The Critical Role of GALNTs-Regulated O-GalNAc Glycosylation in Cancer Malignancy.

1
Introduction
According to the International Agency for Research on Cancer (IARC), there were 9.74 million global cancer deaths in 2022, accounting for approximately one‐sixth of all deaths worldwide. In the same year, China reported 2.5742 million cancer‐related deaths. Cancer is widely recognized as one of the most threatening diseases globally [1]. Key challenges in contemporary cancer therapy, including late‐stage diagnosis, tumor recurrence, metastasis, and drug resistance, are clinical manifestations intimately linked to dysregulated protein glycosylation. Moreover, protein glycosylation plays critical roles in diverse intra‐ and extracellular activities such as protein folding, cell adhesion, recognition, and host–pathogen interactions [2, 3, 4]. Hence, it is of paramount significance to identify early cancer biomarkers and specific therapeutic targets derived from protein glycosylation. A comprehensive understanding and characterization of glycosylation alterations within cancer cells may provide critical insights into the mechanisms of tumorigenesis, facilitate the development of biomarkers, and offer innovative strategies for the design of anti‐cancer therapeutics.
O‐GalNAc glycosylation is a widespread protein post‐translational modification, estimated to occur on over 80% of proteins traversing the Golgi apparatus [5], and plays crucial roles in tissue development and homeostasis. On one hand, O‐GalNAc glycans generated through this process are involved in nearly all aspects of biology, including intercellular communication, cell adhesion, signal transduction, immune surveillance, and host–pathogen interactions [6]. On the other hand, advances in mass spectrometry and genetic engineering technologies have enabled quantitative profiling of O‐GalNAc glycosylation and site‐specific analysis of glycoform microheterogeneity in purified proteins [7].
Whereas most protein glycosylation is directed by one or two specific glycosyltransferases that initiate the process by attaching a monosaccharide to defined amino acid residues, mucin‐type O‐glycosylation is catalyzed by a diverse family of 20 GALNTs. These enzymes transfer N‐acetylgalactosamine (GalNAc) from UDP‐GalNAc to serine or threonine hydroxyl groups [8]. This process is closely associated with epithelial developmental defects and various malignancies [9, 10]. Genetic and epigenetic alterations in genes encoding GALNTs members during development and disease can lead to pathological changes [11]. For instance, regulators such as SAM pointed domain‐containing ETS transcription factor (SPDEF) and miR‐125a modulate GALNT7 expression, thereby influencing cancer stemness and malignant phenotypes [12, 13]. Furthermore, the GALNT family selectively regulates the initiation and elongation of O‐GalNAc glycan formation, leading to the generation of Tn antigen, which can be further modified to form sialyl‐Tn or T antigens. These structurally distinct glycans exhibit domain‐specific functional characteristics that contribute significantly to cancer progression.
Accumulating evidence indicates that aberrant glycosylation of glycoproteins, including truncated glycan structures, highly branched N‐glycans, core fucosylation, and increased terminal sialylation, is closely associated with malignant cell transformation and metastasis [14]. Distinct alterations in serum glycoprotein profiles between cancer patients and healthy individuals hold significant potential for identifying tumor biomarkers [15]. Several atypical glycoproteins, including core‐fucosylated alpha‐fetoprotein (AFP) [16] and sialyl Lewis A antigen (SLeA) carried on carbohydrate antigen 19‐9 (CA19‐9) [17], distinct alterations in serum glycoprotein profiles between cancer patients and healthy individuals hold significant potential for identifying tumor biomarkers utilized as serological cancer markers in clinical practice. In addition, recent studies suggest that certain members of the GALNT family may also serve as promising novel biomarkers.
Despite these advances, research on GALNT enzymes and their regulatory mechanisms in protein O‐GalNAc glycosylation remains at an early stage, with limited clinical applications and many aspects of the underlying processes still not fully understood. This review comprehensively summarizes the molecular architecture of GALNT enzymes and their precise regulation of the initiation and elongation of O‐GalNAc glycans. It further outlines the molecular mechanisms by which GALNTs modulate O‐GalNAc glycosylation in tumors and highlights the critical roles of this modification in cancer progression. Finally, we discuss the promising potential of GALNTs and O‐GalNAc glycosylation as novel biomarkers and therapeutic targets in oncology.

2
Overview of Glycosylation
Protein glycosylation refers to the process by which carbohydrate molecules are covalently attached to functional groups of proteins through chemical bonds. This modification occurs in various intracellular compartments (e.g., endoplasmic reticulum, Golgi apparatus) or on the cell surface and is catalyzed by numerous enzymes. It is widely present in eukaryotes, prokaryotes, and certain viruses [18]. Based on the linkage between the glycan and the substrate molecule, glycosylation is primarily classified into N‐glycosylation, O‐glycosylation, and glycosylphosphatidylinositol (GPI)‐anchor types. Among these, N‐linked and O‐linked glycosylation are the two most prominent forms.
In N‐linked glycosylation, an oligosaccharide chain is covalently bound to the side‐chain amide group of an asparagine (Asn) residue via a β‐glycosidic bond, typically occurring within the conserved sequon Asn‐X‐Ser/Thr (X ≠ Pro). It is noteworthy that the first sugar molecule directly linked to asparagine is almost invariably N‐acetylglucosamine (GlcNAc). In contrast, O‐linked glycosylation is a process that primarily modifies the hydroxyl groups of serine (Ser) or threonine (Thr) residues. This reaction is catalyzed by specific glycosyltransferases. In human cells, the first step is carried out by a family of about 20 polypeptide GalNAc‐transferases. These enzymes recognize a wide range of protein substrates and attach an initial N‐acetylgalactosamine (GalNAc) sugar to specific Ser/Thr sites, which then initiates the elongation of the glycan chain [19].
Glycosylation is a fundamental post‐translational modification, sharing the distinction of occurring in approximately 50% of all proteins. It plays essential roles in numerous cellular activities [20], including protein folding, intercellular recognition, immune response, and signal transduction [21]. Growing evidence continues to deepen our understanding of the functional implications of protein glycosylation in disease contexts, particularly in immune system dysfunction and cancer [22, 23, 24].

3
GALNTs: A Large and Diverse Enzyme Family
3.1
Structure of GALNTs
The enzymatic activity of GALNTs initiates O‐glycosylation by transferring an α‐GalNAc moiety from UDP‐GalNAc to Ser/Thr residues, resulting in the Tn antigen (Figure 1). This antigen serves as a precursor for further elongation; it can be sialylated at C6 to form sialyl‐Tn or galactosylated at C3 to generate the T antigen [25]. GALNT enzymes are type II transmembrane proteins composed of approximately 600–800 amino acids and typically contain several key domains, each playing distinct roles in catalysis and substrate recognition. The canonical structure of GALNTs consists of the following major components: Catalytic Domain: Comprising the GT1 and GT2 subdomains. The GT1 domain serves as the core of the glycosyltransferase, responsible for transferring N‐acetylgalactosamine (GalNAc) to glycoproteins and determining the catalytic activity of the enzyme. The GT2 domain, adjacent to GT1, facilitates auxiliary catalytic functions and enhances the efficiency of the transfer reaction; R‐type Glycoprotein Domain: This domain recognizes and binds specific sequences or regions of target proteins, enabling GALNTs to selectively identify substrates and ensure precise O‐glycosylation site placement. Its lectin domain, which is composed of three homologous repeats (α, β, and γ), functions in part through the α and β repeats. These interact with existing substrate O‐GalNAc moieties to boost catalytic activity toward nearby Thr/Ser acceptor sites [26]. In a 2024 study, Collette et al. demonstrated that in GalNAc‐T1, both α and β repeats play unique and coordinated roles in directing O‐glycosylation across diverse glycopeptide substrates. Glycosylation direction is orchestrated by the α and β repeats: the α repeat guides it toward carboxyl‐terminal sites near existing GalNAc, and the β repeat toward amino‐terminal sites. Through multiple substrate‐binding modes enabled by GalNAc‐T1, this concerted mechanism collectively sharpens the recognition of acceptor sites positioned between two established O‐glycans [27]; C‐Terminal Domain: Typically includes a transmembrane region that anchors the enzyme to the endoplasmic reticulum (ER) or Golgi membrane. This feature not only supports the regulation of O‐GalNAc glycosylation via the GALNT activation (GALA) pathway but also reflects the fact that most GALNTs are single‐pass transmembrane enzymes functioning within the ER lumen or Golgi apparatus [28].
Structural variations in the catalytic and R‐type domains determine substrate preferences among GALNT family members. For example, studies have shown that differences in surface charge between Thr and Ser residues influence substrate specificity [29, 30]. Additionally, while the active site of GALNTs is highly conserved, structural diversity in the substrate‐binding pocket further contributes to substrate selectivity [31].

3.2
Functional Roles of GALNTs Through Regulation of O‐GalNAc Glycosylation
The functional repertoire of the GALNT family arises from the partially overlapping but distinct substrate preferences of its individual isoforms. The expansion of the family thereby increases the scope of potential substrates for O‐glycosylation. A positive correlation exists between the diversity of GALNT isoforms and tissue complexity, indicating their role in modifying an extensive array of proteins. In humans, this is reflected in the distinct expression profiles of different GALNTs across cellular and tissue types during specific developmental and differentiation stages [8]. For instance, GALNT1 and GALNT2 are widely expressed in human organs [32], a pattern well‐documented through extensive profiling [33]. Western blot analyses have revealed that GALNT3 is predominantly localized in the pancreas and testes, with minimal expression in other organs [34]. Furthermore, cell‐type‐specific expression patterns have been identified: GALNT11 and GALNT14 are detected only in renal tubules but not in glomeruli [35], while GALNT4 and GALNT6 are highly expressed in mucous cells and weakly in serous cells of salivary glands [8, 36]. These observations highlight that GALNT expression is precisely regulated under normal physiological conditions.
Accumulating evidence indicates that the pathological roles of GALNTs in cancer do not necessarily arise from de novo expression, but rather from quantitative upregulation, spatial or temporal mislocalization, altered substrate accessibility, or dysregulated coordination with downstream glycosylation enzymes. Such context‐dependent alterations enable aberrant O‐GalNAc glycosylation, thereby reprogramming signaling pathways that favor tumor initiation and progression. Aberrant O‐glycosylation mediated by the GALNT family can activate multiple proteins [37], which regulate interactions between stationary and motile cells, influence organogenesis, modulate responses to external stimuli, and promote excessive proliferation and metastasis of tumor cells [38, 39, 40]. GALNT2 acts as a key mediator of malignant features in hepatocellular carcinoma (HCC) by altering the glycosylation of epidermal growth factor receptor (EGFR), thereby driving malignant behavior in HCC cells [41]. Prostate cancer is the most prevalent malignancy in men, resulting in more than 350 000 global deaths each year. The enzyme GALNT7, which alters O‐glycosylation in prostate cancer cells, is linked to the regulation of the cell cycle and immune signaling. Consequently, it promotes tumor malignancy by modulating key processes such as proliferation and immune evasion [15]. Meanwhile, GALNT12 enhances O‐glycosylation of Bone morphogenetic protein receptor 1A (BMPR1A), activating the Bone morphogenetic protein (BMP) pathway. Activated BMP signaling suppresses the expression of integrin αVβ3, thereby reducing bone‐specific metastasis of prostate cancer cells [42]. Pancreatic ductal adenocarcinoma (PDAC) is a notoriously aggressive cancer of the digestive system, characterized by dismal survival outcomes [43]. Studies indicate that reduced expression of GALNT3 alters O‐glycans on EGFR and Her2 in pancreatic cancer cells, enhancing their invasive capacity [44]. Global cancer statistics rank lung cancer as the second most frequently diagnosed form of cancer worldwide [45]. Non‐small cell lung cancer (NSCLC) is the predominant form of the disease, representing the majority of all cases. Despite various therapeutic strategies, the prognosis of NSCLC patients remains poor. High‐throughput sequencing and bioinformatic analyses have revealed frequent overexpression of GALNT2 in NSCLC tissues, which correlates with poor prognosis. Breast cancer is the most fatal malignancy in women. GALNT6 increases O‐glycosylation of α2M, activating the PI3K/Akt signaling pathway and promoting migration and invasion of breast cancer cells [46]. Lung adenocarcinoma remains a threat due to its high recurrence rate and distant metastasis. GALNT6 drives lung cancer cell Epithelial–mesenchymal transition (EMT) by directly binding to the O‐glycosylated chaperone GRP78, which in turn amplifies the MEK1/2/ERK1/2 signaling pathway [47]. In terms of cancer mortality, colorectal cancer holds the position of the second most common cause of death. GALNT2 modifies O‐glycans on AXL receptor tyrosine kinase (AXL) and alters its expression level via a proteasome‐dependent pathway, thereby modulating tumor metastasis and invasiveness [48]. Bladder cancer (BCa) holds the dual distinction of being the most frequent urinary tract malignancy and a globally prevalent cancer. Bladder cancer stem cells (BCSCs) are considered fundamental to BCa initiation and recurrence. GALNT1 mediates O‐linked glycosylation of Sonic hedgehog (SHH) to promote its activation, which is essential for BCSC self‐renewal and bladder tumorigenesis [49].
Beyond epithelial malignancies, aberrant O‐glycosylation has also been implicated in the pathogenesis of retinoblastoma. Studies have shown that the tumor suppressor pRB and its related family members undergo dynamic O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) modification, which is enhanced when pRB is in its active, hypophosphorylated state during the G1 phase of the cell cycle. This modification cooperates with phosphorylation to regulate pRB–E2F–dependent transcription and cell cycle progression. Although mechanistically distinct from GALNT‐mediated O‐GalNAc glycosylation, these findings highlight a broader principle whereby O‐glycosylation serves as an important post‐translational regulatory mechanism contributing to tumor development, including retinoblastoma [50].
In summary, although GALNTs are essential regulators of physiological O‐GalNAc glycosylation in healthy tissues, their dysregulation in cancer leads to context‐dependent remodeling of glycosylation patterns that selectively enhance oncogenic signaling, tumor cell plasticity, and immune evasion. Through O‐GalNAc glycosylation of key regulatory proteins, the GALNT family influences proliferation, invasion, metastasis, resistance to apoptosis, and the establishment of an immunosuppressive tumor microenvironment. Collectively, these findings underscore the critical importance of GALNT‐mediated O‐GalNAc glycosylation in cancer biology and highlight the GALNT family as a promising focus for future mechanistic and translational research (Table 1).

4
Biosynthesis and Spatial Regulation of O‐GalNAc Glycosylation
The biosynthesis of O‐GalNAc glycosylation operates at two primary levels: first, the selection of specific proteins and their modification sites for O‐GalNAc glycosylation, and second, the subsequent processing of GalNAc into mature O‐glycans to exert biological functions. Both processes are tightly regulated by the GALNTs family. This section focuses on how GALNTs regulate the initiation of O‐glycan synthesis and how the GALA pathway [53] modulates the expression levels and spatial localization of GALNTs in a three‐dimensional context, thereby influencing the initiation and elongation of O‐glycans (Figure 2).
4.1
Regulation of O‐GalNAc Glycan Initiation
The initiation of O‐GalNAc glycans is a critical step in glycosylation, involving complex interactions among multiple enzymes and substrates. The initiation of O‐GalNAc glycosylation is catalyzed by the GALNT enzyme family. These enzymes transfer a GalNAc moiety from the donor substrate UDP‐GalNAc to Ser/Thr residues on target proteins, forming an α‐O‐glycosidic bond. This initiation is typically followed by the elongation of the initial GalNAc into diverse core O‐glycan structures in normal cellular contexts. These core structures can undergo elongation, branching, or capping with sialic acids, often through linkages to bridging glycoproteins, thereby enabling diverse glycosylation pathways (Figure 3) [54].
UDP‐GalNAc is synthesized from glucosamine‐1‐phosphate (GlcNAc‐1‐P) and uridine triphosphate (UTP). The synthesized UDP‐GalNAc is transported into the Golgi lumen, where GALNTs transfer the GalNAc moiety to specific serine or threonine sites on glycoproteins. Insufficient cellular levels of UDP‐GalNAc can inhibit the initiation of glycosylation [55]. Although mucin‐type O‐GalNAc glycosylation is initiated directly by GALNTs using UDP‐GalNAc and does not involve lipid‐linked oligosaccharide intermediates, it operates within the broader ER–Golgi glycosylation network. Dolichol and dolichol phosphate function as essential carriers for oligosaccharide assembly in N‐glycosylation and related pathways, maintaining ER–Golgi homeostasis and the availability of glycan precursors. Perturbations in dolichol metabolism can broadly disrupt glycosylation efficiency, protein folding, and intracellular trafficking, indirectly influencing O‐GalNAc glycosylation. These findings highlight the importance of the glycosylation infrastructure in ensuring proper O‐glycan initiation and processing [56, 57, 58]. During protein maturation, O‐GalNAc glycosylation is tightly coordinated with other post‐translational modifications, including N‐glycosylation [59] and phosphorylation [60]. This regulatory network includes not only positive mechanisms but also negative and competitive regulation [61], such as interference from phosphorylation or acetylation modifications and enzymatic competition. Moreover, complex signaling pathways can indirectly regulate the initiation of O‐GalNAc glycosylation by modulating the activity or expression of GALNTs. For example, the Akt/ERK signaling pathway acts through GALNT5 to mediate the development and progression of cholangiocarcinoma [62].
In summary, the expression of GALNTs is co‐regulated at multiple levels: miRNAs exert transcriptional control, while the GALA pathway fine‐tunes their spatial distribution via subcellular localization. Additionally, the initiation of O‐GalNAc glycans is influenced by the availability of glycosylation precursors, crosstalk with other PTMs, and complex signaling pathways. Thus, the initiation of O‐GalNAc glycans is a highly complex process driven by the integration of multiple regulatory factors.

4.2
Regulation of O‐GalNAc Glycan Elongation by the GALNTs Family
O‐GalNAc glycans can be elongated through various mechanisms. The elongation process typically begins with further modification of the Tn antigen, leading to the formation of distinct core structures. Among these, four major core structures have been identified [63], with Core 1 and Core 2 being the most prevalent and thus described in detail here.
A key step in O‐glycan synthesis is the formation of the Core 1 structure, where Core 1 β1,3‐galactosyltransferase 1 (C1GALT1) adds galactose to the C3 position of the Tn antigen's GalNAc. The product of this reaction is the T antigen (Galβ1‐3GalNAcα1‐Ser/Thr) [64]. This structure is expressed in nearly all cell types, particularly in those rich in mucins and circulating glycoproteins [63]. The formation of the Core 2 structure is initiated by GCNT1, which attaches GlcNAc to the C6 position of GalNAc to create a branched glycan. This branching serves as a key structural feature for generating complexity. Core 2, along with Core 3 and Core 4, is predominantly associated with mucosal glycoproteins, especially those from the gastrointestinal and respiratory epithelia and salivary glands.
After the formation of core structures, O‐GalNAc glycans undergo further elongation and modification. During elongation, various glycosyltransferases sequentially add sugar residues to the four core structures, generating increasingly complex polysaccharide chains with diverse functional roles. For example, additional galactose units can be added to the terminal Gal of Core 1 or Core 2 structures to form extended glycan chains. Sialyltransferases (e.g., ST3Gal or ST6Gal families) catalyze the addition of sialic acid (N‐acetylneuraminic acid, Neu5Ac) to terminal galactose or GalNAc residues, producing sialylated O‐glycans. Fucosyltransferases incorporate fucose (Fuc) into O‐glycan chains to form functional structures such as sialyl Lewis X (SLe^x). Glycosyltransferases like GCNT1 can introduce N‐acetylglucosamine to form branched glycan structures, enhancing structural complexity and functional diversity.
In summary, the GALA pathway plays a critical role in elongating O‐GalNAc glycan chains during O‐glycosylation. Through the coordinated action of multiple glycosyltransferases, various sugar moieties are sequentially added to the initial O‐GalNAc structure, resulting in highly complex and functionally diverse O‐glycans.

4.3
Spatial Regulation of the GALNTs Family via the GALA Pathway
The GALA pathway modulates the initiation of O‐GalNAc glycans by regulating the spatial localization of the GALNT family. Below, we elaborate on the spatial regulatory mechanisms of this pathway.
Src kinase promotes the redistribution of GALNTs from the Golgi apparatus to the endoplasmic reticulum (ER), thereby regulating the O‐glycosylation of mucin‐type proteins [65]. As a tyrosine kinase, Src is closely associated with various membrane structures [66] and can be activated by cell surface receptors such as Epidermal growth factor (EGF) or Platelet‐derived growth factor (PDGF). A subset of Src localizes to the Golgi apparatus [67], and previous studies have confirmed its role in modulating Golgi organization [68, 69]. Through lectin staining and metabolic labeling, David J. Gill et al. demonstrated that Src‐dependent relocalization appears specific to the GALNTs family. This mechanism, referred to as the GALA pathway (GALNT Activation), depends on the COPI transport machinery [70]. All tested GALNT enzymes were affected, whereas various other glycosyltransferases were not redistributed from the Golgi. COPI is well‐known for mediating retrograde transport of ER‐resident proteins between the Golgi and ER [71] and can transport multiple molecules, including Basic salivary proline‐rich protein 2 (PRB2) [17]. Upon Src activation, the COPI coat also redistributes into punctate structures that colocalize with GALNT enzymes. The heptameric COPI coatomer complex, Arf small GTPases, guanine nucleotide exchange factors (GEFs), and other components facilitate this process [72]. These findings indicate the involvement of a Src–COPI–GALNTs transport axis in regulating O‐glycosylation.
Using RNAi and imaging‐based screening of 948 signaling genes, Joanne Chia et al. identified 12 negative regulators of O‐glycosylation, all influencing the subcellular localization of GALNT family members. ERK8, an atypical MAPK with high basal kinase activity partially localized to the Golgi, was shown to suppress GALNTs translocation upon its inhibition without affecting KDEL receptor trafficking, suggesting the existence of two independent COPI‐dependent pathways [73]. After Src‐triggered activation and release, the GALNT redistribution proves permanent. Baseline Tn levels are restored hours later, concurrent with GALNT reaccumulation in the Golgi, implying continuous GALNT cycling between these compartments. This dynamic equilibrium results from COPI‐mediated Golgi export being counterbalanced by COPII‐dependent ER‐to‐Golgi transport [74].

4.4
Transcriptional Regulation of the GALNTs Family by microRNAs
Emerging evidence suggests that the expression of certain GALNTs is regulated by microRNAs. Bioinformatics analyses focusing on non‐coding RNA regulation of glycosylation‐related genes have identified GALNT1, GALNT7, and GALNT3 among the top ten GALNT family members most susceptible to miRNA‐mediated regulation [75].
In cervical cancer, high expression of GALNT7 is implicated in enhanced tumor cell proliferation and invasion. The microRNA miR‐125a‐5p acts as a tumor suppressor by directly binding to GALNT7 and reducing its expression post‐transcriptionally. This suppression leads to the subsequent inactivation of the oncogenic EGFR/PI3K/AKT signaling pathway [12]. Similarly, miR‐30e, which is downregulated in clinical cervical cancer specimens and cell lines, functions as a tumor suppressor by transcriptionally regulating GALNT7 [13]. MiR‐30b‐5p acts by directly targeting GALNT7, and through this downregulation, it ultimately inhibits the EGFR/PI3K/AKT pathway, thereby hindering the development of papillary thyroid carcinoma [76]. In colorectal cancer, elevated levels of small nucleolar RNA host gene 7 (SNHG7) act as a molecular sponge for miR‐216b, leading to increased GALNT1 expression and exerting oncogenic effects during colorectal carcinogenesis [77]. Overexpression of miR‐885‐5p suppresses the metastasis and proliferation of cholangiocarcinoma by directly inhibiting GALNT3 and indirectly promoting downregulation of insulin‐like growth factor 2 mRNA‐binding protein 1 (IGF2BP1) [78].
In non‐oncological contexts such as heart failure, GALNT1 and GALNT2 mediate glycosylation‐dependent regulation of Pro‐brain natriuretic peptide (proBNP) secretion. Their elevated expression promotes inhibition of microRNA‐30 transcription, thereby attenuating the compensatory role of BNP during heart failure progression [79].
A recent systematic study integrating lectin microarray data with miRNA expression profiles across 60 cancer cell lines constructed a comprehensive miRNA–glycan regulatory network. This study identified a cluster associated with Tn and TF antigen expression, encompassing 15 miRNAs with numerous predicted GALNT targets [80]. Focusing on miRNA‐200 family targets—key regulators of EMT, the same research group identified “pre‐mesenchymal glycosylation enzymes” potentially involved in TGFβ‐induced EMT, suggesting that miRNA networks can pinpoint glycosylation enzymes critical in disease progression (Table 2).
Collectively, these studies provide compelling evidence for the transcriptional regulation of the GALNT family by microRNAs, indicating that non‐coding RNAs can influence the initiation of O‐GalNAc glycans by modulating GALNT expression levels.

5
Roles of O‐GalNAc Glycosylation in Tumor Initiation and Progression
As the most ubiquitous form of glycosylation, O‐GalNAc glycosylation plays critical roles in various biological processes and regulates protein function at multiple levels, including proteolytic activity, energy metabolism, subcellular localization, and intermolecular interactions. Recent studies have revealed that aberrant O‐GalNAc glycosylation is closely associated with the pathogenesis and progression of numerous diseases, such as cancer, cardiovascular disorders, neurodegenerative diseases, and immune system dysfunctions. Evidence indicates that dysregulated O‐GalNAc glycosylation represents a hallmark of tumor cells, promoting malignant behaviors including proliferation, migration, invasion, protein stability, and signal transduction (Figure 4). The following sections provide a detailed discussion of these mechanisms.
5.1
O‐GalNAc Glycosylation Promotes Malignant Phenotypes in Tumors
5.1.1
O‐GalNAc Glycosylation Enhances Malignant Proliferation of Tumor Cells
The PI3K/AKT signaling pathway plays a crucial role in regulating cell proliferation and survival. Studies have shown that hyperactivation of this pathway occurs in many cancers, leading to alterations in multiple biological functions and ultimately promoting abnormal proliferation of tumor cells. In NSCLC, elevated O‐GalNAc glycosylation has been reported to activate the PI3K/AKT pathway, enhance AKT phosphorylation, and drive malignant proliferation of NSCLC cells [81]. Similarly, in breast cancer, aberrant O‐GalNAc glycosylation indirectly modulates the PI3K/AKT pathway, thereby influencing tumor cell proliferative capacity [46].
GALNT1 is commonly upregulated in gastric cancer, a frequent digestive system cancer, and its high expression is linked to poorer patient outcomes. This enzyme contributes to tumor aggressiveness by facilitating the proliferation, migration, and invasion of gastric cancer cells in cultured systems and living organisms. One key mechanism involves GALNT1‐mediated abnormal O‐glycosylation of CD44, leading to consequent activation of the Wnt/β‐catenin pathway and the promotion of malignant characteristics [39].
O‐GalNAc glycosylation also modulates cell cycle progression. For instance, MUC4, a highly glycosylated mucin aberrantly expressed in PDAC, contributes to cell cycle arrest in PDAC cells expressing branched or truncated O‐GalNAc glycans upon MUC4 knockdown [82]. Furthermore, GALNT2‐mediated O‐glycosylation of ITGA5 influences the activation of PI3K/Akt and MAPK/ERK pathways, thereby regulating cancer cell growth and metastasis [81].
These findings collectively demonstrate that O‐GalNAc glycosylation promotes malignant proliferation by activating proliferation‐related signaling pathways and disrupting cell cycle regulation.

5.1.2
O‐GalNAc Glycosylation Promotes Tumor Cell Invasion and Migration
EMT is a key mechanism underlying tumor cell invasion and migration, primarily driven by inductive signals, transcriptional regulators, and downstream effectors, with the TGF‐β signaling pathway serving as a major mediator of EMT [83]. Evidence indicates that in breast cancer, knockdown of GALNT4 reduces O‐GalNAc modification of type I and II TGF‐β receptors, leading to enhanced receptor dimerization and activation. Notably, a peptide derived from TβRII was identified as a naked peptide substrate of GALNT4, and Ser31 within its extracellular domain was confirmed as an O‐GalNAc glycosylation site upon in vitro glycosylation, ultimately inhibiting breast cancer cell migration and invasion [84].
The Tn antigen, an O‐GalNAc glycan, is commonly overexpressed in a wide range of cancers. Research indicates that the aberrant relocation of GALNT enzymes from the Golgi to the endoplasmic reticulum is a key factor driving elevated Tn levels, a phenomenon documented in cancer cell models and nearly 70% of human breast carcinomas. This glycosylation alteration functionally promotes enhanced adhesion to the extracellular matrix, along with increased migratory and invasive capacities [85]. The redistribution of GALNTs from the Golgi apparatus can induce O‐glycosylation within the ER, a change that potently enhances cell migration and is constitutively activated in more than 60% of breast cancers. The kinase ERK8 serves as a brake on this GALNT relocalization process. Consequently, the loss of ERK8 expression may promote cancer invasiveness by unleashing this mechanism and increasing cellular motility [73].
Recent studies indicate that GALNT6 enhances the stability of Coiled‐coil domain‐containing protein 88C (CCDC88C) by facilitating its O‐glycosylation. The stabilized CCDC88C protein then transcriptionally upregulates Cell migration‐inducing and hyaluronan‐binding protein (CEMIP) mRNA through the transcription factor c‐JUN, thereby driving metastasis in a manner that is functionally dependent on CEMIP [52]. The regulation of O‐GalNAc glycosylation by GALNT3 is maintained across diverse cellular environments, encompassing trophoblast stem cells, blastocyst trophectoderm, and human mammary epithelial cells. In these systems, the loss of GALNT3 results in diminished O‐glycosylation and consistently triggers the onset of epithelial–mesenchymal transition [51]. Other mechanisms, as previously described, include GALNT6‐mediated promotion of EMT through O‐glycosylated chaperone GRP78 and activation of the MEK1/2/ERK1/2 signaling pathway [47].
Collectively, these studies demonstrate that O‐GalNAc glycosylation promotes tumor cell invasion and migration by facilitating EMT, activating the expression of invasion‐ and metastasis‐related genes, altering the subcellular localization of GALNTs, and modulating Tn antigen levels.

5.2
O‐GalNAc Glycosylation Regulates Protein Stability in Tumor Cells
Extracellular proteolysis serves as a key modulator of cellular and tissue functions, though its dysregulation is common in pathology. Recent research reveals that a functional interface exists between proteolytic networks and other major PTMs, including O‐GalNAc glycosylation. This crosstalk represents an essential regulatory layer that influences protease function and supports tissue homeostasis [86].
O‐GalNAc glycosylation influences protein folding and three‐dimensional structure. Glycan chains form interactions, such as hydrogen bonds, with amino acid residues of target proteins, thereby stabilizing their tertiary structure and preventing misfolding and aggregation. For example, Katrine T.‐B. G. Schjoldager noted that loss of O‐GalNAc glycosylation may lead to protein misfolding, triggering endoplasmic reticulum stress and even apoptosis [87].
The interplay between proteolytic processing and O‐GalNAc glycosylation serves as an important regulatory mechanism for fine‐tuning protease activity and maintaining cellular homeostasis. A study employing Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to quantitatively assess the N‐terminome revealed enhanced proteolysis within the extracellular proteome of MDA‐MB‐231 breast cancer cells expressing Tn antigen, highlighting the role of O‐GalNAc glycosylation in modulating protein susceptibility to proteolytic degradation and its key function in regulating proteolytic processing and proteome homeostasis [86].
In tumor cells, however, dysregulated expression, localization, or activity of GALNT enzymes alters O‐GalNAc glycosylation patterns, leading to protein‐specific changes in stability that are not observed in normal tissues. For instance, GALNT6 promotes the degradation of Gasdermin E (GSDME) via O‐GalNAc glycosylation [88], altering the three‐dimensional structure of GSDME and thereby influencing tumor initiation and progression. Colorectal cancer, the second leading cause of cancer‐related deaths, exhibits altered metastasis and invasiveness through GALNT2‐mediated modification of O‐glycans on AXL, which modulates AXL protein stability via a proteasome‐dependent pathway [48].
These observations indicate that while O‐GalNAc glycosylation supports general protein stability in normal cells, tumor‐specific dysregulation selectively reinforces the stability of oncogenic proteins, contributing to malignant progression and distinguishing cancer cells from healthy tissue.

5.3
O‐GalNAc Glycosylation Modulates Signal Transduction in Tumor Cells
At the forefront of cancer research, dysregulated signal transduction has been identified as a critical driver of tumor initiation and progression. As a key form of post‐translational modification, O‐GalNAc glycosylation plays a pivotal role in modulating signal transduction pathways in tumor cells.
The liver is crucial for maintaining core metabolic functions. In hepatocellular carcinoma, a functionally significant Single‐nucleotide polymorphism (SNP) in GALNT14 predicts clinical outcomes in mid‐advanced stages. Research demonstrates that GALNT14 glycosylates Prohibitin‐2 (PHB2), which promotes PHB2‐ Insulin‐like growth factor‐binding protein 6 (IGFBP6) complex formation and initiates IGF1R signal transduction. This signaling axis critically regulates hepatocellular carcinoma growth, migration, and drug tolerance [40].
PDAC is a notoriously aggressive pancreatic malignancy with a very poor survival outlook. The enzyme GALNT6, which is commonly upregulated in PDAC, serves as a key regulator of pyroptosis by acting through both the NF‐κB/NLRP3/GSDMD and GSDME pathways [88].
Elevated expression of GALNT2 is associated with poor prognosis and higher tumor grades in human glioma. It promotes malignant features by modulating O‐GalNAc glycosylation of EGFR and subsequent activation of the PI3K/Akt/mTOR axis [89].
In hepatocellular carcinoma, overexpression of C1GALT1 activates HGF signaling by regulating MET O‐GalNAc glycosylation and dimerization [90].
These tumor‐specific modifications highlight a key distinction from normal physiology: although O‐GalNAc glycosylation is broadly required for signal transduction in tumor cells, its dysregulation selectively stabilizes or activates oncogenic proteins, thereby enhancing malignant phenotypes.
Collectively, these studies highlight that O‐GalNAc glycosylation in tumors is not merely a reflection of normal protein regulation, but is selectively dysregulated to modulate oncogenic signaling, protein stability, and metastasis. Based on our integrative analysis, we propose that specific GALNT isoforms act as tumor‐context–dependent modulators of these processes, suggesting a general principle that may unify diverse cancer‐specific glycosylation phenomena. This perspective, synthesizing isoform‐specific functions across multiple malignancies, represents a novel insight into the role of GALNT‐mediated O‐GalNAc glycosylation in cancer progression.

6
Role of GALNTs Family‐Regulated O‐GalNAc Glycosylation in Cancer Diagnosis and Treatment
Cancer biomarkers play pivotal roles throughout clinical management, including diagnosis, prognostic assessment, disease staging, tracking treatment response, and informing therapeutic decisions. The fundamental criteria for an ideal biomarker encompass high sensitivity and specificity, detectability during early pathogenesis, and concentration dynamics that reflect tumor burden to support accurate staging, therapy monitoring, and recurrence identification. However, only approximately 20 proteins have been approved by the FDA for clinical use, with merely 5 validated for diagnostic purposes [91]. Many existing biomarkers suffer from suboptimal specificity and sensitivity, necessitating their integration with other diagnostic modalities, such as imaging and tissue biopsies, to support informed clinical decision‐making.
Furthermore, the GALNTs family and O‐GalNAc‐glycosylated proteins hold significant potential to enhance the efficacy of cancer therapeutics, including immunotherapy, vaccine development, inhibitor design, combination therapies, and antibody‐based treatments (Figure 5). The following sections elaborate on these applications.
6.1
Glycosylation‐Based Tumor Biomarkers
Current biomarkers often suffer from limited specificity and sensitivity [92], which may be attributed to their reliance solely on protein‐level information. However, human protein levels do not always strongly correlate with disease states, as they can fluctuate with pathological conditions and exhibit individual variability [93]. Interestingly, many reported glycoproteins, such as AFP in liver cancer [94], Carcinoembryonic antigen (CEA) in colorectal cancer [95], and Prostate‐specific antigen (PSA) in prostate cancer [96], which have already been clinically approved as tumor biomarkers.
Aberrant expression of glycosyltransferases and glycosidases plays a key role in dysregulated glycan and glycoprotein profiles. Members of the GALNTs family exhibit distinct substrate specificities and expression patterns, which are frequently altered in various cancers. Those isoforms predominantly or exclusively expressed in cancer cells may serve as reliable biomarkers for disease onset or progression. Research by Masanori Oshi et al. identified a connection between GALNT1 expression and both angiogenesis and EMT processes. The association supports its possible role as a prognostic indicator, notably in the adolescent and young adult (AYA) breast cancer population [97]. Another study revealed that GALNT1 mRNA expression is 11.2‐fold higher in bladder cancer cells than in normal cells, indicating a positive correlation with bladder carcinogenesis and positioning GALNT1 as a potential biomarker for human bladder cancer [98]. In an investigation across multiple independent clinical cohorts, Scott E. Hodgson and colleagues established that GALNT7 expression is elevated in prostate tumors. Their work further indicated that detecting GALNT7 in urine and blood samples can identify prostate cancer patients with superior diagnostic precision compared to relying solely on serum PSA [15].
Glycoproteins dominate the landscape of clinically employed biomarkers, as exemplified by PSA in prostate cancer and CEA in colon cancer. These proteins exhibit altered glycosylation, which particularly in O‐GalNAc glycan patterns and in malignant tissues [99, 100]. GALNT‐mediated O‐GalNAc glycosylation generates specific glycoproducts whose expression and distribution change in cancer patients. Detecting aberrant O‐GalNAc glycosylation in tumor cells or tissues can thus facilitate early cancer diagnosis and disease monitoring [101].
O‐GalNAc glycans, including specific types such as Tn, sialyl‐Tn (STn), Thomsen‐Friedenreich (TF), sialyl‐Lewis X (SLex), and sialyl‐Lewis A (SLea), have been proposed as potential cancer biomarkers. However, the development of stable antibodies targeting these glycans continues to present considerable challenges. The STn antigen results from premature termination of O‐glycosylation, while SLea is a terminal glycan modification. Both are rarely expressed in normal tissues but are significantly upregulated in advanced gastrointestinal cancers, indicating poor prognosis [2]. Lectins, which target most of these glycans, function as key reagents in detection methodologies. Multiple lectin‐based approaches have been validated and put into practice, utilizing methods like immunohistochemistry, blotting, liquid chromatography, and microarray assays [27, 102].
Importantly, the clinical performance of serum glycoprotein biomarkers, such as AFP and CA19‐9 (SLeA), depends not only on absolute concentrations but also on normalization strategies applied in routine clinical testing. In standard practice, these markers are measured using calibrated immunoassays with internal standards and interpreted relative to population‐based reference ranges while accounting for patient‐specific confounders such as liver dysfunction, inflammation, and benign disease conditions.
From a glycosylation perspective, conventional clinical assays primarily quantify total protein levels and do not resolve glycoform‐specific changes. This represents a critical limitation, as cancer‐associated alterations in O‐GalNAc glycosylation may occur independently of overall protein abundance. Emerging strategies, including lectin‐based assays, glycoform‐specific antibodies, and mass spectrometry–based glycoproteomics, allow relative or site‐specific quantification of aberrant O‐glycoforms normalized to total protein levels, thereby enhancing analytical specificity and reducing false‐positive signals.
Importantly, the clinical translation of O‐GalNAc glycosylation–based biomarkers requires rigorous standardization and normalization strategies. Conventional assays often measure total protein levels without resolving glycoform‐specific changes, which can obscure cancer‐associated modifications. Emerging approaches address this limitation by incorporating internal standards, site‐specific glycoform quantification, and normalization to total protein abundance or stable housekeeping glycoproteins. These strategies enhance analytical specificity, reduce inter‐sample variability, and mitigate confounding effects from physiological or pathological fluctuations in protein expression. Furthermore, cross‐laboratory calibration and reference materials are critical to ensure reproducibility and clinical reliability. Integrating these standardized methodologies with high‐throughput mass spectrometry or lectin‐based platforms could facilitate accurate, sensitive, and reproducible detection of O‐GalNAc glycoforms in patient samples, supporting early diagnosis, disease monitoring, and therapeutic decision‐making in oncology (Table 3).

6.2
Therapeutic Applications
O‐GalNAc glycans play a critical role in host–microbe interactions, and alterations in their composition are closely linked to specific diseases and metabolic conditions. This characteristic positions them as promising targets for novel diagnostic and therapeutic interventions. Combined advances in mass spectrometry and mammalian cell line engineering have empowered the high‐throughput systematic analysis of protein O‐glycosylation. This capability enables the creation of comprehensive O‐glycosylation maps and is paving the way for identifying new targets for therapy. These approaches also aid in preventing proteolytic cleavage and modulating binding affinity or receptor function [103].
For instance, Sun et al. reported that elevated GALNT6 levels in bladder cancer are negatively correlated with CD8+ T cell infiltration in the tumor microenvironment and patient prognosis [104]. Similarly, Han Wang et al. developed a prognostic model based on glycosylation‐related genes and demonstrated that GALNT6 and GALNT15 are closely associated with the abundance of tumor‐infiltrating lymphocytes (TILs) [105]. Tanja Ninkovic revealed that O‐glycosylated mucin‐type peptides serve as efficient substrates for the immunoproteasome, highlighting epitopes on human MUC1 tumor‐associated glycans as major targets for anticancer immunotherapy [106]. These findings suggest that the GALNTs family influences immune cell function and shapes the tumor immune microenvironment.
Moreover, O‐GalNAc glycans can alter the structure and properties of tumor cell surface antigens, affecting antigen presentation and subsequent immune responses. Consequently, research is increasingly focused on engaging the immune system against tumor‐associated O‐glycans and GALNTs enzymes through specific antibodies and vaccines. A range of antibodies targeting O‐glycoforms such as Tn, STn, and mucins has been developed. These agents hold promise by mediating both antibody‐dependent cell‐mediated cytotoxicity (ADCC) and direct cytolytic effects, with several showing promising results in vivo [107]. A recent study by Sun describes an innovative approach to selectively engage tumor‐associated Tn antigens, demonstrating the feasibility of Tn‐targeted therapeutic interventions. This work highlights the potential of combining glycan‐targeted therapies with conventional or immune‐based treatments, providing a promising avenue for precision oncology. Integrating such approaches into the broader framework of O‐GalNAc glycosylation–based interventions may enhance both diagnostic specificity and therapeutic efficacy in cancers characterized by aberrant Tn expression [108].

7
Conclusions and Future Perspectives
The GALNTs family plays a central role in regulating O‐GalNAc glycosylation. These enzymes only catalyze the addition of GalNAc residues to specific serine or threonine residues of target proteins, initiating and elongating O‐GalNAc glycosylation via the GALA pathway. Alterations in the expression and activity of GALNT members also influence the structure and function of glycosylated proteins, thereby impacting cancer initiation and progression. O‐GalNAc glycosylation modulates biological processes in tumor cells, including proliferation, invasion, metastasis, and angiogenesis. Aberrant O‐GalNAc glycosylation is closely associated with cell recognition, signal transduction, protein stability, activity, and trafficking, as well as with various diseases such as cancer, inflammation, and metabolic disorders.
Due to the critical role of O‐GalNAc glycosylation in cancer and its regulation by the GALNTs family, it has emerged as a promising target for cancer diagnosis and treatment. Further investigation into the expression regulation, enzymatic activity, and molecular interactions of GALNTs with other molecules is essential to fully elucidate the mechanisms underlying O‐GalNAc glycosylation in cancer.
Although O‐GalNAc glycosylation‐based diagnostic approaches hold significant potential, they currently face challenges related to limited sensitivity and specificity. Future research should focus on the development of advanced analytical platforms, including high‐resolution mass spectrometry, glycoprotein microarrays, and integrated multi‐omics technologies that combine genomics, transcriptomics, proteomics, and metabolomics to enhance diagnostic accuracy and reliability.
Finally, therapeutic strategies targeting O‐GalNAc glycosylation and the GALNT family are still in the exploratory stage. Approaches such as immunotherapy, vaccine development, inhibitor design, combination therapies, and antibody‐based treatments directed against GALNTs or O‐GalNAc glycosylation‐related molecules require continued innovation and validation.

Author Contributions
J.L. and S.W. designed figures, collected relevant literature and drafted the first manuscript. G.Z., J.L., Y.Y. and A.U. provided critical suggestions and edited. S.P. and Y.F. developed the concept of the manuscript and performed revisions.

Funding
We would like to thank the support of the Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University (QNCXTD2023022) and the Joint Fund of Henan Provincial Research and Development Program for Science and Technology (242301420008).

Conflicts of Interest
The authors declare no conflicts of interest.