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Protein Glycosylation and Tumor Microenvironment Alterations Driving Cancer Hallmarks

Protein Glycosylation and Tumor Microenvironment Alterations Driving Cancer Hallmarks REVIEW published: 14 May 2019 doi: 10.3389/fonc.2019.00380 Protein Glycosylation and Tumor Microenvironment Alterations Driving Cancer Hallmarks 1,2,3,4 1 1,2 1,5 Andreia Peixoto , Marta Relvas-Santos , Rita Azevedo , Lúcio Lara Santos and 1,2,6 José Alexandre Ferreira * 1 2 Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal, Tumour and Microenvironment Interactions Group, INEB-Institute for Biomedical Engineering, Porto, Portugal, Instituto de Investigação e Inovação em Saúde, Universidade do 5 6 Porto, Porto, Portugal, Department of Surgical Oncology, Portuguese Institute of Oncology, Porto, Portugal, Porto Comprehensive Cancer Center, Porto, Portugal Decades of research have disclosed a plethora of alterations in protein glycosylation that decisively impact in all stages of disease and ultimately contribute to more aggressive cell phenotypes. The biosynthesis of cancer-associated glycans and its reflection in the glycoproteome is driven by microenvironmental cues and these events act synergistically toward disease evolution. Such intricate crosstalk provides the molecular foundations for the activation of relevant oncogenic pathways and leads to functional alterations driving invasion and disease dissemination. However, it also provides an important source Edited by: of relevant glyco(neo)epitopes holding tremendous potential for clinical intervention. Leonardo Freire-de-Lima, Therefore, we highlight the transversal nature of glycans throughout the currently Federal University of Rio de Janeiro, Brazil accepted cancer hallmarks, with emphasis on the crosstalk between glycans and the Reviewed by: tumor microenvironment stromal components. Focus is also set on the pressing need Feng Guan, to include glycans and glycoconjugates in comprehensive panomics models envisaging Northwest University, China Monica M. Burdick, molecular-based precision medicine capable of improving patient care. We foresee Ohio University, United States that this may provide the necessary rationale for more comprehensive studies and *Correspondence: molecular-based intervention. José Alexandre Ferreira Keywords: cancer, microenvironment, glycans, protein glycosylation, cancer hallmarks Specialty section: This article was submitted to INTRODUCTION Molecular and Cellular Oncology, a section of the journal Genetic and epigenetic alterations are considered primary causes of cancer development, with Frontiers in Oncology downstream phenotypic changes at the protein level being amongst the driving forces of cancer Received: 15 March 2019 progression and dissemination. Specifically, post-translational modifications, as glycosylation, Accepted: 23 April 2019 impact on protein trafficking, stability and folding, ultimately altering its biochemical, and Published: 14 May 2019 biophysical properties (1, 2). Moreover, glycans dictate proteolysis patterns and directly mediate Citation: ligand-receptor interactions, oncogenic signaling transduction, immune recognition, migration Peixoto A, Relvas-Santos M, and both cell-cell and cell-matrix adhesion (3–5). In addition, intracellular O-GlcNAc glycosylation Azevedo R, Santos LL and Ferreira JA (in Ser/Thr residues) of proteins plays a major role in cell physiology and signaling by direct (2019) Protein Glycosylation and competition with phosphorylation (6). As such, several studies have so far disclosed a plethora Tumor Microenvironment Alterations of glycans that confer selective advantage to tumor cells, while providing important surrogate Driving Cancer Hallmarks. biomarkers for specific biological milieus (7, 8). Moreover, while there are few evidences of Front. Oncol. 9:380. doi: 10.3389/fonc.2019.00380 mutations in genes involved in glycosylation pathways, it is well known that transcriptional Frontiers in Oncology | 1 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment and metabolic reprograming of cancer cells has tremendous more frequently, complex or hybrid type structures, which impact on their glycome and glycoproteome, leading not only frequently experience further elongation. Both O- and N- to the overexpression but also to the de novo expression of glycan chains are generally branched and/or elongated and specific glycoepitopes (9, 10). Despite its sour side, cancer-specific may present sialic acids, Lewis blood group related antigens or alterations in protein glycosylation provide a unique opportunity ABO(H) blood group determinants as terminal structures (8). for clinical intervention. The uniqueness of the created molecular Further glycan diversity results from several modifications in features may be explored to selectively target tumor cells or may individual sugars, including O-Acetylation of sialic acids and O- provide non-invasive biomarkers after secretion or shedding into Sulfation of galactose and N-acetylglucosamine residues. Mature body fluids from tumor sites (11, 12). glycans may still experience structural remodeling at the cell- Building on these findings, the glycobiology field has surface by extracellular glycosyltransferases and glycosidases been progressing toward a more functional understanding of freely circulating in the plasma or carried by platelets, further glycosylation impact on cancer biology, disease progression, and increasing the glycome’s structural complexity and dynamic dissemination. While specific details on the biosynthesis and nature (14–16). In addition, other less abundant and far less diversity of cancer-associated glycans may be found in recent studied classes of protein glycans can be found at the cell reviews (7, 8), the following sections attempts to highlight the membrane, including O-Fucosylation, O-Mannosylation, O- transversal nature of glycans, glycoproteins, and glycan-binding glucosylation, and C-Mannosylation (17–19). This provides a proteins throughout currently accepted cancer hallmarks, wide array of potential posttranslational modifications that with emphasis on the crosstalk between glycans and the decisively contribute to define protein functional roles. stromal components of the tumor microenvironment (Figure 2). In addition to the structural modification of extracellular These comprehend: (i) sustained proliferative signaling; (ii) and cell membrane proteins, intracellular proteins can resistance to cell death; (iii) deregulated cellular energetics; also be glycosylated with functional implications. Namely, (iv) evasion of growth suppressors; (v) genome instability intracellular glycosylation results from the reversible attachment and mutations; (vi) replicative immortality; (vii) induction of of a N-acetylglucosamine moiety (β-linked GlcNAc) to angiogenesis; (viii) activation of invasion and metastasis; (ix) Ser or Thr residues in cytoplasmic and nuclear proteins tumor-promoting inflammation; and (x) immune escape (13). (20–22). The GlcNAc residue is generally not elongated Moreover, we highlight the significance of the most promising or modified to generate complex structures (23). The protein glycosignatures in cancer arising from the cancer cells- dynamic cycling of O-GlcNAcylation is catalyzed by two microenvironment crosstalk, its relevance and main milestones ubiquitously expressed and highly conserved enzymes: facing clinical translation and personalized medicine, as well as uridine diphospho-N-acetylglucosamine:polypeptide β-N- the opportunities provided by high-throughput glycomics and acetylglucosaminyltransferase (O-GlcNAc transferase, OGT), glycoproteomics toward molecular-based precision oncology. which adds GlcNAc to the hydroxyl side chain of Ser and We foresee that this may provide the necessary rationale for more Thr, and N-acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA), comprehensive studies and molecular-based intervention. the enzyme that removes O-GlcNAc. This posttranslational modification has regulatory functions akin to phosphorylation, modulating protein conformation, stability, and reversible multimeric protein assembly (24). Moreover, it functions as a PROTEIN GLYCOSYLATION IN CANCER nutrient sensor, providing a biochemical switch to enable the Glycosylation is the most common, structurally diverse and cell adaptation to glucose level alterations and hormonal cues, complex posttranslational modification of membrane-bound while regulating a myriad of cellular processes like cellular proteins, being a non-templated but highly regulated process that adhesion, DNA transcription, translation, nuclear transport, and rapidly changes in response to physiological and pathological cytoskeletal assembly (25, 26). Interestingly; different isoforms contexts. Glycans result from the highly coordinated action of OGT and OGA vary in length and subcellular localization, of nucleotide sugar transporters, glycosyltransferases (GTs) and suggesting that they target distinct subsets of the proteome (27). glycosidases in the endoplasmic reticulum (ER) and Golgi It has been long known that advanced tumors present severe apparatus (GA). Two main classes of glycans can be found dysregulations in glycosylation pathways, with tumor-associated in membrane and extracellular glycoproteins: (i) O-GalNAc carbohydrates arising from incomplete or neo-synthesis glycans, initiated in the GA by the attachment of a GalNAc processes (28). Of note, incomplete synthesis originating to the hydroxyl groups of serine (Ser) or threonine (Thr) truncated structures is more common in early carcinogenesis residues, forming the simplest O-glycan Tn antigen (GalNAcα- (29, 30), while the de novo synthesis of neoantigens is more Ser/Thr). The Tn antigen may be further elongated into different frequent in advanced stages of several cancers (31). The most core structures that serve as scaffolds for more complex O- reported alterations associated to cancer include the over- and/or GalNAc glycans; (ii) N-glycans, whose biosynthesis starts in de novo expression of short-chain O-GalNAc glycans (Tn, T, the ER with the addition of an oligosaccharide chain to an Sialyl-T, and Sialyl-Tn), Lewis blood group related antigens asparagine (Asn) residue in a peptide consensus sequence of and their sialylated counterparts [sialyl-Lewis A (SLe ) and Asn-X-Ser/Thr (X denotes any amino acid except proline). N- X (SLe )], as well as complex branched N-glycans (32–34) glycans experience further structural maturation in the GA (Figure 1). Many of these structural features are common to to yield either partially unprocessed oligomannose antenna or, most advanced solid tumors and often associate with poor Frontiers in Oncology | 2 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment FIGURE 1 | Main classes of glycans modulating cancer hallmarks. N-glycans, whose biosynthesis starts in the endoplasmic reticulum (ER) with the addition of an oligosaccharide chain to an asparagine (Asn) residue, experience further structural maturation in the golgi apparatus (GA) to yield complex bisected and branched structures. O-GalNAc glycans, initiated in the GA by the attachment of a GalNAc to the hydroxyl groups of serine (Ser) or threonine (Thr) residues, forming the simplest O-glycan Tn antigen (GalNAcα-Ser/Thr), may be further elongated into different core structures that serve as scaffolds for more complex O-GalNAc glycans. Both O- and N-glycan chains are generally branched and/or elongated and may present sialic acids, Lewis blood group related antigens and/or their sialylated counterparts as terminal structures. Proteoglycans constitute another class of functionally complex glycoconjugates found as transmembrane, basement membrane and extracellular matrix (ECM) components, exhibiting one or several high molecular weight glycosaminoglycan (GAG) chains covalently attached to a protein core. The figure highlights the structures of some of the most relevant glycans and glycoconjugates driving cancer hallmarks. prognosis, suggesting common molecular mechanisms, which Overall, the most widely occurring glycosylation is yet to be proven. Nevertheless, distinct proteome signatures, modifications in cancer stem from alterations in glycan glycosylation density, and glycosite distribution may ultimately length, often toward shorter O-glycans and more branched dictate organ, cell-type and cancer-specific molecular signatures N-glycans. This is accompanied by critical changes in and clinically relevant glycoforms. glycans sialylation and fucosylation that impact on the Another class of cell-surface glycoconjugates that populate nature of terminal epitopes at glycan chains. In addition, the cell surface and extracellular matrix are proteoglycans, several changes in glycan chains have been reported for generally composed of one or several high molecular weight glycosaminoglycans (GAG). The structural nature of glycan glycosaminoglycan (GAG) chains, and composed of sulphated alteration in cancer and underlying biosynthesis mechanisms disaccharide repeating units of chondroitin sulfate (CS), heparan have been comprehensively reviewed in recent years (7, 8, 37) sulfate (HS), or dermatan sulfate (DS) covalently attached to and will not be covered in detail here. Aberrant glycosylation a protein core (Figure 1). These polymers can be found as actively contributes to tumor progression by regulating tumor transmembrane, basement membrane and extracellular matrix proliferation, invasion, metastasis, and angiogenesis (7, 38), (ECM) components, presenting high affinities for various ECM being frequently cited as a hallmark of cancer (39). As such, constituents and cell adhesion molecules. As such, proteoglycans we reinforce this notion by highlighting aberrant glycosylation largely contribute to the acquisition of cancer hallmarks by as an integral part of all recognized cancer hallmark traits. playing a role in intercellular and ECM interactions, as well as Furthermore, we include the cabal contribution of stromal in cellular signaling, especially as co-receptors for growth factors cells and microenvironmental features for tumor progression and tyrosine kinase receptors (35, 36). and aggressiveness. Frontiers in Oncology | 3 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment TUMOR MICROENVIRONMENT AND proteoglycans mainly act as positive regulators of sustained proliferative signaling. In line with this, adipocyte-derived GLYCOSYLATION CROSSTALK TOWARD ECM collagen VI affects early mammary tumor progression THE HALLMARKS OF CANCER in vivo via signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on tumor cells (55). Thereby, The glycocalyx, combining glycoproteins and sugar moieties stromal adipocytes also constitute active players in driving located on the external side of the plasma membrane, drives the tumor cell proliferation. Of note, the mechanisms through which interplay between cancer cells and the tumor microenvironment proteoglycans enforce their action are not fully elucidated and the (TME), a complex scaffold of extracellular matrix (ECM) true implications of GAG chains are yet to be fully clarified. Given and various cell types. Both glycans, glycoconjugates and these insights, the reciprocal communication between neoplastic the TME actively contribute to the acquisition of cancer and stromal cells is essential to maintain mitogenic factors supply hallmarks, adding another dimension of complexity to cancer to sustain cellular proliferation. progression by influencing cell adhesion and cell-cell recognition, Glycosylation adds a second level of proliferation regulation as well as intracellular signaling and ECM interactions by mediating growth factor receptor activation and structural (8, 40, 41). Herein, we will highlight the glycosylation- alterations (Figure 2A). Namely, the O-GlcNAc modification mediated promotion of cancer hallmarks, including the role of of transcription factors involved in cell cycle progression, stromal cells. such as factor forkhead protein M1 (FoxM1), cyclin D1, and c-MYC, stabilizes them and contributes to oncogenesis Sustained Proliferative Signaling (56, 57) (Figure 2A). Moreover, numerous cell-surface tyrosine Malignant cells are characterized by uncontrolled proliferation, kinase receptors (RTK), including EGFR, FGFR, PDGF, c- largely due to the loss of homeostasis in the production, MET, ERBB2/HER2, and IGFR are known to be regulated release, and affinity for growth-promoting signals. That said, by cancer-associated glycans (58–60), glycosyltransferases (61), cancer cells may rely on autocrine proliferative signaling or and proteoglycans (62–65). For instance, the degree of N- stimulate stromal cells to supply them with mitotic factors to glycan branching of several RTKs contributes to its capability sustain proliferation. For instance, endothelial and infiltrating to induce or arrest cellular proliferation (66, 67). Showcasing immune cells secrete growth-promoting factors that paracrinaly this, studies with CHO cells demonstrated that the Asn418- stimulate neoplastic cells proliferation independently from linked N-glycan in ERBB3 plays an essential role in regulating blood-borne factors (42, 43). Moreover, tumor and immune receptor heterodimerization with ERBB2 (59), providing a cells-promoted ECM remodeling uncages mitogenic agents pivotal checkpoint where N-glycans may regulate key cellular while disabling growth suppressing adhesion complexes, thereby processes involved in cell proliferation and transformation. maintaining the proliferative potential of cancer cells (44). Moreover, core 1 β1,3-galactosyltransferase (C1GALT1, Furthermore, several ECM proteoglycans, mainly produced responsible for Tn antigen biosynthesis) overexpression in by cancer-associated fibroblast (CAF), regulate proliferative hepatocellular carcinoma activates hepatocyte growth factor signaling in adjacent tumor cells (Figure 2A). For instance, (HGF) signaling via modulation of MET kinase O-glycosylation CAF-derived proteoglycans syndecan-1 and versican promote and dimerization, thereby enhancing cell proliferation in vivo proliferation of human breast cancer cells (45–47) and myeloma and in vitro (61). Contrastingly, overexpression of β1,4-N- tumors (48), mainly by influencing EGF receptor signaling. acetylglucosaminyltransferase III (MGAT3), which adds β1,4 Likewise, transmembrane syndecan-2 expression appears to bisecting branches to N-glycans, appears to inhibit EGFR be critical for colon carcinoma cell behavior by mediating sensitivity to EGF in glioma cells (58), thereby reducing increased adhesion and proliferation (49). Also, the ECM cellular response to the proliferative effects of EGF. In turn, multifunctional heparan sulfate proteoglycan perlecan strongly β1,6-N-acetylglucosaminyltransferase V (MGAT5) knockout augments the binding and mitogenic activity of basic fibroblast mice were shown less prone to mammary tumor growth growth factor (bFGF), contributing to sustained tumor cell and metastasis, while showing poor PI3K/AKT activation, proliferation by FGF pathway activation (50). In line with this, emphasizing the importance of β1,6-GlcNAc-branched N- fibroblast-derived hyaluronic acid (HA) paracrinally enhances glycans in proliferative signaling pathways (68). Also, ABO the in vitro proliferation of melanoma cells, while proteins glycosyltransferase mRNA downregulation in normal and secreted by tumor cells further increase HA synthesis in CAFs malignant urothelium is associated with EGF stimulation, in a phosphatidylinositol 3/mitogen-activated protein-kinase- resulting in decreased cell proliferation (69). Together, these dependent manner (51). On the other hand, the small leucine- findings highlight the relevance of glycosyltransferases in tumor rich proteoglycan decorin, expressed primarily by myofibroblast, cell proliferative signaling. autocrinally, and paracrinally reduces tumor growth and In turn, the short-chain O-GalNAc STn antigen is mainly metastasis in murine xenograft models by downregulating EGFR observed in non-proliferative tumor areas of highly proliferative and Met receptors (52), while inhibiting tumor growth factor bladder tumors (70), while being overexpressed in less β (TGF-β) signaling (53). Decorin also activates ERBB4, which proliferative hypoxic bladder cancer models (29), suggesting blocks the phosphorylation of heterodimers containing either a yet unknown indirect regulation of proliferation by O- ERBB2 or ERBB3, thereby suppressing cell growth in mammary glycosylation in bladder cancer. In ovarian cancer cells, the carcinoma cells (54). These findings suggest that CAF-derived knockout of core 1 synthase chaperone Cosmc, resulting in Frontiers in Oncology | 4 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment A/X FIGURE 2 | Role of glycans, glycoproteins, glycan-binding proteins, and proteoglycans across currently accepted cancer hallmarks. Glycans (sTn, sLe , Neu5Gc,β1,6-branched N-glycans), glycoproteins (Fas, TRAIL-R, integrin α3β1, VEGFR2, ATM, p53, Rb), proteoglycans (decorin, neuropilin-1,-2, hyaluronic acid, versican, perlecan, hyaluronic acid), lectins (Gal-3 and Gal-1), and O-GlcNAcylated transcription factors (c-Myc, Fox M1, cyclin D1, NF-κB) are mechanistically implicated in cancer hallmarks acquisition and thus represented. Overall, the illustrations focus on particular molecular mechanisms driving hallmark acquisition; namely (A) sustaining proliferative signaling, (B) resistance to cell death, (C) deregulated cellular energetics; (D) evasion of growth suppression; (E) genome instability and mutation; (F) angiogenesis, (G) invasion and metastasis, (H) tumor-promoting inflammation, and (I) Immune scape. Stromal and immune cells providing the soluble factors driving cancer hallmarks are also highlighted, namely tumor-associated macrophages, dendritic cells, adipocytes, and fibroblasts. Tn and STn O-glycans expression, leads to a reduction in responsive to conventional chemotherapy that mostly targets cellular proliferation compared to the parental cell lines (71). highly proliferative clones (73). Moreover, the use of O-glycan inhibitors in colorectal cancer In addition to alterations in core O- or N-glycans, changes cell lines promptly blocks proliferation in a so far unexplored in terminal glycan structures may likewise induce changes in manner (72). Overall, short-chain O-glycans expression seem cell proliferation. For instance, in aggressive non-small cell lung to reduce tumor cell growth. This process might actually confer cancer cell lines, knockdown of α1,6-fucosyltransferase 8 (FUT8), selective advantage to tumor cells which are rendered less catalyzing the addition of fucose in alpha 1-6 linkage to GlcNAc Frontiers in Oncology | 5 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment residues, significantly inhibits cell proliferation (74). Moreover, from effector cells (92, 93). For instance, the tumor necrosis overexpression of sialyltransferases and α1,3-fucosyltransferases factor–related apoptosis-inducing ligand (Apo2L/TRAIL) (FUT4 or FUT6) would suppress EGFR dimerization and promotes tumor cell apoptosis through the death receptors phosphorylation upon EGF treatment, decreasing lung cancer TRAIL-R1 and TRAIL-R2, whose O-glycosylation status cells proliferation (60). In line with this, enhanced α2–6 determines its sensitivity to the ligand. Specifically, the O- sialylation, secondary to overexpression of ganglioside-specific glycosylation initiating enzyme GALNT14 showed a strong ST6GalNAcV, inhibits glioma growth in vivo (75, 76). Altogether, link to TRAIL sensitivity in pancreatic carcinoma, NSCLC these findings demonstrate the pleiotropic and occasionally and melanoma, whereas expression of GALNT3, along with opposing effects of altered glycosylation in cell proliferation. the O-glycan processing enzymes FUT3 and FUT6, correlated In summary, these examples demonstrate how the with responsiveness in colorectal cancer cells, rendering helpful microenvironment and glycosylation can sustain proliferative data for identifying cancer patients who are more likely to signals. Overall, the crosstalk between neoplastic cells and respond to TRAIL-based therapies (93). Consistent with these the TME ensures the positive feedback look of growth factors observations, a lower degree of fucosylation, which occurs supply and ECM remodeling, while glycosylation promotes the by mutation of the GDP-mannose-4-6-dehydratase (GMDS) exposure and interaction of protein domains with RTKs as well gene, increases resistance to TRAIL-induced apoptosis in as the constitutive activation of oncogenic pathways through colon cancer cells, followed by immune escape (94). Moreover, kinases modification. N-glycosylation also plays an important regulatory role in TRAIL-R1-mediated apoptosis, but not for TRAIL-R2, which Resistance to Cell Death is devoid of N-glycans. In this context, defective apoptotic The TME aids programmed cell death evasion by providing signaling by N-glycan-deficient TRAIL receptors was associated survival signals and offering a physical barrier against pro- with lower TRAIL receptor aggregation and reduced death- apoptotic factors such as chemotherapy. First, endothelial cells inducing signaling complex (DISC) formation, but not with establish vasculature to attenuate cell death that would otherwise reduced TRAIL-binding affinity (95). result from hypoxia and lack of serum-derived nutrients (77). In turn, the death receptor Fas (CD95/APO-1) has two N- However, when neovascularization cannot keep up with nutrient glycosylation sites at N136 and N118 moderately affecting Fas- demand, an hypoxic microenvironment is established where induced apoptosis. Specifically, the addition of sialic acids by HIF-1α drives antiapoptotic changes (78). In addition, infiltrating ST6Gal-I in an α2-6 linkage to the N-glycans of Fas provides macrophages circumvent apoptosis of cancer cells by shielding protection against Fas-mediated apoptosis in colon carcinoma them from external apoptotic factors and chemotherapy (79). cells. Namely, α2-6 sialylation of Fas prevents FasL-induced Similarly, CAFs are highly implicated in apoptotic signaling apoptosis by decreased activation of caspases 8 and 3, blockage of evasion by secreting paracrine survival factors and inducing Fas–Fas-associated death domain (FADD) association with Fas ECM remodeling (80–82). Moreover, CAF-derived chondroitin cytoplasmic tails, and inhibition of Fas internalization (96). In sulfate proteoglycan serglycin (SRGN) induces lung cancer line with this, high-grade tumors, which are known to express chemoresistance and anoikis-resistance, promoting malignant high levels of O-6 sialylation, significantly overexpress Fas, but phenotypes through interaction with tumor cell receptor CD44 are insensitive to Fas-ligand, thereby avoiding immune cell- (83). In addition, ECM proteoglycans as the small leucine-rich mediated apoptosis (30, 97, 98). Moreover, N-deglycosylation of lumican promote melanoma cells apoptosis, ultimately inhibiting Fas leads to the slowing down of procaspase-8 activation at the metastasis to the lungs (84). Consistent with the changes in DISC complex, with no impact on DISC formation or FADD ECM composition and topography, expression of many ECM recruitment (99). Overall, these findings demonstrate that, in remodeling enzymes is often deregulated in human cancers as contrast to the TRAIL-R O-linked glycan moiety, the Fas N- tumor cells acquire anchorage independence for survival (85). In glycan structure contributes to a smaller extent to the initiation this context, tumor cell-ECM interactions control malignant cells of the apoptotic signaling leading to cell death. subversion of positional information and basement membrane Glycosyltransferases, as N-acetylgalactosaminyltransferase dependence to evade apoptosis upon ECM detachment during 1 (GALNT1), also contribute to activate survival signals cancer progression (86, 87). Furthermore, the ECM also aids that supress apoptosis. Specifically, overexpression of N- tumor cells chemotherapy-induced apoptosis evasion (88–90). acetylgalactosaminyltransferase 1 (GALNT1) contributes Likewise, cancer-associated adipocytes are an abundant source to aberrant glycosylation of integrin α3β1, changing the of pro-survival factors and extracellular matrix components, conformation of integrin heterodimers, and initiating signal specially collagen VI which confers resistance to cisplatin- transduction to induce focal adhesion kinase (FAK) activation induced death in ovarian cancer cells (90, 91). in bladder cancer cells (100). Accordingly, both the knockdown Glycosylation mostly influences the extrinsic apoptotic of FAK and suppression of FAK phosphorylation were able to program, involving both TRAILR and Fas death receptors, as induce apoptosis in BC cells through caspase-3 recruitment well as integrin and galectin-mediated signaling (Figure 2B). and Src phosphorylation, respectively (101). The suppression Several glycans, glycosyltransferases, and glycosidases play of FAK phosphorylation also inhibited the PI3K/AKT signaling critical roles in programmed cell death (92) by hindering pathway, suggesting it acts downstream of FAK to regulate ligand–receptor interactions, which influences the formation apoptosis (101). Interestingly, FAK is overexpressed in a variety of signaling complexes, and modulating ligand secretion of human tumors where it mediates survival signaling, and Frontiers in Oncology | 6 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment these findings might point an intervention strategy to regulate glucose and glutamine uptake (the Warburg effect) (113). apoptotic stimuli through glycosyltransferases modulation. In this context, adaptation to hypoxia and cellular energetic In addition, several studies suggest that hyper-O- reprograming occurs mostly in a HIF-1α-dependent manner, GlcNAcylation in cancer may play an anti-apoptotic being frequently accompanied by cell dedifferentiation and role (Figure 2B). For instance, human pancreatic ductal acquisition of mesenchymal characteristics (29). Briefly, to adenocarcinoma cells are supported by oncogenic NF-κB compensate the reduction of intracellular ATP levels under transcriptional activity and both NF-κB p65 subunit and hypoxic conditions, HIF-1α upregulates the expression of glucose upstream kinases IKKα/IKKβ are O-GlcNAcylated. As transporters-1 and 3 (GLUT1, GLUT3), allowing the intracellular such, reducing hyper-O-GlcNAcylation decreases NF-κB uptake and phosphorylation of glucose (114–116). Subsequently, transcriptional activity and target gene expression, driving Glc-6-P enters one of several possible biosynthetic pathways, apoptosis (102). Furthermore, increasing O-GlcNAc in namely glycolysis, hexosamine biosynthetic pathway (HBP), pancreatic cancer cells protects against suspension-induced pentose phosphate pathway (PPP), or glycogen synthesis, all of apoptosis (102). Moreover, hyper-O-GlcNAcylation could which substantially regulated by HIF-1α (117–124) (Figure 2C). contribute to cancer cell survival by mitigating ER stress through Simultaneously, HIF-1α decreases O consumption and reactive the inhibition of the folding enzyme chaperone CHOP (103). oxygen species (ROS) generation within the mitochondria (125– Another important molecular mechanism relating protein 127) to circumvent oxidative stress. glycosylation to apoptosis in cancer cells results from the By regulating the flux through the HBP and PPP pathways, crosstalk between lectins and death receptors. Classically, the HIF-1α dramatically affects glycosylation, either by altering effect of Galectin-3 (Gal-3) in the regulation of apoptosis depends precursor production or by governing enzymatic activity. on its subcellular localization. Accordingly, cytoplasmic Gal- Specifically, HIF-1α has significant impact on HBP by inhibiting 3 is anti-apoptotic, whereas nuclear Gal-3 is pro-apoptotic the TCA cycle and suppressing the addition of acetyl groups, (104). Upon extracellular secretion via a non-classical pathway that would otherwise arise from that pathway, to glucosamine, (105), Gal-3 may bind to cell surface glycans, increasing cell leading to an overall reduction in the glycosylation precursor signaling and cell-matrix interactions (106, 107). Interestingly, UDP-N-Acetylglucosamine (UDP-GlcNAc) production (128– overexpression of STn results in decreased Gal-3 at the cell 130). Another branch of the HBP, the CMP-NeuAc nucleotide surface in colon cancer cells, promoting an accumulation of sugar biosynthesis pathway, is activated under hypoxia through Gal-3 in the cytoplasm and reducing chemotherapy induced the epimerization of UDP-GlcNAc by UDP-GlcNAc 2-epimerase apoptosis (108). Moreover, it has been shown that O-6-sialylation (GNE), ultimately enabling cell surface sialylation in a HIF-1α- of integrin β1 N-glycans, mediated by ST6Gal-I, completely dependent manner (131) (Figure 2C). blocked its recognition by Gal-3; conversely O-3-sialylation did Moreover, during acute hypoxia, the production of not affect Gal-3 recognition in gastric cancer (108, 109). These ATP, GTP, UTP, and CTP nucleotides through the PPP is observations suggest that Gal-3 binding to glycans is dependent decreased, compromising the addition of UDP to GlcNAc (132). on sialylation and that decoding the sialome of cancer cells may Interestingly, while hypoxia causes downregulation of the rate bring new insights on programmed cell death pathways. limiting enzyme of the PPP Glucose-6-phosphate dehydrogenase Together, these findings demonstrate that both glycosidic (G6PD) in several cancers (133), glycosylation promotes G6PD and microenvironmental cues aid tumor cells to circumvent activity and increases glucose flux through the PPP, providing apoptosis. Interestingly, the tumor microenvironment mostly precursors for nucleotide and lipid biosynthesis, and reducing provides factors to evade intrinsic apoptotic signaling, while equivalents for antioxidant defense. Particularly, G6PD is glycosylation mostly regulates the extrinsic signaling pathway dynamically O-GlcNAcylated in response to hypoxia, and initiated by binding of a death ligand to a death receptor on the blocking G6PD glycosylation reduces cancer cell proliferation cell surface. in vitro and in vivo (134), most likely through energetic unbalance. On the same note, blockage of hypoxia induced Deregulated Cellular Energetics O-GlcNAcylation at serine 529 of phosphofructokinase 1 (PFK1) The microenvironmental modulation of tumor cell energetics is reduced cancer cell proliferation in vitro and impaired tumor crucial to drive metabolic adaptation and survival of neoplastic formation in vivo (135). Of note, it has been reported that cells. As such, CAFs and endothelial cells are able to create elevated O-GlcNAcylation in cancer cells stabilizes HIF-1α in an collaborative metabolic domains by activating complementary indirect manner, thereby reinforcing the Warburg effect (103) in metabolic pathways to buffer and recycle metabolites of tumor what appears to be negative feedback loop toward homeostatic cells in order to maintain stromal and tumoral growth (110, O-GlcNAcylation levels. 111). Adipocytes also engage in this metabolic crosstalk by In addition to intracellular glucose metabolism modifications, providing fatty acids utilized by cancer cells to generate ATP decreased 1,2-fucosylation of cell-surface glycans, galectin via mitochondrial β-oxidation in metastatic ovarian cancer overexpression, and glycosyltransferases as well as glycosidases (112). Another pivotal microenvironmental feature driving modulation toward the expression of short-chain sialylated energetic adaptation is hypoxia, resulting from uncontrolled O-glycans are some consequences of the hypoxic tumor proliferation and inefficient neovascularization. Hypoxic stress microenvironment. Additionally, increased expression of within a tumor leads to a shift from aerobic oxidative gangliosides carrying N-glycolyl sialic acids can also be phosphorylation to anaerobic glycolysis, with high rates of significantly affected by hypoxia (29, 136). For all these Frontiers in Oncology | 7 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment reasons, it is possible to realize that hypoxia strongly alters In summary, cancer cells circumvent growth suppression glycobiologic events within tumors, resulting in increased by negatively regulating the two canonical suppressors of O-GlcNAcylation and sialylation; thereby leading to more proliferation p53 and RB through glycosidic modifications, while aggressive phenotypes (136–138). stromal cells and hypoxia aid tumor cell growth by abrogating the Besides regulating glycolytic enzymes in the context suppressive role of adhesion complexes and selecting for more of hypoxia, O-GlcNAcylation also governs transcription proliferative clones. factors activity (ChREBP, carbohydrate-responsive element- binding protein, Sp, and c-MYC) toward increased aerobic Genome Instability and Mutations glycolysis, anaplerotic resupply of TCA intermediates used in During uncontrolled cell division, random mutations, and biosynthesis, nucleotide metabolism and lipogenesis (139–144). chromosomal instability promote genomic alterations, which Together, these findings suggest that hyper-O-GlcNAcylation coupled with disruption of genome integrity checkpoints contributes to oncogenicity through metabolic reprograming culminate in selective advantage of tumor cells (152). In this and stabilization of oncogenic transcription factors. context, intratumoral hypoxia leads to increased mutation rates Based on these insights, hypoxia is a major driving force and altered DNA damage response, while HIF-1α interplays with of the energetic reprograming of cancer cells, largely affecting oncoproteins such as c-MYC to drive malignant progression glycosylation in a HIF-1α-dependent manner. As such, both (153–155). In addition, recent evidence shows that oxidative O-GlcNAc modifications and HIF-1α transcriptional activity stress in CAFs induces genomic instability in adjacent breast emerge as key metabolic modulators, while stromal cells promote cancer cells via mutagenic evolution, potentially increasing their a metabolic symbiosis with tumor cells envisaging tumor survival aggressive behavior (156). Together, these findings suggest that and growth. tumor progression is prompted by the orchestrated interaction of malignant cells and the TME, which promotes genetic instability toward more aggressive phenotypes. Evasion of Growth Suppressors It is known that the tumor suppressor p53 plays a central role To prevail, cancer cells not only induce and maintain stimulatory in genomic stability maintenance (157). However, stabilization growth signals but also develop the ability to evade the negative of previously mutated p53 by O-GlcNAcylation is not expected regulation of tumor suppressor genes (145). Even though tumor to lead to tumor suppression (149). Nevertheless, SILAC-based growth suppression is mostly regulated by intrinsic mechanisms quantitative proteomics of O-GlcNAc transferase wild-type and involving p53 and retinoblastoma (RB) pathways, some stromal Null cells has demonstrated the O-GlcNAcylation regulation of and microenvironmental components have been implicated the ATM (ataxia-telangiectasia mutated)-mediated DNA damage in growth arrest evasion by inhibiting adhesion complexes response pathway through ATM and its downstream targets and promoting clonal selection. Namely, proteolytic enzymes H2AX, and Chk2 (158) (Figure 2E). Other molecular studies produced by stromal cells are able to disrupt cell-cell or cell-ECM have reinforced that ATM interacts with O-GlcNAc transferase, adhesion complexes significantly contributing to uncontrolled with its activation and recovery states being affected by O- cell proliferation and progressive distortion of normal tissue GlcNAcylation (159). architecture (85, 146, 147). Moreover, tumor hypoxia selects Importantly, genetics is not the only factor contributing clones expressing mutant p53, facilitating the clonal expansion of to genetic instability. Epigenetic modifications through DNA cells that have a dominant-negative effect on the wild-type cells, methylation, posttranslational modification of histone proteins, thus evading growth suppression (148). and interactions of non-coding RNAs with proteins or other Interestingly, the two canonical suppressors of cell nucleic acids also largely drive cancer progression (160, 161). proliferation, p53 and RB, are regulated by O-GlcNAcylation Interestingly, histones H2A, H2B, and H4 are O-GlcNAcylated (149, 150) (Figure 2D). Particularly, it was demonstrated in vivo, making O-GlcNAc modifications a part of the histone that p53 O-GlcNAcylation on Ser149 limits both ubiquitin- code regulating gene transcription (162). Although no specific dependent proteasome degradation and the interaction with links between hyper-O-GlcNAcylation and cancer cell epigenetic E3 ubiquitin-protein ligase MDM2 (149). Contrariwise, contribution to transformation have been established, some overexpression of O-GlcNAcase (OGA) results in increased clonal expansions may well be triggered by these non-mutational MDM2 phosphorylation at Ser166, stimulating MDM2-p300 changes affecting the regulation of gene expression. interactions and resulting in p53 degradation (151). In turn, RB In summary, tumor microenvironmental features and stromal activity is regulated by the dynamic crosstalk between O-GlcNAc cells contribute to a mutagenic environment through the modification and phosphorylation (150). Retinoblastoma binds production of oxygen and nitrogen reactive species, while E2F-1 transcription factor preventing co-activator complexes altering transcription and translation of several DNA damage from binding E2F-1, thereby arresting cell cycle in the G1 response and repair genes. In turn, glycosylation modulates phase. Particularly, RB is densely modified with O-GlcNAc in DNA damage response pathway components and possibly non- the G1 phase, which prevents its phosphorylation and sustains mutational changes affecting the regulation of gene expression. its activity. During mid- to late-G1, a shift toward increased phosphorylation leads to the release of E2F-1 from RB and Replicative Immortality E2F-1-dependent transcriptional activation of essential S-phase The maintenance of telomerase lengths by DNA polymerase genes, allowing cell cycle progression (150). telomerase is a key event contributing to the unlimited Frontiers in Oncology | 8 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment replicative potential of cancer cells (163). Recently, hotspot hydrolysis of carbohydrate chains in proteoglycans by HYAL point mutations in the regulatory region of the telomerase hyaluronidase, also display pro-angiogenic properties in several reverse transcriptase (TERT) gene, encoding the core catalytic cancer models (179–182). Contrastingly, stromal decorin component of telomerase, was identified as a novel mechanism angiogenic role seems to be context dependent. Namely, it to activate telomerase in cancer (164, 165). Interestingly, there blocks tumor cell-mediated angiogenesis by downregulating is currently no substantive evidence of microenvironmental VEGFA production, as well as Met and downstream angiogenic contributions to telomere stabilization in cancer cells. However, networks in some tumor models (183, 184), while being required there is evidence that hypoxia up-regulates telomerase activity for efficient tube formation by EC and inflammation-induced in cancer cells via MAPK cascade signaling activation as a angiogenesis in others (185). In turn, the basal lamina lumican, stress response against hypoxia-induced genotoxicity (166). a class II small leucine-rich proteoglycan, inhibits melanoma Moreover, hypoxia induces c-MYC activation, which, in turn, angiogenesis by compromising the migratory capacity of EC transactivates TERT (167). So far, TERT has not been described and pseudotubes formation, supressing lung metastasis (84). as a glycoprotein; nevertheless, there could be an indirect link Moreover, lumican affects angiogenesis by interfering with between glycosylation and telomerase activation through c-MYC α2β1 integrin receptor activity and downregulating proteolytic O-GlcNAcylation regulation (57). As such, future studies should activity associated with surface membranes of EC (186). In investigate whether O-GlcNAc-mediated stabilization of c-MYC line with this, several studies highlight that lumican inhibits can indirectly influence telomerase activation and contribute to EC invasion, angiogenic sprouting, and vessel formation, while replicative immortality. enhancing Fas mediated EC apoptosis (187–190). Collectively, In conclusion, both glycosylation and microenvironmental these findings provide new insights into how ECM remodeling factors allow successive cell cycles mostly by circumventing regulates angiogenesis activation and resolution, as well as cell death, while having little to do with avoiding senescence identify proteoglycans as effectors modulating angiogenesis both and regulating telomere length. However, tumor hypoxia in vitro and in vivo. might contribute to immortalization by indirectly influencing Glycans and glycan-binding proteins, as galectins, add kinase cascades and transcriptions factors, while glycosylation another level of positive regulation of angiogenesis by modifications have a more modest impact in transcription modulating EC migration, branching, survival, and vascular factor regulation. permeability (191–193). For instance, a glycosylation-dependent pathway that preserves angiogenesis in response to VEGF Angiogenesis blockade was identified, in which galectin-1 (Gal-1) binds The formation of neovasculature through angiogenic processes β1-6GlcNAc branched N-glycans present on VEGFR2 in EC is vital for cancer cell proliferation and tumor progression surface to activate a VEGF-like signaling (Figure 2F). Moreover, to metastasis (168). Historically, tumor angiogenesis was vessels within anti-VEGF-sensitive tumors exhibited high levels perceived as being primarily regulated by cancer cells of α2-6-linked sialic acids, which prevented Gal-1 binding expressing proangiogenic factors; however, now it becomes and VEGFR2 activation (192). Moreover, interruption of β1- increasingly clear that the tumor microenvironment is a key 6GlcNAc branching in EC or silencing of tumor-derived Gal-1 factor inducing and sustaining chronic angiogenesis, including converted refractory tumors into anti-VEGF-sensitive (192). in a glycosylation-dependent manner. First, tumor hypoxia Importantly, this could allow pinpointing patients better served upregulates multiple pro-angiogenic pathways mediating key by anti-VEGF therapy and targeting glycosylation-dependent aspects of stromal, endothelial cell (EC) and vascular support lectin-receptor interactions envisaging increased treatment cell biology to influence neovessel patterning, maturation, and efficacy in refractory patients (194, 195). function (169). Concomitantly, stromal innate immune cells In addition, reduced O-GlcNAcylation in prostate cancer and CAFs synthesize or release through ECM remodeling cells has been associated with decreased expression of several several angiogenic soluble factors driving the expansion of angiogenic factors, such as matrix metalloproteinases MMP-2 the pre-existing vascular supply (170–174). In line with this, and MMP-9, and VEGF, resulting in inhibition of angiogenesis stromal cells-derived proteoglycans and ECM molecules are (196). Moreover, glycosydic cues as O-glucose, O-GlcNAc, and also active angiogenesis regulators (Figure 2F). For instance, O-GalNAc glycans affect Notch signaling, thereby regulating heparan sulfate (HS) proteoglycans inhibition hampers pro- angiogenesis (197). Also, α2,6-sialic acids are necessary for angiogenic signaling and neovessel formation by effecting the the cell-surface residency of platelet endothelial cell adhesion bioactivity, diffusion, half-life and interaction of VEGF with molecule (PECAM), a member of the immunoglobulin its tyrosine kinase receptors (175, 176). In ovarian cancer, superfamily that plays multiple roles in EC adhesion, mechanical HS has also been shown to impact angiogenesis through stress sensing, anti-apoptosis, and EC-mediated angiogenesis EGF receptor signaling and by influencing the expression (198). Together these finding highlight the glycosylation of angiogenic cytokines (177). Particularly, CAF-derived HS modulation of tumor angiogenesis. proteoglycan syndecan-1 expression stimulates breast tumor In summary, the tumor microenvironment ensures the supply angiogenesis, being correlated with both vessel density and of pro-angiogenic factors, while upregulating multiple pro- total vessel area (178). Furthermore, Neuropilin-1 (NRP-1) angiogenic pathways governing the maturation and survival and Neuropilin-2 (NRP-2) transmembrane proteoglycans, as of endothelial cells. In turn, glycans and glycoconjugates well as hyaluronic acid (HA) fragments resulting from the can be angiogenic per se or alter the affinity of angiogenic Frontiers in Oncology | 9 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment factor receptors for their ligands toward a pro-angiogenic tumor through electrostatic repulsion of negative charges, phenotype of EC. physically disrupting cell adhesion (215, 216). In line with this, the STn antigen reduces cell adhesion in prostate cancer Invasion and Metastasis (217), while increasing migration and invasion in bladder Throughout the course of disease, cancer cells often acquire (29), breast (218), and gastric (219, 220) carcinomas in more motile phenotypes, as well as the capability to invade a ST6GalNAc.I-dependent manner. Also, the increased and surrounding tissues and adjacent organs. Subsequently, cancer de novo expression of the STn antigen in bladder cancer cells reach lymph and blood vessels, entering circulation cells is part of an array of molecular events underlying the and eventually metastasizing to distant locations. Interestingly, establishment of mesenchymal traits (29). Moreover, STn was metastatic tumor cells may even travel from the primary site mainly found in densely O-glycosylated adhesion proteins to the secondary location with stromal components, including such as integrins and cadherins (29, 30). It is likely that activated fibroblasts, achieving a very favorable outcome in the the transition from extended to shorter and heavily sialylated colonization step of tumor progression (199). In this context, structures may impair these proteins normal function and stroma, ECM, and microenvironmental cues often facilitate induce molecular and spatial reorganization at the cell-cell and invasion and the establishment of metastatic colonies by tumor cell-matrix interfaces. In agreement with these observations, cells. For instance, tumor hypoxia aids migration and invasion STn expressing cells are frequently simultaneously observed in of tumor cells by influencing angiogenesis, immune tolerance, invasion fronts, near blood vessels and corresponding lymph epithelial-to-mesenchymal transition (EMT), and regulating nodes, as well as in distant metastasis (70, 221). Moreover, adhesion molecules expression and glycosylation (200). At a it has been recently reported that most circulating tumor distance, hypoxia contributes to the production of diffusible cells (CTC) in the blood of metastatic bladder cancer patients factors and exosomes involved in premetastatic niche formation, present a highly undifferentiated and more aggressive basal while regulating metabolic and survival pathways that allow phenotype, while overexpressing the STn antigen (221). As such, cells to adapt to distant microenvironments (201). Within the STn expression seems to confer a competitive advantage to tumor stroma, infiltrating immune cells and CAFs promote ECM neoplastic bladder cells by enabling not only invasion but also remodeling while producing pro-invasive and EMT promoting the necessary mechanisms for successful cancer dissemination. factors (172, 202–204). Namely, the CAF-derived proteoglycans Similarly, ST6Gal.I-mediated α2,6-sialylation of breast cancer versican and serglycin promote tumor invasion and metastasis cells mediates reduced cell-cell adhesion and enhanced invasion in breast, ovarian, and prostate cancer (47, 205, 206), as well as capacity (222). Overall, immature truncated O-glycophenotype NSCLC cells EMT, migration, invasion and liver colonization, of cancer cells directly induces oncogenic features, including respectively (83). Similarly, the ECM hyaluronic acid (HA) and enhanced migration and invasive capacity (223). biglycan are directly involved in the metastatic potential of Reinforcing the key role played by sialic acids in cell- breast and prostate tumor cells (207, 208) as well as melanoma cell adhesion, sialylated α3β1 integrin, displaying numerous cells (209), respectively. Moreover, metastatic tumor cells must sialylated tetra-antennary complex type glycans, exhibited acquire the capability to autonomously synthesize, assemble, significantly lower fibronectin-binding capability than its and process their own “portable” HA-rich microenvironments unsialylated counterpart and showed migration ability to survive in circulation, metastasize to ectopic sites, and through fibronectin in vitro (224). Apart from integrins, E- escape therapeutic intervention. As such, strategies to disrupt cadherin aberrant glycosylation highly affects its function and the HA machinery of primary tumor and circulating tumor cellular localization, frequently culminating in epithelial cells may enhance the effectiveness of current conventional cell invasion in gastric cancer (225, 226). Namely, N- and targeted therapies (210, 211). On the other hand, triple- acetylglucosaminyltransferase III (GnT-III, MGAT3) and negative orthotopic breast carcinoma systemic treatment with the N-acetylglucosaminyltransferase V (GnT-V, MGAT5) proteoglycan decorin induced the tumor suppressor cell adhesion competitively modify E-cadherin N-glycans, adding bisecting molecule 1 (Cadm1), favoring a less metastatic phenotype GlcNAc structures and β1,6-GlcNAc branches, respectively. (212, 213). Altogether, these findings highlight stromal-derived Wild-type E-cadherin positively regulates the metastasis proteoglycans as major players driving the metastatic potential suppressor MGAT3 gene, resulting in increased GnT-III of tumor cells. Concomitantly, in vitro studies suggested expression and bisecting GlcNAc N-glycans addition to the that stromal derived TGFβ-induced EMT alters glycogenes plasma membrane-bound protein (225). Conversely, the addition expression and consequently promotes N-glycan remodeling, of β1,6-GlcNAc branches by GnT-V, specially at Asn-554, drives including decreased bi-, tri- and tetra-antennary complex N- E-cadherin translocation to the cytoplasm, alters cis-dimer glycans and increased expression of hybrid-type N-glycans formation and molecular assembly, and drives instability of and fucosylation (214); thereby showing a correlation between the adherens junctions. Furthermore, preventing Asn-554 microenvironmental soluble factors and glycosylation changes. N-glycosylation, either by a mutation or by silencing GnT-V, In line with glycoconjugate regulation of invasion and resulted in a protective effect on E-cadherin, precluding its metastasis, glycans add another dimension of regulation to functional dysregulation and contributing to tumor suppression the acquisition of this cancer hallmark. Namely, it has been (226, 227). Another study demonstrated a novel pathway of proposed that increased sialylation, accompanying malignant GnT-V-mediated metastasis via the addition of β1,6-GlcNAc transformation, promotes cell detachment from the primary branches to matriptase, thereby stabilizing it and activating Frontiers in Oncology | 10 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment invasion effectors as urokinase-type plasminogen activator Glycome alterations also decisively contribute to the and hepatocyte growth factor (HGF) (228). Overall, these establishment and maintenance of tumor-promoting findings suggest that aberrant N-linked β1,6- GlcNAc branching inflammation. Namely, E-, P-, and L-Selectins interactions a/x occurring during oncogenesis can lessen cell-cell adhesion, with SLe not only control the establishment of metastatic contributing to increased cellular motility and invasiveness cancer cells colonies but also the recruitment of circulating (Figure 2G). However, some glycosydic modifications can lymphocytes into peripheral lymph nodes and inflamed tissues promote tumor cell adhesion and still favor tumor progression. (238, 252, 253) (Figure 2H). Moreover, several inflammatory a/x For instance, tumor cells also overexpress SLe antigens, mediators are regulated by its glycosylation state. Namely, NF-κB which are specific ligands for E- and P-selectins upregulated is activated by O-GlcNAcylation at Ser350 of its c-Rel subunit a/x in activated endothelial cells. Selectins and SLe interactions (254), while the proinflammatory cytokine Cyclooxygenase-2 are key regulators of the metastatic cascade by promoting the (COX-2) turnover depends on Asn570 glycosylation, negatively recruitment of malignant cells to vessels, rolling of tumor cells on affecting the efficacy of certain COX-2 inhibitors (255, 256). the endothelial surface, and arrest of CTCs in distant locations Furthermore, recent studies have described that non-human N- (229–231) (Figure 2G). Besides the establishment of metastatic glycolyl-neuraminic acid (Neu5Gc) can be incorporated into cell colonies, these ligands also mediate tumor growth, invasion, surface glycans instead of N-acetyl-neuraminic acid (Neu5Ac), angiogenesis, and inflammation in numerous other tumor types leading to autoimmune systemic inflammation associated with (232–236). In addition, slightly altered forms of these antigens cancer initiation and progression (257–259). also have important biological features. Namely, the addition Importantly, in the same way glycans govern inflammation, of a sulfate group at the sixth position of GlcNAc generates the inflammatory tumor microenvironment is also able to induce 6-sulfo-sLe , which is considered the physiologic ligand for changes in tumor cells glycosylation. For instance, pancreatic L-selectin (237) but also E-selectin in bladder cancer (238). and gastric carcinomas are characterized by an abundant Herein, it has a dual role by promoting tumor cell adhesion to stroma containing several pro-inflammatory cytokines, as vascular endothelial cells, while favoring lymphocyte recruitment IL-1β and IL-6, which regulate the expression of biosynthetic to enhance anti-tumor immune responses (238). In agreement glycosyltransferases to increase the expression sialylated antigens x a/x with these observations, Le -positive cell lines from invasive as SLe (260, 261). Furthermore, the extracellular matrix bladder tumors with metastatic potential show high levels of proteoglycan versican has been shown to promote bladder alpha1,3-fucosyltransferase VI (FT-VI) and FT-VII, two enzymes cancer-derived lung metastasis through enhanced tumor cell involved in SLe synthesis, and display E-selectin dependent migration and creation of an inflammatory environment adhesion (232). involving macrophages and pro-tumor CCL2/CCR2 signaling Glycosyltransferases may also play a key role in mediating axis (262, 263), providing another the involvement of cancer cell metastization. Namely, the sialyltransferase glycoconjugates in macrophage-mediated inflammation. ST6GalNAcII was identified as a novel metastasis suppressor, These findings highlight the relevance of tumor stromal while ST6GalNAcV and N-Acetylgalactosaminyltransferase cells, glycans, and glycoconjugates as mediators of tumor- GalNT9 identify metastatic potential in breast cancer (239–241). promoting inflammation by providing pro-inflammatory factors In summary, cancer-associated glycosylation changes and allowing the recruitment of circulating lymphocytes into and stromal cells aid tumor cell invasion, distant organ tumor sites. colonization, and metastasis by supplying pro-metastatic factors, compromising vasculature integrity and the stromal barrier to Immune Escape tumor cell migration, promoting EMT and by tethering tumor Several stromal components of the tumor microenvironment cells to improve colonization at distant sites. Concomitantly, the aid tumor cell immune scape, either by recruiting highly regulated balance between loss of adhesive properties and immunosuppressive immune cells or by driving the acquisition the ability to anchor at metastatic sites defines the metastatic of tolerogenic phenotypes. In this context, tumor-infiltrating potential of tumor cells. immune cells frequently develop immunosuppressive activities, differentiating into regulatory T cells (Tregs), immature Tumor-Promoting Inflammation monocytes, and alternatively activated macrophages, mast cells, Tumor-associated stromal cells have been found to secrete a neutrophils, dendritic cells (DC), and T helper 2 (TH2)-CD4 variety of pro-inflammatory cytokines, chemokines and matrix- T cells, all of which producing a multitude of factors aiding remodeling enzymes favoring the establishment of immune cell tumor growth and survival (264). Specifically, endothelial cells infiltrates (242, 243). Particularly, CAFs and mature adipocytes lining the tumor vasculature can suppress T cell activity, target promote sustained inflammation by producing large amounts them for destruction, and block them from entering the tumor of pro-inflammatory IL-6, IL-1β, TNF-alpha, and CXCL1 to through the deregulation of adhesion molecules (265). Moreover, drive chemoattraction of monocytic immune cells (244), while the CAF secretome can also shape T cell-dependent antitumor favoring tumor growth and metastasis (245–250). Another immune responses by negatively affecting DCs, myeloid-derived pivotal microenvironmental factor driving cancer-associated suppressor cells, TH17, and CD8 T cells functions. Activated inflammation is hypoxia, which is essential for granulocytes and fibroblasts can also drive the switch of CD4 T lymphocytes monocytes/macrophages infiltration and activation in vivo in a from a TH1 to a TH2 phenotype, while expressing some HIF-1α-dependent manner (251). ligands of immune checkpoint receptors (266). CAF-derived Frontiers in Oncology | 11 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment proteoglycans, as decorin, further suppress immunomodulatory (277) (Figure 2I). Consistent with this tolerogenic profile, T genes in triple-negative orthotopic breast carcinoma xenografts, cells primed by DCs pulsed with STn-expressing glycoproteins including Siglec (Sialic acid binding Ig-like lectin), Lipg (IFNγ displayed a FoxP3(high) IFN-γ(low) phenotype and little inducible GTPase), and Il1b (Interleukin 1β) (213). These capacity to trigger protective anti-tumor T cell responses findings suggest that targeting CAFs or their secretome may (277). More importantly, blocking STn-MUC1 and CD44 probably reduce immune effector cell dysfunctions as well glycoforms partially reverted DC maturation, suggesting that as decrease the recruitment of immunosuppressive cells. targeting STn-expressing glycoproteins may allow circumventing Other ECM molecules, as HA, are known to determine the tumor-induced tolerogenic mechanisms. Similarly, sialylation trafficking of tumor-associated macrophages (TAM) through of the T antigen in MUC1 on breast cancer cells creates the tumor stromal areas. In line with this, HA deficiency in tumor MUC1–ST antigen which engages Singlec-9 on tumor-associated stroma impairs not only macrophage trafficking but also tumor macrophages to initiate inhibitory immune signaling through the angiogenesis and lymphangiogenesis, ultimately compromising activation of the MAPK/ERK pathway (278). In line with this, immune cells access to tumor sites and aiding immune scape sialylated ligands of singlec-7 and−9 are expressed on cancer (267). Furthermore, recent studies in myeloma tumors have cells of different histological types and interactions between these demonstrated the immunomodulatory roles of the ECM lectin receptors and its ligands influence NK cell-dependent proteoglycan versican proteolytic processing. In this context, tumor immunosurveillance (279). Moreover, hypersialylation the interplay between stromal cells and myeloid cells generates of tumor ligands for NKG2D receptors, expressed by NK versikine, a novel bioactive damage-associated molecular pattern cells, NK1.1+ T cells, γδ T cells, activated CD8 αβ T cells that may facilitate immune sensing of myeloma tumors and and macrophages, is thought to repulse their interaction via modulate the tolerogenic consequences of intact versican highly negative charge repulsions, hampering immune response accumulation (268). (280, 281). Tumor-derived sialoglycans also inhibit CD8+ T As described in previous sections, advanced stage tumors cell cytotoxicity by interfering with lytic granule trafficking are frequently characterized by profound deregulations in and exocytosis in response to TCR engagement (282). Thus, glycosylation pathways, resulting in the presentation of aberrant hypersialylation often observed on tumor cells may ultimately structures at the cell surface. Importantly, these structures only be amongst the mechanisms by which tumors evade immune render cancer cells mildly antigenic and rarely immunogenic system recognition (30, 70, 216, 283). Also, C2GnT-expressing (269). This may occur because most cancer-associated structures bladder tumor cells express heavily core 2 O-glycosylated MUC1 have an embryonic origin or are mildly expressed in healthy which interacts with Gal-3 to attenuate the interaction of tumor tissues, allowing them to be perceived as “self” by immune system cells with NK cells, allowing tumor cells to survive longer in host effector cells (270). Furthermore, specialized B lymphocytes blood circulation and potentially metastasize (284). Given these producing high-affinity antibodies against these structures might insights, sialylated and fucosylated antigens contribute to create even be eliminated during development (271). However, glycans an immunosuppressive microenvironment toward tumor cell play a key role in the regulation of various aspects of immune escape. Furthermore, the structure and function of well- immune response, ultimately enabling immune suppression by known immune checkpoint molecules as PD-L1 can be stabilized interacting with lectin receptors in immune cells. For instance, by N-glycosylation, reducing its proteasomal degradation and fucosylated blood group related Lewis antigens interact with C- consequently enhancing its immunosuppressive activity over T- type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing cells (285). These findings highlight the disseminated role of non-integrin; also known as CD209) on macrophages and DC glucans and glycoconjugates in tumor cell immune scape. to upregulate the anti-inflammatory cytokines IL-10 and IL- In summary, the tumor microenvironment increasingly 27. This ultimately induces TH2, T follicular helper (TFH) or becomes more immunosuppressive, resulting in tumor cell Treg cells, highlighting the immune suppressive nature of Lewis survival and metastasis. Concomitantly, tumor cells glycosylation antigens (272, 273). Similarly to fucosylation, enhanced tumor promotes immune scape by being simple and “self”-like, by sialylation often culminates in immune suppression and anti- inducing tolerogenic immune cell phenotypes, and by effectively inflammatory microenvironments. Accordingly, the presence of shielding tumor cells from effector immune cells, culminating in sialylated structures on melanoma cells impedes T cell mediated tumor progression. anti-tumor responses while promoting tumor-associated Treg cells and decreased NK cell activity (274) (Figure 2I). Moreover, sialoglycans interact with sialic acid-binding immunoglobulin- SIGNIFICANCE OF GLYCOSIGNATURES like lectins (SIGLECs) to induce an antigen-specific tolerogenic FOR PERSONALIZED MEDICINE programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells (275). For instance, The previous sections have highlighted that changes in glycans macrophage associated Siglec-15 preferentially binds the STn and glycoconjugates drive several biological processes in tumor antigen in myeloid tumor cells, resulting in increased TGF- cells, culminating in the acquisition of cancer hallmarks and β secretion into the tumor microenvironment and tumor increasingly aggressive disease. Glycosylation changes reflect not progression (276). Moreover, in bladder cancer, STn expression only the genomic, transcriptomic, proteomic and metabolomic has led to impaired DC maturation while significantly reducing state of cells but also its external microenvironment, making the production of Th1-inducing cytokines IL-12 and TNF-α glycosignatures highly context-specific and attractive targets for Frontiers in Oncology | 12 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment personalized medicine affecting tumor and stromal cells. At a addressing conventionally accepted glycan-biomarkers and systemic level, glycosignatures provide a global reflection on an involving small and often biased patient cohorts. Heterogeneous individual’s health/disease status and can function as predictive protocols, including different sample processing and detection indicators for treatment success. In this context, several methods, as well as the lack of endpoint standardization have serological markers have emerged, with several FDA-approved also constituted major drawbacks. Moreover, most studies cancer glycobiomarkers currently used in clinical practice fail to provide complementary functional assays capable of recently revised by kirwan et al. (286). To circumvent relatively pinpointing clinically relevant glycobiomarkers. These aspects low specificity and sensitivity issues, more comprehensive are often further aggravated by the lack of untargeted approaches approaches propose combinations of glycobiomarkers achieving capable of broadening our understanding on the glycome remarkable sensitivity and specificity values (287). Another and glycoproteome. Moreover, few efforts were undertaken strategy to improve specificity consists in narrowing the cancer to incorporate glycans in broad biomarker panels of different cell proteome to clinically relevant glycoforms. Showcasing molecular natures, envisaging highly sensitive and specific this aspect, a recent targeted investigation of the bladder detection methods. Facing these challenges, significant efforts are cancer glycoproteome highlighted that specific MUC16 ongoing to standardize glycomics and glycoproteomics protocols glycoforms (CA125 antigen) could be used to define subsets and implement robust high-throughput mass spectrometry- of chemoresistant patients, whereas no associations could based glycoanalytical platforms (290, 291). As such, it is now be found based solely on the presence of the protein (30). possible to extract significant structural information from Moreover, the field of liquid biopsies is rapidly evolving from minute amounts of clinical samples (nanomolar-fentomolar classical approaches, focusing on a single or few protein range), including from challenging starting materials such biomarkers, toward multiplex settings that will likely improve as formalin-fixed paraffin-embedded (FFPE) tissues available on these preliminary findings (Figure 3). The detection of in many hospital archives (30, 292), which will enable large minor amounts of circulating tumor nucleic acids, exosomes, scale retrospective analysis of well characterized clinical circulating tumor cells (CTC) and stromal components, which samples. Moreover, advances in MALDI Imaging Mass decisively contribute to the pre-metastatic and metastatic Spectrometry has allowed obtaining structural information niches, will pave the way for improving the management of from glycans with significant spatial resolution (293). Important advanced stage patients. In this context, deeper insights on their bioinformatics tools and databases are already available and molecular nature may provide the necessary means for real-time novel improvements are emerging for supporting glycans and disease monitoring and early intervention, guiding therapeutic glycopeptide mass spectrometry data interpretation, which decision and, more importantly, designing novel therapeutics is a critical matter facing big datasets (294). Altogether, the (Figure 3). Accordingly, explorative studies have demonstrated technological set-up and structural knowledge envisaging the that exosomes, responsible by pre-metastatic signaling, present engagement in multicenter randomized glycan-based trials have distinct glycosylation patterns (288, 289). Furthermore, pioneer been overcome; nevertheless, a more ambitious focus should be work using a recently developed microfluidics device has set on integrative panomics applications (295). This knowledge demonstrated that over 90% of bladder cancer CTC yield the will foster the development of glycan-based therapeutic STn antigen (221). More importantly, the STn antigen was not strategies and novel immunotherapeutics, including inhibitors detected in blood cells from healthy individuals, reinforcing of glycosyltransferases catalytic activity (296) and theragnostic its cancer-associated nature. Downstream molecular analysis antibodies against cancer-specific glycoepitopes. The later confirmed the basal nature of STn-positive CTC in molecular should be capable of inducing antibody-dependent cellular mimicry of the primary tumor and corresponding metastasis cytotoxicity and/or overcoming the immunotolerance generated (221). Therefore, the STn may allow targeting bladder CTC, by cancer-associated glycoconjugates and microenvironmental which has been a challenging enterprise given the scarce cues (297). Moreover, glycan-based antibodies may be used knowledge about their molecular nature. to guide emerging nanotherapies (298, 299) or serve has Despite these promising advances, current diagnostic basis for developing genetically modified T cells expressing strategies are based on measuring protein marker concentrations, chimeric antigen receptors (CAR-T) (300), while allowing cancer disregarding its glycosylation status, even though it might detection and identification of patients better-served by these provide key information to improve diagnosis and stratify therapies. In addition, blocking tumor-associated glycan–lectin patients. This might be due to the lack of user-friendly tools interactions could prevent the activation of inhibitory immune allowing health care technicians to obtain this information in receptors toward more efficient immunotherapies. Regarding sufficient specificity and sensitivity within the standard capacities personalized immunotherapies, in recent years, the targeting of of a clinical laboratory. Moreover, the glyco-heterogeneity of DCs has emerged as an interesting approach for the induction protein markers, arising from multiple glycosylation sites and of antitumor immunity. Namely, glycopeptides targeting DC- glycosylation patterns, might further hamper selectivity. As such, SIGN in DCs are easily internalized and cross-presented to + + the profound knowledge of cancer-specific glycan signatures stimulate tumor-specific CD4 and CD8 T cell responses. and glycosites, as well as its status within a healthy population Finally, anticancer multicomponent glycoconjugate vaccines, represent the first crucial steps toward including glycosylation in based on glycan antigens coupled to T-cell peptide epitopes or the diagnostic process. From the bench side, current glycobiology immunostimulant epitopes, have been demonstrated effective in rationale is mostly built on immunoaffinity-based studies circumventing cancer immunotolerance (301, 302), providing Frontiers in Oncology | 13 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment FIGURE 3 | Glycan-based therapeutic strategies. Successful clinical implementation of glycan-based therapeutic strategies could include inhibitors of glycosyltransferases catalytic activity, as well as theragnostic antibodies against cancer glycoepitopes capable of cancer detection, antibody-dependent cellular cytotoxicity induction, and abrogation of immunotolerance generated by cancer-associated glycoconjugates. Moreover, glycan-based antibodies may be used to guide emerging nanotherapies or serve has basis for developing genetically modified T cells expressing chimeric antigen receptors (CART-T). In addition, + + glycopeptides can be used for in vivo targeting of dendritic cells (DCs) to induce tumor-specific CD4 and CD8 T cells. Finally, glycan antigens coupled to T-cell peptide epitopes or immunostimulant epitopes can form fully synthetic multicomponent glycoconjugate vaccines able to circumvent cancer immunotolerance. an appealing option for the much-awaited development of new is sustained by stromal cells that supply mitogenic factors, glycan-based therapeutic agents. while glycosylation promotes growth factor receptor activation In summary, analytical hurdles related with sample and positively regulates intracellular kinases pathways. Besides preparation, data acquisition and automated analysis that sustained growth, tumor cells must circumvent programmed can also be handled by non-glycobiologists represent key steps to cell death to ensure cancer progression. Envisaging this, overcome to introduce glycomics and glycoproteomics as routine stromal cells and ECM remodeling provide diffusible paracrine clinical parameters. To achieve this goal, the development of new survival factors and non-diffusible survival signals, while and clinic-friendly techniques, as well as glycobiology-focused offering a physical barrier against pro-apoptotic factors such as bioinformatics tools open new avenues to predict the tumor chemotherapy. In line with this, glycosylation determines the glyco-code. In addition, stratification and large-scale validation sensitivity of death receptors to their ligands and drives the of potential diagnostic targets will also be indispensable initiation of pro-survival cascades, while altering transcription to successfully translate promising research results into solid factor activity. Concomitantly, sustained proliferation and clinical tests. In a distant future, an inclusive approach combining programmed cell death evasion culminate in highly energy the increasing amount of glycomics and glycoproteomics data demanding tumors that establish symbiotic relationships with with patient’s genomics, transcriptomics, proteomics, and stromal cells that activate complementary metabolic pathways metabolomics will have a major impact on the unraveling of to buffer and recycle tumor-derived metabolites. Moreover, to novel targets and strategies for early diagnosis, prognosis, patient sustain growth and survival in the face of hypoxia, HIF-1α stratification and improved cancer management. strongly regulates glucose metabolism throughout the several biosynthesis pathways, culminating in altered glycosylation precursor expression as well as increased sialylation and O- CONCLUDING REMARKS GlcNAcylation toward more aggressive clones. Simultaneously, tumor cells evade growth suppression by abrogating the As thoroughly described in the previous sections, tumor stromal suppressive role of adhesion complexes with the ECM, mostly by cells and ECM components have a preliminary regulatory role in the action of stromal-derived proteolytic enzymes. At the same the acquisition of hallmark capabilities, mostly by supplying the time, the two canonical suppressors of proliferation p53 and RB soluble factors that drive adaptation or shielding tumor cells from are negatively regulated through O-GlcNAc modifications. All external stress. Glycosylation ads a second level of regulation by the above-mentioned events are largely driven by the genomic governing structural alterations in major receptors, by modifying instability of cancer cells, culminating in advantageous random soluble factors and/or by modulating intracellular kinase mutations. This variability thrives much as a consequence cascades (Figure 4). Showcasing this, proliferative signaling Frontiers in Oncology | 14 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment FIGURE 4 | Transversal nature of glycans, glycoproteins, glycan-binding proteins, and proteoglycans throughout the 10 currently accepted cancer hallmarks. Aberrantly expressed glycans (O-GlcNAc, Le, SLe, Neu5Gc, Sialic acids (Sia), fucose residues (Fuc), ST, STn), glycoproteins (integrin α3β1, VEGFR2), and proteoglycans (Perlecan, Decorin, Neuropilin-1,-2, Syndecan-1,−2, Hyaluronic acid fragments (HA), Versican, Lumican, Serglycin) are mechanistically involved in cancer hallmarks acquisition. The illustration highlights the most common glycosylation modifications throughout the cancer hallmarks, transmitting the empiric notion that a great number of glycosylation aberrations mostly contribute to disease dissemination through increased angiogenesis and potentiation of invasion and metastasis. Moreover, the post-translational modification β-O-N-acetyl-d-glucosamine (O-GlcNAc) emerges as a key regulator of cellular activities through the modulation of signal transduction and protein stabilization. In conclusion, glycans and glycoconjugates are not bystanders to malignant transformation but major players, making then attractive targets to drive molecular-based clinical intervention. of the DNA damage promoted by the mutagenic/oxidative microenvironment and glycosylation have little to do with the microenvironment indorsed by stromal cells. Also, hypoxia replicative immortality of tumor cells, their contribution is alters the transcription and translation of several DNA damage mainly based on the indirect regulation of the transcription response and repair genes. In turn, glycosylation modulates factor c-MYC and kinase cascades. Importantly, to sustain DNA damage response pathway components, reinforcing the proliferation and the energetic demands of ever-growing tumors, genomic instability of tumor cells. Interestingly, both the tumor a pro-angiogenic environment must be established. As such, to Frontiers in Oncology | 15 May 2019 | Volume 9 | Article 380 Peixoto et al. Glycans and the Tumor Microenvironment ensure neovascularization, stromal cells supply pro-angiogenic contribution to key aspects of neoplastic transformation as the factors and upregulate multiple angiogenic pathways culminating acquisition of genomic instability and replicative immortality, in the maturation and survival of endothelial cells. In turn, opening an avenue for novel research (Figure 4). The sweet angiogenic glycans and glycoconjugates alter the affinity of side to this sour end resides on the possibility of exploring angiogenic factor receptors for their ligands toward a pro- the extracellular nature of glycans for targeting tumor and angiogenic phenotype of EC. Advanced stage tumors frequently stromal cells using more effective non-invasive tools. As such, we progress to invasion and metastasis, which is facilitated by intend to reinforce the need to concentrate efforts to incorporate the compromised vascular and stromal barriers to tumor cell glycans in broad biomarker panels of different molecular natures, migration. Moreover, stromal cells can promote EMT in tumor envisaging highly sensitive and specific detection methods for cells and tether these cells to improve colonization at distant sites. disease monitoring and early intervention. Moreover, by Concomitantly, glycosylation changes in tumor cells physically integrating microenvironmental information, glycosignatures disrupt cell adhesion by upregulating sialylated antigens and N- will most likely provide the necessary key for designing highly linked β1,6-GlcNAc branches, contributing to increased cellular specific cancer ligands envisaging theragnostic applications; motility and invasiveness. On the other hand, glycosylation can thereby allowing guiding therapeutic decision and, more promote adhesion of tumor cells and still favor the establishment importantly, designing novel therapeutics. Notwithstanding, a/x of metastatic colonies. Namely, tumor cells overexpressing SLe significant room lays beyond targeted approaches, specially are able to roll on the endothelial surface and extravasate into facing the recent advances in glycomics and glycoproteomics. circulation, while arresting its movement in distant locations Therefore, it is now possible to engage on a comprehensive by interacting with selectins expressed by endothelial cells. study of the glycome and glycoproteome envisaging the Some glycosyltransferases expression also defines the metastatic necessary glycobiology landscape for intervention. Of note, potential of tumor cells, acting as metastasis suppressors selectin and galectin antagonists, including glycomimetic or enablers. compounds, antibodies, aptamers, and peptides are currently In the meantime, tumor-associated stromal cells contribute in FDA clinical trials and near-clinical trials for the treatment to tumor-promoting inflammation by supplying several pro- of blood-related cancers and solid tumors metastasis (303). inflammatory cytokines and chemokines, ultimately driving Moreover, the high sensitivity and resolution of new generation tumor growth, neovascularization, immune cell recruitment, mass-spectrometers will allow obtaining structural information and glycosyltransferases expression. Furthermore, glycosylation almost to a single-cell level, enabling the analysis of exosomes, changes not only contribute to the recruitment of circulating CTC, and stromal components, which will be crucial for lymphocytes into peripheral lymph nodes and inflamed tissues addressing metastatic disease. Overall, we believe that the but also regulate the activity of several inflammatory mediators necessary context has been created to foster more in-depth and the polarization of immune cells into immunosuppressor studies on the glycobiology of tumors and its microenvironment phenotypes. In line with this, the tumor microenvironment envisaging molecular-based precision medicine and improved increasingly becomes populated with immunosuppressive patient care. immune cells. Concomitantly, tumor cells glycosylation, mostly characterized by hypersialylation, promotes immune scape by AUTHOR CONTRIBUTIONS being simple and “self”-like, by inducing tolerogenic immune cell phenotypes, and by effectively shielding tumor cells from AP and JF wrote the manuscript. AP, MR-S, and RA produced the effector immune cells, culminating in tumor progression. artwork. MR-S, RA, and LS revised it. Based on these insights, glycosylation changes reflect not only the genomic, transcriptomic, proteomic, and metabolomic state of cells but also its external microenvironment, making ACKNOWLEDGMENTS glycosignatures highly context-specific and attractive targets The authors wish to acknowledge the Portuguese Foundation for personalized medicine. Several evidences support the existence of a unique repertoire of glycans associated with for Science and Technology (FCT) for the human resources grants: PhD grant SFRH/BD/111242/2015 (AP), and FCT disease progression and dissemination, decisively reflecting on virtually all cancer hallmarks (Figure 4). Changes in O- auxiliary researcher grant CEECIND/03186/2017 (JF). FCT is co-financed by European Social Fund (ESF) under Human GlcNAcylation is the most common glycosylation modification throughout cancer hallmarks, providing a dynamic but Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge highly regulated sensor driving protein stabilization and signal transduction. Sialic acids and, particularly sialylated the Portuguese Oncology Institute of Porto Research Centre (CI-IPOP-29-2014; CI-IPOP-58-2015), the PhD Program in short-chain O-glycans are also amongst the most common structures driving invasion and immune escape, clearly marking Biomedical Sciences of ICBAS-University of Porto, and the more aggressive tumor cell phenotypes. Moreover, the major Early stage cancer treatment, driven by context of molecular imaging (ESTIMA) framework (NORTE-01-0145-FEDER- bulk of glycosylation modifications accompanying malignant transformation seem to contribute to disease dissemination 000027). The authors were also supported by the CANCER project (NORTE-01-0145-FEDER-000029) co-funded through through increased angiogenesis and potentiation of invasion and metastasis. Notwithstanding, little is known about glycosylation the NORTE-45-2015-02. Frontiers in Oncology | 16 May 2019 | Volume 9 | Article 380 Peixoto et al. 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Protein Glycosylation and Tumor Microenvironment Alterations Driving Cancer Hallmarks

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