Altered glycosylation in cancer: A promising target for biomarkers and therapeutics.

Decades of research on carbohydrates helped scientists develop glycan-based therapeutics, a promising approach in the treatment modalities for cancer. It is believed that the introduction of glycans themselves as therapeutics in the clinic is a significant breakthrough in the fight against cancer. Several recent reviews are available on the topic of glycan based therapeutics against cancer. Recently, Ferreira et al. have discussed glycan-based therapeutic opportunities in detail with particular reference to immunotherapy, antibody-based therapy, glycan mimetics, and nanovehicles [ 223 ]. This section briefly discusses some of the recent glycan-based therapeutics, including vaccine design, development of glycoconjugate drugs, glycan-based targeted nanotherapies, and glycosylation inhibitors that have clinical relevance. During cancer development, the characteristic change in glycosylation machinery pertains to an increase in the branched heavily glycosylated structures often accompanied by the differential glycosylation of mucin core proteins as MUC1 and MUC16 [ 224 ]. Tumor-associated carbohydrate antigens (TACA) have been considered a principal target for cancer vaccination development. The covalent linking of carbohydrates to an immunologically active protein exceptionally enhance their immunogenicity. Since the linker molecule that is used to couple carbohydrate to protein can impact the immunological properties, it is essential to use immunologically inactive linkers for the synthesis of the conjugate [ 225 ]. Based on the number of TACA used to synthesize a conjugate, glycoconjugate vaccines can be classified as mono-epitopic vaccines, mono-epitopic cluster vaccines, multi-epitopic vaccines containing single type TACA, cluster of one type of TACA, and multiple kinds of TACA respectively. Among these, mono-epitopic vaccines are highly explored and extensively used in clinical trials [ 226 ]. The synthesis of the Globo H hexasaccharide vaccine conjugated to keyhole limpet hemocyanin for prostate cancer patients is considered a revolutionary move in developing glycan-based cancer therapeutics [ 227 , 228 ]. The synthetic Globo H-keyhole limpet hemocyanin (KLH) conjugates plus the immunologic adjuvant QS-21 was found as a well-tolerated vaccine for breast cancer patients in phase 1 clinical trial [ 229 ]. A phase 1 clinical trial of Thomsen-Friedenreich (TF)-KLH-QS21 vaccine showed a promising effect with higher titers of IgM and IgG antibodies in patients with relapsed prostate cancer [ 230 ]. Since multiple TACA is associated with tumor progression, scientists have tried the possibilities of designing multi-epitopic cancer vaccines to target different cell populations. Therefore, the same research group has later modified the vaccine containing five different carbohydrate antigens, Globo-H, TF, GM2, TF, STn, and Tn-to maleimide-modified carrier protein KLH that are specifically associated with prostate and breast cancer progression [ 231 ]. In a parallel study, another research group has synthesized a three-component vaccine composed of toll-like receptor 2 (TLR2) agonist, T-helper epitope, and a tumor-associated glycopeptide, which has been observed to elicit high titers of IgG antibodies that specifically recognize TACA [ 232 ]. Interestingly, the Sialyl-Tn antigen, a carbohydrate associated with MUC1, was identified as an ideal candidate to incorporate in the vaccine, as it is not expressed on healthy tissues. The vaccine synthesized by coupling synthetic STn antigen to KLH protein has successfully reached phase 3 clinical trial for breast cancer [ 233 ]. However, unfortunately, none of the vaccines has met the success rate of preventing disease progression and overall survival in clinical trials. Studies demonstrated that semi-synthetic conjugate vaccines hold some inherent drawbacks, such as heterogenicity and imprecise structures acquired by carbohydrate-protein conjugation methods, resulting in conjugates with the difference in physical, chemical and immunological properties which may affect the reproducibility of the immune response. Therefore, researchers have focused on fully-synthetic conjugate vaccines that possess well-defined structures with no immunosuppression that enhance the immunogenicity of vaccines. Toyokuni and colleagues [ 234 ] have reported a synthetic carbohydrate with a robust immune response without using a carrier protein or linker molecule for the first time. Then, a glycopeptide containing a cluster of three Le(y)-serine epitopes were found as superior in action to the one containing a single Le(y) epitope [ 235 ]. Several synthetic carbohydrate vaccines have been developed with minimal structural features and useful T cell-dependent immune response. A synthetic vaccine using Tn antigen, YAF peptide, and lipopeptide Pam3Cys has been synthesized and found was effective in immune response for cancer treatment [ 236 ]. Another study provided evidence that covalent coupling of a TLR2 agonist, peptide T-helper epitope, and a TCGA gives a compound that exhibits high titers of IgG antibodies against breast cancer [ 237 ]. A MUC1 glycopeptide and Pam(3) Cys lipopeptide prepared by solid-phase synthesis provides an oligovalent synthetic anti-cancer vaccine with a more immunogenic response by click chemistry [ 238 ]. Several other synthetic glycan-based vaccines were also highly effective with a more immunogenic response to kill cancer cells [ 239 , 240 , 241 , 242 , 243 ]. Immunization with several of these synthetic vaccines resulted in a reduction of tumor size in vivo . However, more efforts are still warranted to analyze its clinical relevance. Chemotherapeutic drugs face significant challenges, including increased toxicity and poor selectivity, to better clinical outcomes. Several efforts have been made to overcome this, and one such method is the glycoconjugation of drugs. For the first time, Pohl et al. [ 244 ] have synthesized sugar conjugated drugs in 1995. The glycoconjugate drug (glufosfamide) exhibited 4.5-fold decreased toxicity in rats. Later, glufosfamide had a good effect in phase II clinical trial in 20 patients with refractory solid tumors [ 245 ]. There are several other informative examples of such drug modifications with carbohydrate moiety to improve the efficacy of chemotherapeutic drugs.. For instance, Adriamycin’s cardiotoxicity and multidrug resistance have been shown significantly downregulated upon conjugation with 2-amino-2-deoxy-d-glucose and succinic acid [ 246 ]. Similarly, galactose and lactose conjugation to Geldanamycin, HSP90 inhibitor, resulted in a forty-fold increase in its anti-cancer activity [ 247 ]. Many other anti-cancer drugs such as Paclitaxel, Azomycin, Ketoprofen, Cadalene, Docetaxel, Chlorambucil, etc. have also been reported promising effects in cancer therapeutics upon conjugation with monosaccharides [ 248 , 249 , 250 , 251 , 252 ]. Small molecule inhibitors of glycosylation machinery are being exploited for therapeutic benefit against cancer. Mechanisms of action of most of the glycosylation inhibitors are either by interfering with metabolism of precursors or by interfering with intracellular activities. Since most of the inhibitors are small molecules that can be easily taken up by various cell types, it provides an excellent opportunity for the researchers to design drugs to treat multiple diseases correlated with aberrant glycosylation. Several of such glycosylation inhibitors are being explored in clinical trials as a promising therapeutic strategy against cancer progression. For example, the use of Uproleselan (GMI-1271) and E-Selectin Antagonist along with chemotherapies was shown to result in well-tolerated, high remission rates and promising survival outcomes in patients with Acute Myeloid Leukemia in a phase 1/2 clinical trial [ 253 ]. Galectin inhibitor GR-MD-02 is being used in combination with chemotherapeutic drug Pembrolizumab against melanoma, non-small cell lung cancer, and squamous cell carcinoma head and neck [ 254 ]. Similarly, another galectin inhibitor GM-CT-01, and chemotherapeutic drug 5-Fluorouracil is in phase2 clinical trial for colorectal cancer [ 255 ]. GM-CT-01 with Fluorouracil also in phase 1 clinical trials for lung, breast, prostate, head, and neck cancers [ 256 ]. The fucosylation inhibitor SGN-2FF is currently in phase 1 clinical trial for several cancers, including breast, urinary bladder, renal, gastric, colorectal, squamous cell carcinoma, head and neck, and non-small cell lung cancer [ 257 ]. Several other preclinical studies have also highlighted glycosylation inhibitors’ potential to prevent cancer progression, which was reviewed in detail previously [ 258 , 259 , 260 ]. Glycan based polymers such as hydrogels, micelles, and nanoparticles have been considered as effective delivery vehicles of chemotherapeutics. Puranik et al. [ 261 ] have demonstrated that nanoscale hydrogels have desired physicochemical properties and possess feasible biocompatibility for oral delivery of hydrophobic chemotherapeutics. Recent research approaches led to the development of carbohydrate-based nanomaterials, especially for the controlled release of chemotherapeutics. Our recent study demonstrates that the use of polymeric nanogel significantly enhanced efficacy and systemic administration of a combination of cisplatin and gemcitabine in the treatment of pancreatic cancer. We found that encapsulation of cisplatin in polymeric nanogel with cross-linked ionic cores enhances cisplatin accumulation at the tumor site and improves its safety profile. To attain the targeted delivery, we have decorated nanogel encapsulated cisplatin with the TKH2 monoclonal antibody, which shows high specificity towards the STn antigen expressed explicitly on cancer cells. We found that combined simultaneous treatment with Gemcitabine and TKH2-functionalized cisplatin loaded nanogels significantly attenuated tumor growth both in vitro and in vivo with no detectable toxicity [ 262 ]. Another research group has developed a biocompatible nanoparticle for the targeted delivery of 5-fluorouracil and Paclitaxel to SLe A -expressing gastric cancer cells with minimal affinity for the healthy cells [ 263 ]. The need for controlled and targeted drug action has prompted researchers to use several polysaccharides such as chitosan, chondroitin sulfate, alginate, hyaluronic acid (HA) to synthesize nanoparticles as they provide unique physiochemical characteristics [ 264 ]. Using carbohydrate-based polymers enhances the solubility of low soluble or insoluble drugs, maximizes therapeutic efficacy, and minimizes side effects by affecting absorption, distribution, and metabolism of therapeutic drugs. Hence, carbohydrate-based polymers have been broadly considered promising materials in synthesizing effective nano-carriers for anti-cancer drug regimens [ 265 ]. Taken together, aberrant protein glycosylation in cancer provides scientists with additional opportunities to identify novel biomarkers. The presence of glycan structures on various biomolecules exhibits pathological importance, and therefore glycan-based therapies are gaining more attention in the therapeutic interventions against cancer progression ( Figure 6 ).

Cancer is a devastating disease that comprehends numerous cellular modifications. Most of the cancer-associated deaths are due to the lack of early diagnosis and effective targeted therapies. Current treatment regimens mostly rely upon conventional chemotherapy with a few targeted therapies. However, emerging evidence highlighted that altered glycans are key drivers throughout tumorigenesis and tumor progression. Glycoproteins that are found in cells and their glycan structures are commonly modified upon neo-transformation. Though glycosylation is complex and challenging, research efforts have been commenced to target altered glycosylation in cancer. Since altered glycans are abundant in tumor cells and specific glycan structures are uniquely expressed on tumor cells, these may serve as valuable targets for disease diagnosis, prognosis, and therapy. Glycosylation research encounters several challenges, such as identifying particular biological consequences of expression patterns and levels of glycosyltransferases/glycosidases; identification of glycan structures at specific sites, and its abundance in a protein. For example, the biomarkers CA125 and CA19.9 has been identified three decades ago; however, the specific sites and their abundance in proteins remain elusive. Apart from these, the development of effective synthetic tools for studying the effect of glycosylation on structure and functions of biomolecules is highly challenging because multiple isoforms of glycosyltransferases can encode a single glycan epitope. While the development of many glycan based therapeutics have failed to get a significant result against tumor progression, several research attempts have been made to tackle major problems associated with glycosylation machinery. Some of the emerging therapies possess promising outcomes. Further investigations of cancer-specific glycans are hoped to develop more useful prognostic, diagnostic, and therapeutic targets in the fight against cancer.

The diversity of the monosaccharide building blocks and the multiple ways of assembling these building blocks to an oligosaccharide within the cell makes glycosylation a complex cellular event. Unlike the biosynthesis of proteins or nucleic acids, oligosaccharides are assembled through a systematic enzyme-catalyzed non-template driven machinery that generates glycans with diversity and heterogenicity [ 1 ]. Among the four major classes of biomolecules (nucleic acids, proteins, carbohydrates, and lipids), carbohydrates are the least understood biomolecule for their pathobiological functions. However, the past two decades of research provide remarkable progress in understanding the biology and pathology of glycosylation [ 2 ]. In addition to their metabolic role, oligosaccharides are often added to the proteins or lipids to form glycoconjugates such as glycoproteins, proteoglycans, and glycosphingolipids in the endoplasmic reticulum (ER) and Golgi apparatus. The formation of these glycan structures on proteins or lipids provides a vital stabilizing force for proteins within their microenvironment, thereby modulating their biological functions. Glycosyltransferases and glycosidases mainly orchestrate the sequential events of glycosylation. Functionally, the glycan biosynthetic ability largely depends upon the bioavailability and abundance of glycosylation enzymes, substrates, and sugar donors [ 3 ]. The direct precursors used in the biosynthesis of glycoproteins are nucleoside diphosphate or monophosphate sugars. Most of the cell surface and secreted proteins are co-translationally translocated and pre-manufactured in the lumen of ER. The partially glycosylated proteins make their way to the multiple stacks of the Golgi apparatus for further processing or trimming. The fully glycosylated proteins are then being distributed to various destinations. In the secretory pathway, two significant glycosylation types are known according to the nature of linkages between oligosaccharides: N-glycosylation and O-glycosylation. This glycosylation machinery allows biological tuning of proteins for selective functions such as protein folding, intracellular trafficking, mediating cell-cell and cell-matrix interactions, altering half-life of proteins, cell adhesion, host-pathogen recognition, etc. [ 4 ] ( Figure 1 ). The cellular functions of a glycan mainly depend upon the structure and function of the carrier protein on which it expresses. Cumulative evidence shows that alteration in the glycosylation patterns of the carrier proteins including incomplete synthesis of glycan structures, enhanced expressions of a complex branched N-glycans, expressions of truncated O-glycans (Tn and Sialyl Tn antigen), overexpression of ‘core’ fucosylation, altered expressions of sialylated glycans, etc. facilitate the acquisition of cellular features necessary for the malignant transformation of cells [ 5 ]. Such protein-specific, site-specific, and cell-specific tumor-associated glycans modifications increase molecular heterogeneity within the cell populations and the microenvironment. Variations in glycosylation seem to affect cell growth and survival directly and facilitate tumor-associated immune response and metastasis. Since cancer is a ‘microevolutionary’ process where there is the survival of only the fittest cells, the specific glycan changes may also be selected, and a limited subset of changes may be associated with tumor progression and metastasis [ 6 ]. The possibility of distinguishing the glycosylation pattern of proteins between healthy and cancer patients underscores glycobiology as a promising research area for cancer biomarker identification. This review provides a brief understanding of the fundamental concepts of glycobiology in health and disease. We discuss how altered glycosylation machinery is associated with cancer progression and metastasis. We also provide insights into the influence of aberrant glycosylation in the development of cancer biomarkers and targeted therapeutic interventions.

Aberrant glycosylation due to cellular and metabolic changes leads to abnormal expressions of membrane-localized glycans that trigger malignant transformation of cells. The principle mechanisms underlying tumor-associated alterations in the carbohydrate structures are increased branching of complex and hybrid N-glycans, increased levels of sialyl lewis antigens, truncated O-glycan expression, and complex core fucosylation. Moreover, glycans dictate proteolysis and directly mediate oncogenic signal transduction, ligand-receptor interaction, cell-cell, and cell-matrix adhesion in cancer cells. Here, we discuss the glycosylation-mediated promotion of cancer hallmarks, including tumor cell-cell interaction, cell-matrix interaction, oncogenic signaling cascades, and metastasis. Cell-cell communications in epithelial cells are acquired by forming stable connections between adjacent cells in a well-organized manner. Two types of cell-cell communications are documented in mammals, which include tight junctions and adherens junctions. Tight junctions are responsible for cell-cell communications through the barrier, and adherens junctions function as adhesion regulators between adjacent cells. The calcium-dependent transmembrane protein E-cadherin actively regulates cell-cell adhesion, cell motility, and cell growth differentiation by forming a cadherin-catenin complex between the neighboring cells. A disturbance in the cadherin-catenin complex leads to the modulation of cell-cell interaction and cellular integrity in cancer cells. An increasing body of evidence suggests that glycosylation affects the stability of cadherins and cadherin-mediated cell-cell adhesion. For example, the upregulated expressions of GnT-V that regulate β 1,6-GlcNAc branching alter function of N-cadherin, which resulted in the loss of cellular adherence and tumor invasion in human fibrosarcoma cells [ 49 ]. In contrast, removing specific N-glycans from E-cadherin was reported to increase the interaction of the cadherin-catenin complex and thereby stabilized cell-cell adhesion [ 50 ]. Similarly, the reduction of E-cadherin N-glycosylation promoted the stabilization of adherent junctions in healthy and cancer cells. It was shown that increased interaction of such hypo glycosylated E-cadherin with protein phosphatase 2A (PP2A) promotes tight junction assembly in cancer cells [ 51 ]. In addition to this, hyper-glycosylation of E-cadherin was found to mediate the destabilization of adherens junction proteins; and dysregulated N-glycosylation of E-cadherin stabilizes protein on adherens junctions as well as tight junctions that cause disturbances in intracellular adhesion in oral cancer [ 52 ]. β-catenin is a critical element of the Wnt canonical signaling pathway in various cancers, that is directly involved in the intercellular junction integrity in association with E-cadherin. Increased O-GlcNAcylation of β-catenin enhances overexpression of E-cadherin, and its nuclear translocation triggers the invasion and metastasis in colorectal cancer [ 53 ]. Intercellular adhesion molecule 1 (ICAM-1) and activated leukocyte cell adhesion molecule (ALCAM) are responsible for aggregation and sphere formation of tumor cells. Silencing of MAN1A1 (Golgi mannosidase) inhibited the tumor aggregate formation by affecting the N-glycosylation of ALCAM resulted in the reduction of cell adhesion and motility in ovarian cancer cells [ 54 ]. A recent study demonstrates that treatment with tunicamycin inhibited the N-glycosylation, which resulted in the increased dissociation of the desmosome complex, thereby inhibits cell-cell adhesion in primary keratinocyte cells [ 55 ]. The biological functions of E-cadherin is inhibited by the modification of N-glycan structure β1,6-GlcNAc at the Asn-554 site [ 56 ]. Earlier studies revealed that α−1,6 fucosyltransferase inhibition improves the E-cadherin function as cell-cell adhesion, thereby reducing the tumor invasion in lung cancer [ 57 ]. Also, knockdown of FUT8 suppresses the calcium-dependent E-cadherin mediated cell-cell adhesion in pancreatic acinar cell carcinoma [ 58 ]. In addition to this, FUT8 deficient MCF-7 cells showed decreased cell migration and invasion by suppressing the fucosylated modulation of E-cadherin followed by inhibition of integrin-mediated FAK signaling with the decreased localization of β-catenin to the nucleus [ 59 ]. Aberrant expression of N-acetylgalactosaminyltransferase 3 (GALNT3) increases the O-glycosylation of MUC1, which resulted in enhanced stabilization of E-cadherin and β-catenin complex and thereby promotes cell proliferation and migration in ovarian cancer cells. Further, inhibition of GALNT3 destabilizes the MUC1, which results in the suppression of cell proliferation and invasion [ 60 ]. Another study showed that binding of MUC1 with β-catenin enhances the destabilization of adherent junctions, cell invasion, and disturbs the cytoskeletal architecture of tumor cell invasion in breast cancer cells [ 61 ]. It has been reported that overexpression of CD82 decreased the expression of β-galactoside α 2, 3-sialyltransferase (ST3GAL4), thereby inhibits sialyl lewis antigen and also diminished the adhesion of tumor cells to the blood vessel that reduced metastasis [ 62 ]. Along with this, overexpression of ST3Gal IV was found to increase the levels of sialyl lewis x antigen with the decreased levels of α−2, 6 sialic acids. It promoted the migratory properties of PDAC cells [ 63 ]. Dysregulation of cell adhesion molecule E-cadherin and integrins influences the cancer cell invasion and metastasis. A previous study reported that overexpression of ST3Gal III alters the sialylation on the α2β1 integrin that resulted in diminished cell-cell aggregation and increased invasiveness in pancreatic cancer [ 64 ]. MUC16, a transmembrane protein, facilitates cell-cell contact in epithelial cells through its association with the actin cytoskeleton in the extracellular matrix (ECM). The genetic ablation of MUC16 perturbs the binding of actin cytoskeleton to the cytosolic domain of MUC16, which increases the migration and invasion in epithelial cells with altered expression of ZO-1 [ 65 ]. In summary, altered glycosylation affects the adherent junction complex with the simultaneous dissociation of tight junction proteins, which regulates integrin and Wnt/β-catenin signaling mediated invasion and metastasis of cancer cells ( Figure 3 ). Atypical glycosylation influences the cell-matrix interaction by modifying the functions of anchoring proteins and extracellular basement membrane proteins, including integrins, laminin, fibronectin, and collagen in the ECM. The transmembrane protein Integrins regulate cell-cell and cell-matrix interaction to maintain cytoskeleton architecture and promote cell growth and proliferation through direct association with ECM proteins. Alterations in anchoring protein glycosylation would result in various pathological conditions like muscular dystrophy [ 66 ], cardiovascular diseases [ 67 ], neurodegenerative diseases [ 68 ], and cancer [ 69 , 70 ]. An earlier study has reported that the genetic deletion of N-acetylglucosaminyltransferase III (GnT-III) inhibited the formation of a β−1,6 branch of complex N-glycans that affects the expression of integrins, which further resulted in the inhibition of invasion and metastasis in mouse melanoma cells [ 71 ]. Moreover, β−1,6 branched sialylated complex-type N-glycans alters the function of α5β3 integrins in tumor cells. Highly N-glycosylated α5β3 integrin alters the cell adhesion properties of cancer cells in association with vitronectin, a substrate for α5β3 integrin that proceeds the invasion of melanoma cells [ 72 ]. Overexpression of ST6Gal-I enhances the α−2, 6 sialylations on β1 integrin and talin that increases the expression of collagen IV, and promotes tumor invasion and motility in colon cancer cells [ 73 ]. Further, caveolin-1 triggers ST6Gal-I expression that enhances α−2, 6 sialylations of α5β1 integrin, promoting the adhesion of epithelial cells to the fibronectin in hepatocellular carcinoma [ 74 , 75 ]. Previously, we have reported that overexpression of core 3 synthase alters the expression of α2β1 integrin through the modified glycosylation in MUC1, which further down-regulated the expression of phospho-FAK and its downstream targets, thereby decreases the tumor growth and metastasis in pancreatic ductal adenocarcinoma [ 76 ]. Inhibition of O-glycosylation and N-glycosylation by chemical inhibitors such as benzyl-α-GalNAc and tunicamycin enhanced the cellular adhesion to fibronectin and galectin-3 through the integrin-dependent manner in the ECM [ 77 ]. Attachment of β1,6-GlcNAc-branched N-glycans on the β4 integrin increases its cross-linking with gelatin-3 that subsequently enhances PI3K/Akt signaling mediated cell migration, invasion, and tumor growth. Whereas, the deletion of N-acetylglucosaminyltransferase III (GnT-III) inhibits bisecting of N-glycans that suppressed the β4 integrin/AKT mediated cancer cell invasion and motility [ 78 ]. Recent studies have shown that α−2,6 sialylation on the surface of α5β1 and α2β1 integrins reduced the binding of MDA-MB-231 breast cancer cells to the basement membrane protein fibronectin and Collagen IV [ 79 ]. Collectively, modified glycans on the integrins and recruitment of galectins disturb the matrix proteins in the extracellular space through FAK/Integrin signaling in tumorigenesis. Aberrant and modified glycosylation causes detrimental metabolic and cellular signaling, promoting cancer progression, and its exact molecular mechanism remains unclear. Numerous cellular factors impact tumorigenesis, including altered glycan expression, synthesizing enzymes and its localization, mutations in the tumor suppressor gene, and its instability. All such anomalies are leading to the activation of oncogenic signaling cascades such as Wnt/β-catenin, Hippo signaling, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), Janus kinase/signal transducer, and activator of transcription (JAK/STAT), Transforming growth factor-beta (TGFβ/Smad), and Notch signaling. Altered glycosylation patterns on the cell surface molecules, transmembrane proteins, and growth factors cause tumor cell proliferation, invasion, and metastasis through the activation of these signaling cascades and its downstream targets [ 80 ]. Functional dysregulation and genetic mutations in the Wnt signaling components have a significant impact on various cancer cells. Glycosylation has been shown to affect the function of Wnt signaling components. Overexpression of MUC-13, a transmembrane glycoprotein, destabilizes the β-catenin/APC/Dshvelled complex by phosphorylating β-catenin at Ser552 and Ser675 and enhances its nuclear translocation. Nuclear binding of β-catenin to TCF/LEF transcription factors upregulated the expression of c-myc, Axin-2, and E-cadherin, enhancing tumor growth in hepatocellular carcinoma [ 81 ]. Overexpression of O-glycosylated MUC5AC increases the cell proliferation, invasion, and metastasis properties with significant upregulation of β-catenin and its downstream molecules in the gastric cancer cells. In contrast, the silencing of MUC5AC downregulated the expression of β-catenin, resulted in decreased cancer progression [ 82 ]. Moreover, knockdown of MUC16 resulted in the destabilization of the E-cadherin/β-catenin complex. It reduces the cell adhesion with the simultaneous increase in phosphorylated EGFR, Akt, and ERK expression to promote tumor cell invasion and metastasis in ovarian cancer cells [ 83 ]. It was shown that the N-glycosylation of Wnt ligand and its receptors and E-cadherin promoted the expression and nuclear translocation of β-catenin/γ-catenin that upregulated the transcriptional activity of dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1), which resulted in tumor progression and metastasis. Genetic deletion of DPAGT1 reduced the glycosylation of E-Cadherin and downregulated the canonical Wnt signaling pathways and inhibited tumor cell invasion and metastasis [ 84 , 85 ]. Altogether, the evidence mentioned above clearly demonstrates that the altered pattern of glycosylation in the Wnt signaling components modulates the upregulation of β-catenin and its nuclear localization. Human epidermal growth factor receptors (EGFR) are tyrosine kinase family members (ErbB1, ErbB2, ErbB3, and ErbB4) regulates the oncogenic signal transduction and cancer progression [ 86 ]. Homo or heterodimerization of receptors induced by ligand binding triggers downstream signaling through PI3K/Akt, JAK/STAT, and MAPK pathways. EGFR has 11 typical and 4 atypical N-glycosylation consensus sequences [ 87 ]. The N-glycans in the extracellular region of ErbB receptors could directly modulate their biological activity and intracellular transport. Bisecting GlcNAc was reported to inhibit EGFR and integrin signaling through the MAPK pathway [ 88 ]. The silencing of β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) was found to inhibit EGF mediated phosphorylation of EGFR by modifying the N-glycan structure. Further, it downregulated the phosphorylation of AKT and ERK due to the degradation of EGFR and thereby suppressed colorectal cancer stem cells [ 89 ]. Also, inhibition of N-glycosylation by tunicamycin (a well-known N-glycan inhibitor) decreased ALK’s phosphorylation and expression of its downstream target genes AKT, ERK, and STAT3 in melanoma cancer cells, which resulted in enhanced apoptosis [ 90 ]. Increased GalNAc-type O-glycosylation on the EGFR by abundantly expressed Core 1 β1,3-galactosyltransferase (C1GALT1) was found to promote tumor invasion and progression. In contrast, inhibition of C1GALT1 by the specific inhibitor Itraconazole reduced the elongation and binding of O-glycans to the EGFR, resulting in reduced tumor malignancy [ 91 ]. Aberrant glycosylation of EGFR by Le y carbohydrate enhanced the phosphorylation of EGFR with the concomitant upregulation of phosphorylated AKT that induced tumor cell migration and invasion in oral cancer cells; whereas the absence of Ley glycosylation of EGFR significantly reduced the cancer cell migration [ 92 ]. Therefore, impaired glycosylation mediated oncogenic EGFR signaling and its downstream pathways substantially contribute to the tumor progression and metastasis in various cancers. TGF-β is a pleiotropic cytokine that contributes to various cellular functions such as tissue development, ECM regulation, proliferation, apoptosis, and tumorigenesis. Binding of TGF-β ligand to its receptors on the cell membrane triggers subsequent downstream signaling activation through Smad dependent or Smad independent manner in various pathological conditions, including cancer. The glycosylation of TGF-β and other cell surface proteins is of central importance in regulating signal transduction during cancer progression. Interestingly, cell surface proteoglycans such as endoglin and beta glycan act as co-receptors for TGF-β ligand, facilitating the binding of TGF-β to the receptor complex [ 93 ]. Several studies have highlighted the positive correlation of O-glycosylated mucins and TGF-β oncogenic signaling cascade. The interaction of highly glycosylated MUC-1 CT with TGF-β to promote tumor invasion of pancreatic cancer cells has been reported [ 94 ]. Clinical studies revealed that overexpression of TGF-β is correlated with MUC1 expression in association with CD10 in stromal cells of colorectal cancer tissues [ 95 ]. It was also reported that overexpression of N-acetylgalactosaminyltransferase14 (GALNT14) catalyzes the first step in the mucin-type O-glycosylation, significantly increased EMT markers to promote tumor progression and metastasis [ 96 ]. Conversely, a recent study reported that the fucosylation of TGF-β receptors enhanced the phosphorylation of Smad3 and promoted its nuclear translocation to promote tumor metastasis [ 97 ]. Taken together, the modulation of TGF-β and its receptors glycosylation significantly affect the oncogenic properties of cancer cells. Notch signaling family consists of Notch ligands (Delta/serrate/Dsl) and Notch receptors (notch1–4) in mammalian cells. Notch receptors are predominantly O-glycosylated rather than N-glycosylation by glycosyltransferases. Changes in the glycosylation of Notch associated receptors induce oncogenic signaling in cancer progression [ 98 ]. Mammalian Notch 1 was shown to possess N-glycans based on its sensitivity towards N-glycosidases. It has been shown that silencing of ST6Gal-I decreased the expression of Notch1 and its downstream molecules jagged1, Hes1, MMP-2, MMP-9 and thereby decreased the cell proliferation, invasion, and metastasis in non-small cell lung carcinoma [ 99 ]. An increasing amount of evidence has reported aberrant expression of Notch-modifying glycosyltransferases in various types of cancers. Silencing of protein O-Glucosyltransferase 1 resulted in decreased Notch activation in human myeloid leukemia U937 cells [ 100 ]. Alternatively, overexpression of fucosyltransferases and protein O-Glucosyltransferase 1 might facilitate overexpression of Notch on the cell surface. However, further studies are required to understand how glycosylation affects Notch functions during tumor progression. Hence, glycosylation of cell surface components and their receptors is of great importance in directing oncogenic signaling pathways during tumorigenesis ( Figure 4 ). Therefore, targeting the aberrant glycoforms of glycoproteins and glycolipids could reduce oncogenic potential of glycans in cancer. Malignant transformation and neoplastic progression are the consequences of alterations in several genes and protein expressions. Cumulative evidence shows that changes in protein glycosylation machinery accompany acquisition of cellular features essential for the invasion of tumor cells to distant regions. Remodeling glycans that have been implicated during tumor progression and metastasis is predominantly the consequence of mutation or modifications in the branching enzyme glycosyltransferases [ 101 ]. Altered glycosyltransferases in tumor metastasis have been reported in various cancer types such as colon cancer, pancreatic cancer, breast cancer, oral squamous carcinoma, skin cancer, and hepatocellular carcinoma. N-acetylglucosaminyltransferase, encoded by the MGAT gene that modulates the structure of complex N-glycans, can be associated with cancer progression and metastasis. It has been reported that modification in the branching of α−1, 6 mannose to β1–6-linked N-acetylglucosamine by N-acetylglucosaminyltransferase V (GnT-V) in the growth factors and cell surface receptors increases the metastasis in cancer tissues by regulating Src, EGFR, and TGF-β family oncogenes [ 102 , 103 ]. Previous reports show that N-acetylglucosaminyltransferase V mediates the glycosylation of EGFR in gastric cancer cells. Inhibition of N-acetylglucosaminyltransferase V resulted in decreased ErBb1, ErBb2, and ErBb4 with increased E-cadherin and a concomitant decrease in vimentin expression in gastric cancer [ 104 ]. In contrast, N-acetylglucosaminyltransferase III (GnT-III) catalyzes the synthesis of bisected N-glycans, thereby suppresses tumor metastasis by modulating the turnover of E-cadherin on the cell surface and suppressing β-catenin mediated cell-cell adhesion [ 105 ]. The Golgi resident transmembrane protein N-acetylgalactosaminyltransferases that add GalNAc moieties to the nascent polypeptides at the ser/thr residues are known as one of the active participants in the malignant transformation of cancer cells. Altered localization of GALNT1 increases the O-glycosylation in liver cancer, promoting tumor tissue invasion and growth in association with the increased O-glycosylation of MMP-14 [ 106 ]. Localization GALNT1 is regulated by Late Endosomal/Lysosomal Adaptor, MAPK, and mTOR Activator 5 (LAMTOR5) through Src and c-Jun initiates the abnormal O-glycosylation of MUC1 and osteopontin with enhanced accumulation of Tn antigens in metastatic breast adenocarcinoma cells [ 107 ]. Aberrant O-glycosylation of mucins catalyzed by GALNT3 activates MUC1-PI3K/AKT axis with the upregulation of NFκB promoted tumorigenesis and metastasis in colon cancer cells [ 108 ]. Conversely, GALNT3 deficient PDAC cells showed upregulated expression ErbB family proteins with increased cell proliferation, motility, and metastasis by inhibiting E-cadherin expression in pancreatic cancer cells [ 109 ]. We have recently reported that the aberrant expression of MUC1 downregulates the appearance of the tumor suppressor polypeptide N-Acetylgalactosaminyltransferase 5 (GLANT5) by direct binding to the regulatory elements in the GALNT5 gene that promote pancreatic cancer progression and metastasis [ 110 ]. Core 1 β1, 3-galactosyltransferase (C1GALT1) is one of the critical enzymes involved in synthesizing O-glycans, which transfers galactose residue to the T-antigen favored by COSMC. We have reported that hypermethylation of COSMC resulted in the expression of truncated O-glycan structures Tn and STn antigen in pancreatic cancer that drives cancer-specific phenotype characterized by increased proliferation, compromised adhesion, and invasiveness in cancer cells [ 29 ]. Further, we explored the molecular mechanisms behind malignant potential of truncated O-glycans during tumor progression and metastasis. The genetic deletion of COSMC in pancreatic ductal adenocarcinoma cells resulted in the increased expression of mesenchymal markers with a concomitant reduction in the appearance of epithelial markers. Re-expression of COSMC in knockout cells reversed the induction of EMT. Also, we found that the aberrant expression of truncated O-glycans, maintaining stemness features of cancer cells that promote metastasis through the induction of EMT [ 28 ]. In contrast, modification of O-glycans on FGFR2 by C1GALT1 increases the tumor invasion and metastasis in colon cancer cells [ 111 ]. Also, overexpression of C1GALT1 increases the O-glycosylation of galectin-4, which mediated the activation of EGFR and its downstream molecules Akt/Gsk3β, upregulated expression of vimentin, and twist1 and promoted tumor metastasis in prostate cancer cells [ 112 ]. Further, the upregulated expression of C1GALT1 increases the O-glycosylation of MUC1 in esophagus squamous cell carcinoma patients [ 113 ] and increases the metastasis by modifying O-glycans on integrin β1 in hepatocellular carcinoma [ 114 ]. Altogether, these findings indicate C1galT1 mediated tumor promotion or tumor suppression is tissue / cell specific and context dependent, which needs to be further investigated. MUC1, MUC4, and MUC16 possess increased expression of these tumor-associated antigens Tn, STn, Le x , and SLe a , which promote tumor metastasis by facilitating ligand for different selectins and galectins [ 115 ]. Furthermore, the aberrant O-glycosylation of the ectodomain of MUC1 increased the synthesis of sLe x and sLe a epitopes, which is correlated with invasion and metastasis in colon cancer cells [ 116 , 117 ]. Overexpression of α−1, 6-fucosyltransferase (FUT8) induces EMT like process by increasing the core fucosylation of E-Cadherin with the upregulated expression of phospho-Src Y416 and β-catenin with the concomitant decrease in N-cadherin in lung cancer cells [ 118 ]. Fucosyltransferase IV (FUT4) upregulated the expression of snail and E-cadherin in association with NF-κB and PI3K/Akt-GSK-3β signaling to acquire mesenchymal phenotype that increases the metastasis by the enhanced secretion of MMP-9 in breast cancer cells [ 119 ]. TGF-β is known to be actively involved in tumor invasion and metastasis in several cancers. Herein, a recent study proposed that fucosylation of TGF-β receptor by FUT3 and FUT6 upregulated the expression of TGF-β/Smad signaling molecules leading to EMT in colorectal cancer cells [ 120 ]. In addition to this, the upregulated expression of FUT8 increased the fucosylation of the TGF-β receptor and its downstream signaling, which promoted the breast cancer tissue invasion and metastasis [ 121 ]. Alpha 1, 2 fucosyltransferases, FUT1 and FUT2 catalyzes the synthesis of alpha 1, 2-linked fucose of Lewis y and Globo H on the glycans. Overexpression of FUT1 and FUT2 increases the cancer stem cell potency, which is one of the critical factors for the EMT process. Upregulation of FUT1 and FUT2 increased N-cadherin expression and vimentin with the concomitant downregulation of E-cadherin, which enhances the tumor metastasis in breast cancer cells [ 122 ]. A recent computational biology-based study revealed that FUT8 mediated core fucosylation increased the tumor invasiveness and metastasis by modulating the L1 Cell Adhesion Molecule (L1CAM) [ 123 ]. Similarly, the dysregulation of sialyltransferase expression promoted tumorigenesis and metastasis in various cancers [ 124 ]. α−2,6, sialylation of N-glycans, catalyzed by the enzyme β-Galactoside α2,6-sialyltransferase 1 (ST6GAL1), is associated with tumor metastasis. Overexpression of ST6GAL1 induced TGF-β mediated activation of EMT in cancer cells, whereas the silencing of ST6GAL1 inhibited the TGF-β expression, thereby decreasing cancer progression [ 125 ]. Taken together, aberrant expression of glycosyltransferases affects the various oncogenic signaling molecules which trigger the tumor invasion and metastasis through the activation of EMT ( Figure 5 ). The primary functions of different types of glycans in cancer are summed up in Table 2 .