, and PAP in ChenChen 2014 2014 non-malignant PSA, MUC-1, and PAP in malignant relative to sLexantigensx antigens malignant prostate tissues malignant relative to non-malignant [113[113]] prostate tissues non-malignant prostate tissues was observed prostate tissues was observed

Some studies have also examined the role of core-2-O-linked sLex in prostate cancer, with a particular focus on metastatic disease [113,125,126]. Overexpression of sLex has been correlated with an inferior prognosis, among patients with metastatic prostate cancer, and with castration-resistant, aggressive prostate cancer [113]. Highlighting the role of O- glycans, Chen et al. found an increase in core-2-O-linked sLex antigens on PSA, MUC-1, and prostatic acid phosphatase (PAP) proteins in malignant relative to non-malignant prostate tissues, in patients who had undergone radical prostatectomy [113]. Many prostate cancer clinical trials have focused on mucin-1 (MUC-1), a single pass membrane protein, for which both the expression level of MUC-1 and its glycosylation were frequently altered [127]. An elevated MUC-1 level was detected in 58% of primary tumor tissues and 90% of lymph node metastases, but not in healthy prostate or benign prostate tissues [128]. A recent study by Bai et al. showed that sialyl-T antigen was extensively elevated in all prostate cancer cell lines (VCaP, LNCaP, DU145, PC-3) compared to normal RWPE-1 cells, and it was particularly visible in PC-3 cells. Further research focused on examination of ST3Gal-I function in PC-3 cells; ST3Gal-I silencing studies indicated that ST3Gal-I is correlated with migration, proliferation and apoptosis of PC-3 cells. Subsequently, in vivo studies showed that decreased ST3Gal-I expression was associated with a reduction in tumor size in a xenograft mouse model, demonstrating that sialyl-T antigen could be considered as a biomarker for the prognosis of prostate cancer metastasis (Table3)[124].

11. Conclusions and Future Perspectives Glycoproteins are often considered as prognostic biomarkers for cancer diagnosis and monitoring of tumor progression, as well as predictive biomarkers for disease recurrence [129]. Researchers are constantly working on new approaches to the early diagnosis of prostate cancer, risk prediction and disease treatment, and emphasizing that glycans can be a source of new, non-invasive biomarkers. Glycoprotein markers are characterized by high heterogeneity, which results from multiple glycosylation sites and glycosylation patterns, and it has been emphasized that these features may significantly alter the selectiv- ity of these molecules. Therefore, an attempt to broaden the knowledge of cancer-specific glycan structures and glycosylation sites, and then compare their patterns within a healthy population, can provide the first key step towards determining the importance of glycosylation in the diagnostic process. In view of these aspects, considerable efforts are still being made to standardize a glycomics protocol and implement modern, high-throughput mass spectrometry technology [77,130–132]. The biomarker most frequently used in prostate cancer diagnosis is prostate-specific antigen, but its limitations due to relatively low specificity restrict its use in screening tests and reduce the diagnostic potential [59,133]. The diminished sensitivity of these tests for early prostate cancer screening, along with the emergence of novel methods and technologies for glycan analysis, prompted the search for novel biomarkers based on the detection and identification of specific glycoforms of individual glycoproteins. This approach may lead to the establishment of new biomarkers with higher specificity for early cancer detection or for diagnosis in precancerous lesions [134,135]. Such research has be- come possible to carry out using newly developed, high-throughput platform technologies, Cancers 2021, 13, 3726 16 of 22

which additionally enable the effective analysis of large sample cohorts [79,134]. Numerous studies have investigated whether a cancer-specific glycosylation pattern on PSA can be used to differentiate between BPH and PCa [75,133]. The most recent research on glycan composition in order to identify PCa and establish a prognosis has been conducted on di- verse biological material, including serum [78], urine [77,136], expressed prostatic secretion urine [137], formalin-fixed, paraffin-embedded (FFPE) tissues [138,139], cell lines [124,140], and exosomes [141]. Several studies have reported altered glycosylation, mainly both sialylation and fucosylation, in PSA as specific biomarkers of prostate cancer that are able to distinguish it from benign prostatic hyperplasia [73,133]. A crucial change in prostate cancer glycosylation is the increased level of sialylation, as well as the overexpression of cancer-related sialoglycans. Abnormal sialylation is evidently associated with tumor growth, invasion and enhanced cell survival and the onset of metastasis. Additionally, an increased level of the α2,3-sialylated isomer of PSA was noted as a distinctive feature of aggressive prostate cancer [16,70,76]; thus, analysis more targeted at aberrant sialylation is likely to be of significant therapeutic value [142]. Hence, a high-performance assay has recently been developed that allows for differentiation of α2,3- and α2,6-linked sialic acid isomers of PSA in urine [136], and subsequently a mass spectrometric method for distinguishing α2,3- and α2,6-sialoglycopeptide isomers in seminal plasma PSA was optimized [143], with good prospects for application of both methods in the diagnosis of prostate cancer. Thus, high-performance analysis of glycopeptides presenting prostate cancer-associated glycans has recently opened up new avenues for the discovery of glycoconjugates and glycoforms for the future emergence of cancer biomarkers with potential clinical applications [144]. In summary, composition of glycoproteins and glycans is likely to play an important role in both non-malignant prostate cancer and in prostate cancer. Hence in recent years, impressive progress in understanding their composition and function has been achieved. Numerous studies in this area have contributed to the emergence of glycans as promising biomarkers, highlighting their use in clinical settings as attractive targets for prostate cancer treatments [48,59,145].

Author Contributions: Conceptualization, A.K. and M.F.-S.; data analysis, A.K.; writing—original draft preparation, A.K.; writing—review and editing, A.K. and M.F.-S.; visualization, A.K.; critical review of manuscript, A.K., J.S. and M.F.-S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wroclaw Medical University, Poland, No. SUB.A070.19.034. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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