Glucose consumption is required by living cells. Through glycolysis, glucose is mainly broken down into pyruvate, which enters into the tricarboxylic acid (TCA) cycle for maximum energy production. Cancer cells, however, uptake glucose at a higher rate and produce lactic acid rather than metabolizing pyruvate through the TCA cycle. This adaptive metabolic shift is termed the Warburg effect (1), leading to anaerobic glycolysis, and is thought to provide an evolutionary advantage to cancer cells by providing both increase bioenergetics and biosynthesis (2). Many proto-oncogenes (e.g., Ras and Myc) and tumor suppressors (e.g., p53) influence metabolism, and mutations in these genes can upregulate glucose uptake in cancer cells and promote a metabolic phenotype supporting tumor cell growth and proliferation (3). Elevated glucose uptake in cancer cells can be applied to monitor the location of primary and metastatic tumor sites; for an example, using F-18 fluorodeoxyglucose (FDG), a glucose analog, with a combination of positron emission tomography/computed tomography (PET/CT) (4). In general, most glucose enters into the glycolytic pathway, but a small fraction of glucose goes to the hexosamine biosynthesis pathway (HBP). This pathway generates a nucleotide sugar, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), used for many reactions including a sugar donor, and in multiple glycosylation reactions such as proteoglycan synthesis, N-linked glycosylation, and the formation of O-linked glycoproteins or O-GlcNAcylation (5).

The O-GlcNAc is dynamically regulated by two key enzymes: O-GlcNAc transferase (OGT) (6) and O-GlcNAcase (OGA) (7), for the addition and removal of a single GlcNAc residue from proteins, respectively. Unlike classical glycosylation present in endoplasmic reticulum (ER) and golgi apparatus, O-GlcNAcylation takes place in the cytoplasm, nucleus, and mitochondria, and is implicated in a wide range of effects on cellular function and signaling in metabolic diseases and cancer (8). Almost three decades since its discovery, more than 1,000 O-GlcNAcylated proteins have now been identified (9). Growing evidence reveals that O-GlcNAcylation has extensive crosstalk with phosphorylation either on the same or adjacent sites of various proteins (10). The interplay of these two post-translational protein modifications (PTMs) can work to fine-tune the regulation of target protein functions, stabilization, translocation, complex formation, and enzyme activity, which subsequently affects cellular signaling pathways. Glucose flux into the HBP resulting in increased O-GlcNAcylation is an emerging paradigm of the integration of metabolic and signaling networks. This review emphasizes the recent connection between the HBP and metabolic shifts, O-GlcNAc cycling enzymes, and O-GlcNAcylation and phosphorylation found in most cancers. In addition, O-GlcNAcylated proteins have been identified in primary breast and colorectal cancer (CRC) as well as in their cancer cell lines. Moreover, O-GlcNAcylated proteins are discussed as potential candidates for novel biomarkers of cancer.

The final end product of the HBP is UDP-GlcNAc (Figure 1), which is built up from glucose, glutamine, fatty acid (acetyl-CoA), and uridine. O-GlcNAc level may, therefore, be considered as a nutrient sensor for both normal physiology and disease pathophysiology, such as diabetes and cancer. Approximately 2–4% of glucose uptake into the cells enters into the HBP (11). Oncogenic genes (e.g., c-Myc, Kras) and hypoxia contribute in glucose metabolism. Osthus et al. showed that overexpression of c-Myc directly transactivates genes encoding glucose transporter GLUT1 and increases glucose uptake (12). Ying et al. also reported that activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) is required in stimulating glucose uptake in an in vivo model of pancreatic ductal adenocarcinoma (PDAC) (13). Loss of Kras functions led to the downregulation of glucose uptake. Moreover, the levels of metabolites of the pentose phosphate pathway (PPP) and the HBP, as well as O-GlcNAcylation were decreased upon Kras inactivation. The rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the conversion of fructose-6-phosphate to glucosamine-6-phosphate. Hypoxia has been reported to induce the transcription of GFAT gene through the hypoxia responsive element (HRE) (14). Guillaumond et al. also showed that hypoxia increases the levels of GFAT mRNA expression and O-GlcNAcylation in pancreatic cancer cells (15). Blocking of GFAT activity by azaserine led to a decrease in hypoxic cell number, suggesting that activation of the HBP is required for survival of hypoxic pancreatic cancer cells. Glucosamine, although normally present at low levels in bodily fluids, enters into cells via the glucose transporters (16) and is phosphorylated to glucosamine-6-phosphate by hexokinase, thereby bypassing GFAT and elevating UDP-GlcNAc levels. Recently, Yang et al. demonstrated that radiolabeled glucosamine analogs can be introduced as novel agents to complement FDG imaging to increase specificity and improve the accuracy of lesion size in oncology applications (17). Glucosamine analogs become UDP-GlcNAc analogs and the newly modified O-GlcNAc analog proteins catalyzed by OGT are found in the cytoplasm and nucleus, whereas FDG, a glucose analog, is not metabolized and remains in the cytoplasm of cells. This method can be used to tag transcription factors known to be modified by O-GlcNAc (e.g., Sp1 and NF-κB), which are moved from the cytoplasm into the nucleus upon stimulation or activation in cancer cells. Glucosamine analogs can, therefore, be used in nuclear imaging to observe bio-activity in tumors.

In cancer cells, hyperglycemia is thought to feed the HBP and promote abnormally elevated O-GlcNAcylation of key signaling proteins (18, 19). Phosphofructokinase 1 (PFK1), one of the key enzymes in the glycolysis pathway, is modified by O-GlcNAc at Ser-529 in response to hypoxia in the lung cancer cell line, H1299 (20). This glycosylation suppressed PFK1 activity and redirected glucose flux through the PPP, thus increasing nucleotide metabolism and providing a growth advantage for cancer cells. Blocking of O-GlcNAcylated PFK1 led to a reduction of cancer cell proliferation in vitro and impaired tumor formation in vivo. Other glycolytic enzymes also reported to be modified by O-GlcNAc include triose phosphate isomerase (TPI) (21), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (22–25), enolase 2 (Eno2) (22, 25–27), and pyruvate kinase M2 (PKM2) (25). Although the glycosylation sites of these glycolytic enzymes have not been identified, their modifications could potentially modulate tumor cell metabolism promoting proliferation.

Glutamine is a major source of energy for rapidly dividing cells and is also an amino donor substrate for the conversion of fructose-6-phosphate to glucosamine-6-phosphate by GFAT in the HBP. Glutamine enters into cells via the glutamine transporter, which is found to be overexpressed in various cancers (28, 29). If GFAT is active, glutamine uptake in cancer cells can increase the HBP flux, as well as UDP-GlcNAc, and O-GlcNAcylation. This augmentation, for example, is found in human PDAC (30). In a related context, inhibition of glutaminase, an amidohydrolase enzyme, which generates glutamate from glutamine, led to a lower proliferation rate in human breast cancer cells (31). This inhibition also caused a reduction of GFAT activity and changes in O-GlcNAc targets, such as OGT and transcription factor Sp1. Moreover, removal of glutamine from the culture medium promotes tumor cell differentiation and decreased proliferation; conversely, addition of glutamine protects cells from apoptosis and induces proliferation (32). Thus, metabolic shifts through the HBP flux, hyperglycemia, glutamine consumption, elevation of UDP-GlcNAc, and O-GlcNAcylation contribute in regulating signaling cascades and cell proliferation in cancer.

O-GlcNAcylation is tightly regulated by O-GlcNAc cycling enzymes, OGT and OGA for the addition and removal of sugar in and out of proteins, respectively. In human beings, there is only a single OGT gene localized at chromosome Xq13.1 (33). The alternative splicing of the OGT gene translates into at least three different isoforms of OGT enzyme, including the 110-kDa nucleocytoplasmic isoform (ncOGT), the 103-kDa mitochondrial isoform (mOGT), and the short 78-kDa isoform (sOGT) (34). The different isoforms of OGT differ in the number of tetra-tricopeptide repeats (TRP) located at the N-terminal domain, which is involved in protein interaction and is important for protein substrate recognition (33). OGT is inhibited by various inhibitors such as ST045849 (35). A single OGA encoding gene, identified as meningioma-expressed antigen 5 (MGEA5), is localized on chromosome 10q24.1–q24.3 (33). The two isoforms of OGA from alternative splicing have been identified (7). The 130-kDa isoform localizes predominantly in the cytoplasm, while the 75-kDa isoform, lacking one-third of the C-terminal domain, resides in the nucleus (33). The N-terminal domain of OGA is the catalytic domain, while the C-terminal domain contains a histone acetyltransferase (HATs) sequence (33). This enzyme can be inhibited with inhibitors such as PUGNAc, Thiamet G (36), and GlcNAcstatin (37). A number of studies have revealed complex formation between OGA and OGT. In a yeast-two hybrid screening study, OGA has been reported as a binding partner of OGT (38). The OGA-OGT complex has also been identified in combination with mSin3A, and histone deacetylase-1 (HDAC1) when the estrogen and progesterone signaling are stimulated in CHO cells (39). Therefore, it is possible that the binding between OGA and OGT may affect the regulation of each other under specific conditions.

O-GlcNAcylation in cancer has been increasingly studied for the past decade. Accumulating evidence reveals that the levels of O-GlcNAcylation and its cycling enzymes in malignant tissues are altered in various cancers. Recently, we showed that O-GlcNAcylation and OGT expression levels are increased in both breast and CRC (25, 40). As mentioned above, the O-GlcNAc cycling enzymes OGT and OGA tightly regulate the level of O-GlcNAcylation. Enhanced O-GlcNAcylation level corresponding to increased OGT and decreased OGA expression is commonly observed in various cancers including bone (41), bladder (42), breast (26, 43–45), bile duct (46), colon (47–49), leukemia (50), liver (51), lung (47), ovary (36), pancreas (30), and prostate (52, 53). Alteration of this glycosylation in thyroid cancer, however, occurs in the opposite way (54). Interestingly, no mutations in OGT and OGA genes have been reported in human cancers, suggesting that these enzymes are tightly conserved. Therefore, besides increased flux through the HBP, the altered expression of OGT and OGA also contributes to enhancement of O-GlcNAcylation in most cancers.

Manipulation and regulation of O-GlcNAc cycling enzymes in cancer may be a way of stopping cancer growth. We have shown that OGT silencing led to a reduction of anchorage-independent growth of a breast cancer cell line, MDA-MB-231 (25). Caldwell et al. also reported that reduction of OGT in breast cancer cells caused inhibition of tumor growth, both in vitro and in vivo (43). OGT knockdown did not block cell growth in a non-transformed breast cell line, MCF-10A (43). Consistent with this finding, reduction of O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited human PDAC cell proliferation and anchorage-independent growth, and triggered apoptosis (30). Because cancer cells appear to overexpress OGT, strategies to reduce OGT activity or expression level are attractive as an anti-cancer approach. Recently, the crystal structure of human OGT was solved by Lazarus et al. (55). As mentioned above, there is only one OGT gene in mammals with three alternative splicing isoforms, but more than one thousand O-GlcNAc proteins have been identified so far (dbOGAP: Database of O-GlcNAcylated Proteins and Sites). How does one OGT specifically modify a wide range of target proteins? OGT actually has protein-binding partners, which can form a transient complex under specific stimulation such as by nutrient, stress, and hormone. These partners include p38, OIP106, and OGA (38). The binding between OGT and its adaptor specifically targets the catalytic site of OGT to O-GlcNAcylate its target proteins (55). This knowledge will accelerate the rational design of OGT inhibitors for anti-cancer drug in the future.

O-GlcNAcylation has been studied widely for 30 years. Novel methodologies for enrichment of O-GlcNAc modified proteins are now available, as well as mass spectrometric methods for their characterization. Increasing study of O-GlcNAc proteins suggest extensive crosstalk between O-GlcNAcylation and phosphorylation. Crosstalk between these two modifications occurs not only by sharing their protein substrates but also by regulating each other’s cycling enzymes. Both post-translational modifications share many characteristics including the cycling of their substrates at a similar time scale, the site of modification, and cellular state (33). This becomes more complicated when several studies show O-GlcNAcylation of many kinases (56), as well as phosphorylation of OGT and OGA (10).

The O-GlcNAc attachment sites can be predicted using online software (57) such as Yin-Yang and dbOGAP (Database of O-GlcNAcylated Proteins and Sites). These programs can be used to determine the interplay between O-GlcNAcylation and phosphorylation. The crosstalk between O-GlcNAcylation and phosphorylation in cancer has been observed in various biological signaling regulators, including c-Myc (58, 59), p53 (57, 60), Snail1 (61), and NF-κB p65 subunit (30).

c-Myc is a transcription factor regulating transcription of many genes involved in cell proliferation, cell differentiation, and programed cell death, and displays a reciprocal interplay between both modifications. c-Myc at Thr-58 can be both a target for phosphorylation by GSK3 and O-GlcNAcylation (58, 59). Crosstalk between these two modifications is competitive depending on certain conditions. For example, O-GlcNAc modification at Thr-58 of c-Myc was higher than phosphorylation at the same site when cells are starved of serum. Serum stimulated cells showed the opposite result, since Thr-58 shows enhanced phosphorylation and decreased O-GlcNAcylation. Moreover, point mutation at Thr-58 in the coding region of c-myc is frequently found in human Burkitt lymphomas (59, 62). Therefore, modifications of this site might be crucial for tumor progression.

Yang et al. reported O-GlcNAc modification of p53 at Ser-149 in a breast cancer cell line, MCF-7 and a lung cancer cell line, H1299 (57, 60). O-GlcNAcylation at Ser-149 reduced phosphorylation at Thr-155, leading to disruption of binding between Mdm2 and p53, which consequently reduced p53 ubiquitin-proteasome degradation (57). Another study by Park et al. showed the important role of O-GlcNAcylation of Snail1, a transcriptional repressor of E-cadherin (61). Snail1 is phosphorylated by GSK-3β, promoting its ubiquitination and degradation (63). Elevated O-GlcNAc level caused by OGA inhibitors inhibited the phosphorylation-mediated proteasomal degradation of Snail1 and consequently increased Snail1 half-life in a similar manner to p53.

Nuclear factor-kappa B (NF-κB) is a well-known transcription factor regulating cytokine production, lymphocyte activation, and proliferation. NF-κB activation was found in lymphoma and many solid tumors (64). NF-κB is a dimer of p65 (RelA) and p50 subunits. Ma et al. reported O-GlcNAc modification of NF-κB p65 subunit and IKKα/IKKβ in human PDAC (30). Phosphorylation of the p65 subunit was increased when global O-GlcNAcylation was reduced.

Crosstalk between O-GlcNAcylation and phosphorylation in cancer are not always reciprocal. Examples include vimentin and heat shock protein 27 (HSP27), as well as keratin 8 and 18. O-GlcNAc sites of vimentin are on Ser-7, Thr-33, Ser-34, and Ser-54 (65). Alteration of O-GlcNAc level by OGA overexpression in HeLa cells led to decreased pSer-82 level and increased pSer-71 level in vimentin (66). Guo et al. also showed that nuclear translocation of HSP27 observed in liver cancer cells is regulated by both O-GlcNAc and phosphate groups (67). Another study from Srikanth et al. displayed the synergistic effects of O-GlcNAcylation and phosphorylation on keratin 8 and 18 (68). The more O-GlcNAc modification occurs, the more phosphorylation was observed in soluble keratins compared to filamentous form. Moreover, increased O-GlcNAcylation and phosphorylation of keratin 8 and 18 were observed in heat stress-induced HepG2 cells.

Current information strongly suggests that alteration of cellular metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis provides both bioenergetics and biosynthesis capability for cancer cells. Increasing glucose uptake and the redirection of glucose to the HBP flux can lead to an increase of UDP-GlcNAc and O-GlcNAcylation levels in cancer. Mutation of genes involving glucose uptake also contributes to this. In addition, O-GlcNAc cycling enzymes are altered in most cancers. Changes in O-GlcNAc and OGT levels clearly show positive relationship with the histological grade of breast and colorectal tumors. Greater increases in O-GlcNAc levels correlated with higher grades of tumor development. Importantly, this modification has extensive crosstalk with phosphorylation, which consequently affects cellular signaling. Thus, metabolic shifts through the HBP flux and O-GlcNAcylation contribute to regulation of signaling cascades and cell proliferation in cancer.

Study of O-GlcNAcylation in cancer is rapidly growing in terms of both detection and functional studies using in vitro and in vivo models. Several O-GlcNAc modified proteins have been discovered in breast and CRC. However, more O-GlcNAcylated proteins have been identified in breast cancer in comparison with CRC. This may result from differences in protein expression in each specific organ, as well as the tumor grade examined in the study. In addition, the levels of O-GlcNAc cycling enzymes differ. In breast cancer, major identified proteins are glycolytic enzymes and proteins, which function in biosynthesis (nucleic acid metabolism). The modified protein groups shared in breast and CRC are (1) proteins, which function in the stress responses; (2) hnRNPs and proteins involved in transcription and translation; (3) proteins related to the cytoskeleton and their regulation; and (4) transcription factors (e.g., Snail1 and β-catenin), which are difficult to observe and need to be enriched before detection. In addition, a number of O-GlcNAc targets in breast and CRC are not shown to be modified directly but rather regulate gene and/or protein expression such as FoxM1, Caveolin-1, and IκB-β. Moreover, more O-GlcNAc modified proteins were identified from breast cancer tissues than from breast cancer cell lines, indicating the complexity of O-GlcNAc regulation in vivo (25). O-GlcNAc modified proteins identified from clinical samples are thus more realistic as potential novel cancer biomarkers. Examples are PKM2 in breast cancer (25) and annexin A2 in CRC (40). However, larger scale studies need to be performed to obtain information with greater accuracy and reliability for possible use in clinical detection. On another hand, O-GlcNAc research on cell lines is also needed in order to test mechanisms and functions. A good example is that of O-GlcNAcylation of PFK1, which leads to decrease in activity and redirection of glucose metabolism in cancer cell lines (20). Alteration of OGT expression both in breast cancer cells (in vitro) and in animal model (in vivo) also suggests a promising approach for anti-cancer therapy (43). Research from both specimen samples and cell lines is, therefore, needed to provide a better understanding of O-GlcNAc biology in cancer. Taken together, this review shows the current findings on O-GlcNAcylation in breast and CRC. Many O-GlcNAc modified proteins are promising as potential novel cancer biomarkers or may be used in combination with standard detection (e.g., serum biomarkers) to enhance specificity and accuracy. Ongoing research will aim to detect these modified proteins in large-scale samples specifically and rapidly. Many detection techniques such as advanced mass analyzers with new fragmentation techniques including electron transfer dissociation (ETD) are being developed. Specific O-GlcNAc modified proteins in cancer specimens, therefore, are challenging to discover as potential candidates for cancer diagnosis, especially in breast and CRC.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.