The advancement of proteomics and its different tributaries, like phospho-proteomics, has facilitated the understanding of the structural and functional roles of various proteins in cancer cells. Protein function is influenced by many cellular signals and post-translational modifications (PTMs). Glycosylation is a ubiquitous and prominent PTM, with glycan structures found on secretory and membrane-bound proteins. The reactions generating glycans are catalyzed by enzymes called glycosyltransferases that reside in the endoplasmic reticulum, Golgi apparatus, and some extracellular spaces (1, 2). In humans, the two main glycosylation pathways are N-glycosylation, in which a monosaccharide is attached to the nitrogen atom of Asparagine on proteins, and O-glycosylation, wherein the linkage is via oxygen atoms on Serine or Threonine amino acid residues in proteins (3). Not surprisingly, the pathogenesis of cancer works hand in hand with important glycan alterations on a variety of proteins. For example, N-glycan modifications have been found to stabilize the PD-L1 immune checkpoint and decrease cytotoxic T-cell activity (4). O-glycosylation-based changes are very abundant in the evolution of cancer. One of the most abundantly expressed aberrant O-glycoforms is the Tn antigen and its derivative, the STn antigen (5, 6). These antigens arise from incomplete O-glycan synthesis, leading to a dearth of fully branched O-glycans that can be used to the advantage of tumor cells. The multitude of mechanisms that can lead to the presence of aberrantly glycosylated proteins and important events altered by these Tn and STn antigens to hijack tumor cell signaling in gastrointestinal cancers like pancreatic ductal adenocarcinoma (PDAC) and influence the infiltration of immune cells in the tumor niche are discussed in this review.

The process of O-glycosylation is highly complex and regulated by a series of enzymatic reactions catalyzed by glycosyltransferases initiated in the Golgi apparatus. The most exposed amino acid residues on a folded protein in the Golgi are Serine (Ser) and Threonine (Thr). O-glycosylation begins with the addition of an N-acetylgalactosamine (GalNAc) residue using UDP-GalNAc by the enzyme polypeptidyl α-GalNAc transferase (ppGalNAcT) to a Ser/Thr residue to form a covalent bond at their hydroxyl group. This structure is called the Tn antigen (GalNAc-α1-O-Ser/Thr), also referred to as the Thomsen-nouveau antigen (7). Further addition of a galactose (Gal) residue to Tn forms the T antigen, also known as the Core 1 structure (Galβ1-3-GalNAc-α1-O-Ser/Thr) or the Thomsen-Friedenreich (TF) antigen. This is catalyzed by the enzyme T synthase (synonyms: core1 β3-galactosyltransferase, C1GALT1). T synthase is synthesized in the endoplasmic reticulum (ER), and its proper folding is ensured by a chaperone called COSMC (Core1-Specific-Molecular-Chaperone). In cellular conditions devoid of a functional T synthase, adding one sialic acid (N-acetylneuraminic acid or Neu5Ac or NANA) residue to the GalNAc generates the STn antigen, a reaction catalyzed by the sialyltransferase ST6GalNAc-I (8). On the other hand, elongation of the T antigen or Core 1 structure forms the Core 2 O-glycan branching. Additionally, the Tn antigen can be modified to create the Core 3 and Core 4 structures using a different set of glycosyltransferases, all of which lead to complex O-glycosylation-based branching in normal cells ( Figure 1A ).

The aberrant overexpression of the Tn/STn antigens is observed in many diseases and malignancies. Some of the mechanisms that can mediate this process are as follows: (i) Somatic mutations in COSMC can prevent proper folding of T synthase (C1GALT1) and render it incapable of extending the Tn structure (9); (ii) Epigenetic silencing of either COSMC or T synthase transcription as identified in the “Tn syndrome” in hematopoietic cells and pancreatic cancer (10); (iii) Aberrant expression or subcellular localization of the ppGalNAcT enzymes (11, 12); (iv) Loss of availability of the necessary UDP-sugars to facilitate glycan chain extension post Tn/STn antigen formation (13); (v) Elevated expression of the ST6GalNAc-I enzyme that can outcompete T synthase and promote the terminal generation of the STn antigen, in a way that inhibits further addition of any sugar by the glycosyltransferases (14) ( Figure 1A ).

Cancer models like cell lines have been used to study various mechanisms that govern the pathogenesis of tumors. In one such expedition, researchers studying the molecular basis of breast cancer discovered the overexpression of the Tn/STn antigens in this malignancy for the first time (15). The Tn/STn antigens have since been established as members of the tumor-associated carbohydrate antigen (TACA) group of cancer biomarkers due to their overwhelming presence in various solid tumors (16). Radhakrishnan et al. reported for the first time that pancreatic cancer cells possess a hypermethylated promoter for the gene encoding COSMC in about 40% of PDAC patients studied in their cohort. This leads to the downregulation of T synthase and heightened Tn/STn antigen expression. These cancer cells exhibit a highly invasive and migratory phenotype due to altered cell-cell adhesion structures that disrupt tissue homeostasis and instigate tumorigenic processes (17). The Tn/STn antigen’s tumor-promoting role was further validated in robust cell line and orthotopic models of pancreatic cancer. COSMC knockout led to tumor spread as it enhanced the epithelial-to-mesenchymal transition (EMT), a critical hallmark of cancer, and increased stemness markers, including CD133 and CD44 in PDAC cells (18). In another study, Dong et al. knocked out the gene encoding C1GALT1 in colorectal cancer cells, which prevented the expression of the T-synthase enzyme, leading to the expression of the Tn/STn antigen. Markers like N-cadherin, Snail, and Slug were all elevated in the C1GALT1 knockout cells demonstrating the higher EMT potential of these Tn/STn expressing cells as compared to the counterparts expressing the C1GALT1 ( Figure 1B ) (18). Additionally, the presence of the Tn/STn antigens on the glycoprotein MUC16 increased aggressiveness in PDAC. Tn/STn-MUC16 bound to the α4β1 integrin complex more efficiently than its fully glycosylated counterpart, and augmented integrin-linked kinase (ILK) and focal-adhesion kinase (FAK) mediated cell signaling that increased cell survival and migration (19). A brief description of mucin-associated truncated O-glycans in the context of immune evasion is described in a further section.

The death receptors (DR) 4 and 5 interact with ligands TRAIL/Apo2L and stimulate programmed cell death. There exist crucial conserved O-glycosylation sites on these death receptors, and mutations in these sites lead to disrupted O-glycan presence on the receptor ectodomains, making them ineffective in inducing apoptosis (20). Jiang et al. showed that a similar mechanism was prevalent in cancer cells deficient in COSMC, expressing the Tn/STn antigen. Tn/STn expression attenuated TRAIL-induced apoptosis in cancer cells - more specifically, by impairing homo-oligomerization and structural stability of the DR4/5 receptors. Treating these cells with the TRAIL/Apo2L ligands rendered the cancer cells insensitive to cell death. Thus, the Tn/STn antigens can facilitate the escape of tumor cells from apoptosis (21). Expression of COSMC in such Tn-positive cells via transfection facilitated the production of a functional T-synthase, which enabled the extension of Core 1 and Core 2 O-glycan structures. Treating the cancer cells now containing functionally active COSMC/T-synthase with TRAIL ligand-mediated their apoptosis and decreased neoplastic transformation ( Figure 1B ) (21, 22).

Antibodies are crucial components of our response against pathogens. They bring about cytotoxic effects by various mechanisms: (i) Antibody-dependent cellular cytotoxicity (ADCC); (ii) Complement-mediated cytotoxicity (CDC); (iii) Blocking receptors on the surface of cancer cells that lead to dampening of tumor-promoting signaling. In 1981, the first monoclonal antibody (mAb) against the Tn antigen (B72.3) was generated in mice challenged with membrane fractions isolated from human breast cancer samples (52). One of the epitopes recognized by B72.3 is a mucin-like glycoprotein TAG-72, which is routinely used to assess the presence of the Tn antigen in research. As TAG-72 is an antigen that binds Tn/STn targeting antibody, this protein was purified and then administered to mice, which led to the generation of another mAb called CC49, which also reacts with the Tn and STn antigens (53). The CC49 mAb has been tagged with a radiolabel Lutetium-177 and subsequently tested in clinical trials in combination with interferon-α and paclitaxel for the treatment of ovarian cancer (54). Many other mAbs that react with the Tn and STn antigens have since been developed for cancer screening and therapy. One such antibody, TKH2, targeted the STn antigen and was used to coat polymeric nanoparticles loaded with cisplatin. Targeted delivery of this compound increased gemcitabine sensitivity to STn-high PDAC tumors (55). NC318 is another mAb in phase I/II clinical trials (NCT03665285) that blocks Siglec-15 and is being employed as a therapeutic strategy for metastatic solid cancers, including CRC, cholangiocarcinoma and more. Preclinical studies with NC318 demonstrated its ability to alleviate tumor burden and immunosuppression, partly by re-establishing T cell cytotoxicity (56). Gatipotuzumab is another mAb used in phase I/II clinical trials for solid tumors (NCT03360734, NCT01222624) that binds to STn-bearing MUC1 and prevents its interactions with siglec-9, to instigate ADCC against the tumor cells (57, 58).

The role of the Tn and STn antigens in the pathogenesis of gastrointestinal malignancies discussed in this review is manifold. These antigens are expressed on multiple proteins, especially mucins, through various molecular mechanisms that affect the activity of the T synthase, COSMC, and/or ST6GalNAc-I enzymes. Mostly, the expression of these aberrant O-glycoforms leads to tumor-promoting mechanisms. These mechanisms are initiated through a wide range of cell signaling cascades that tumor cells use for their own benefit. In recent literature, the focus has been directed on the role of Tn/STn antigens in affecting components of the tumor microenvironment. The role of the Tn/STn antigens in modulating the behavior of immune cells has ramifications that need to be combated to boost anti-cancer treatment strategies in Tn-high tumors. These antigens predominantly mediate multiple immunosuppressive schemes that facilitate the development of what we now know as an “immune cold” tumor. Fortunately, many targeted therapies are being developed to attenuate such pro-tumor immune escape strategies. This area of glycoproteomic research has seen relatively slower growth because of factors like the low availability of suitable model systems until the last two decades to monitor the immune fraction and issues met with the development of potent molecules like antibodies to detect the Tn/STn antigens. The challenges associated with these therapies have been acknowledged, and further research to dissect the exact mechanisms involved in disease resistance is being avidly conducted.

The manuscript was written and designed by CR and PR. Both authors reviewed and approved the final manuscript.