Increased Levels of Tetra-antennary N-Linked Glycan but Not Core Fucosylation Are Associated with Hepatocellular Carcinoma Tissue

Infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the major etiology of hepatocellular carcinoma (HCC; refs. 1–4). Both HBV and HCV cause acute and chronic liver infections, and most chronically infected individuals remain asymptomatic for many years (5). Between 10% and 40% of all chronic HBV carriers eventually develop primary liver cancer (HCC), and it is estimated that more than one million people worldwide die of HBV/HCV-associated liver cancer (2, 6, 7). Indeed, HBV and HCV infections are associated with more than 80% of all HCC cases worldwide and can be as high as 96% in regions where these viruses are endemic (3).

The progression from liver disease to liver cancer is often monitored with serum levels of oncofetal glycoprotein, α-fetoprotein (AFP), or the core fucosylated glycoform of AFP (AFP-L3; refs. 8–10). However, AFP can be produced under circumstances other than HCC, including association with other liver diseases (8–10), and it is not elevated in all patients with HCC (11). Therefore, the reliability of AFP levels as a screening tool for HCC has been questioned (12), and more sensitive serum biomarkers for HCC are desired.

Using various proteomic methods to look for biomarkers useful in the early detection of HCC, we identified changes in the levels of N-glycan in total serum and in serum depleted of IgG and/or major acute-phase proteins (13–15). The change in glycosylation observed in the serum associated with HCC was an increase in the level of fucosylation, which has also been reported by others. In this study, we expanded our search by analyzing the glycosylation of HCC tissue as compared with the noncancerous adjacent tissue. In addition, we have examined the N-linked glycosylation of normal healthy livers. Surprisingly, increased levels of fucosylation were not observed in the HCC tissue as compared with either surrounding tissue or “healthy” tissue. Thus, the increased levels of fucosylation that are observed in sera of patients with HCC, but not in those of benign liver diseases, suggests an abnormal secretion of fucosylated proteins in HCC. In contrast, increased levels of tetra-antennary glycan were observed in the HCC tissue as compared with both the surrounding tissue and the tissue from a normal liver. Tetra-antennary glycans result from increased activity of the N-acetylglucosaminyltransferase V (GnT-V) enzyme, which has long been associated with cancer development and metastatic potential (16–19). This is the first report of increased levels of tetra-antennary glycan in HCC tissue. Importantly, this change was found in both AFP-positive and more importantly, AFP-negative tumors, indicating its potential role as a cancer biomarker.

The present study enrolled 16 patients of ages 62.4 ± 4.2 (10 males, 5 females, and 1 NA) with HCC who had undergone surgical resection between January 1992 and December 1997 at Osaka Rosai Hospital (Nakasonecho, Kitaku, Sakai, Japan). For all patients, hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), and HCV antibody were routinely examined by commercially available methods. Positivity for HCV-RNA was confirmed in a subset of patients with HCV. Of the 16 patients enrolled, HBsAg and HCV antibody were positive in 4 and 10 patients, respectively. One patient was double negative for HBV and HCV and another patient had no clinical information. Clinical data of each patients are described in Table 1. All patients with HCC had no history of treatment for HCC before the operation in which liver samples were collected. Tumor samples and nontumor portions of the liver were obtained during the surgical resection and were snap-frozen in liquid nitrogen and stored at −80°C until used. Nontumor tissues were collected at a distance of at least 5 cm from the cancer lesions. All of the liver samples were histologically examined by an experienced pathologist who had no knowledge of the analytic results. This project was approved by the Ethics Committees of Osaka Rosai Hospital hospitals and written informed consent was obtained from patients in this study.

Tissue was sliced into fine pieces and dounce homogenized in a 30 mmol/L Tris-HCl pH 7.4 buffer containing 0.3% SDS, 3% dithiothreitol (DTT), 4 mmol/L MgCl₂, and a Protease Inhibitor Cocktail (Roche) and placed on ice for 1 hour. The lysate was spun down at 14,000 rpm for 30 minutes and buffer exchanged into PBS at pH 7.2. Protein concentration was assayed using a NanoDrop (ThermoFisher).

Four hundred micrograms of protein lysate or 5 μl of human serum was absorbed into a 3 mm dehydrated gel plugs. The protein gel plugs were then reduced by boiling in 35 mmol/L DTT for 5 minutes, and alkylated at room temperature in 100 mmol/L iodoacetamide. The gel plugs were fixed, 3 × 30 minutes in a solution of 40% methanol and 7% acetic acid. A washing step is conducted by dehydration in acetonitrile, rehydration in 20 mmol/L ammonium bicarbonate, and dehydration with acetonitrile, then dried in a speedVac. Ten microunits of peptide:N-glycosidase F (Prozyme) was diluted with 7 μL of 20 mmol/L ammonium bicarbonate, pH 7, and allowed to adsorb into the gel plug. The gel plug was then covered with buffer and allowed to incubate overnight at 37°C. The glycans were eluted from the gel plug by sonication in Milli-Q water 3 times; the pooled elutant was dried down and labeled with a 2-aminobenzamide dye (Ludger) according to the manufacturer's instructions. Excess dye was removed with paper chromatography and the glycans were passed through a 0.22-μm syringe filter. Fluorescently labeled glycans were subsequently analyzed by high-performance liquid chromatography (HPLC) using a TSK-Gel Amide 80 column (Tosoh). The mobile phase consisted of solvent A (50 mmol/L ammonium formate, pH 4.4) and solvent B (acetonitrile). The gradient used was as follows: linear gradient from 20% to 58% solvent A at 0.4 mL/min for 152 minutes followed by a linear gradient from 58% to 100% solvent A for the next 3 minutes. The flow rate was increased to 1.0 mL/min, and the column was washed in 100% solvent A for 5 minutes. Following the washing step, the column was equilibrated in 20% solvent A for 22 minutes in preparation for the next sample run. HPLC analysis was conducted with the Waters Alliance HPLC System, complemented with a Waters fluorescence detector, and quantified with the Millennium Chromatography Manager (Waters Corporation). Glycan structures were identified by calculating the glucose unit value and exoglycosidase digestion, as previously described (20).

Descriptive statistics for patients were compared by scatter plots that included the outliers. All values were reported as mean values ± SD unless otherwise stated. Because the data did not follow a typical Gaussian distribution, a nonparametric test (2-tailed, 95% confidence interval, Mann–Whitney U test) was used to determine statistical differences between groups. To determine the optimal cutoff value for each marker, the receiver operating characteristic (ROC) curves were constructed using all possible cutoff values for each assay. The area under the ROC (AUROC) curves were constructed and compared as described previously. A 2-tailed P value of 0.05 was used to determine statistical significance. All descriptive analyses were conducted using a GraphPad Prism. For combinatorial analysis, AFP values were log transformed to bring the values of all markers into a similar scale, and a centering and scaling approach was taken to normalize data before analysis using a multivariate logistic regression method, using the R package, version 2.8.1.

In previous work, we and others, have observed increased levels of core fucosylated glycan in the serum of patients with HCC (13–15, 21–25). In an effort to determine whether the cancerous tissue is the source of the increased level of serum core fucosylation we have conducted N-linked glycan analysis of normal, tumor, and adjacent nontumor (fibrotic) tissue. Sixteen tumor samples, along with adjacent tissue obtained from patients undergoing tumor resection are described in Table 1. In addition, “normal, healthy” tissue was obtained from 3 independent “control livers” from commercial sources. For all tissue, total protein lysates were made and the amount of total protein was quantified before the N-linked glycans attached to total protein were removed enzymatically using peptide:N-glycosidase F and labeled with a fluorescent dye before analysis of the N-linked glycans via sequential exoglycosidase digestion (26–28).

Figure 1A and B shows the simplified desialylated glycoprofile for a representative patient set (HCC and surrounding tissue) following treatment with neuraminidase (Arthrobacter ureafaciens). Each peak corresponds to a different glycan structure (or multiple structures) and as this figure shows, little difference is observed between the adjacent and HCC tissue from the individual patient shown. Sequential exoglycosidase digestion (data not shown) was used to identify the core fucosylated N-linked glycan. Two major types of core (α-1,6 linked) fucosylated glycan were observed in the liver tissue, a core fucosylated biantennary glycan and a core fucosylated triantennary glycan. The levels of each of these structures in the adjacent and HCC tissue are shown in Fig. 1C and D. As this figure shows, the level of core fucosylation is not substantially increased in the patients with HCC as compared with the adjacent tissue or normal tissue from a donor liver. Figure 1C shows the relative levels of the core fucosylated biantennary glycan in all 16 tissue pairs. As summarized in Table 2, the core fucosylated biantennary peak had a mean value of 13.34% of the total glycan pool in the adjacent tissue, and 14.81% in the HCC tissue. This compares with an average of 11.6% in the control tissue (Table 3). None of these differences were statistically different. Pairwise statistical analysis was also used to determine whether difference in core fucosylation could be observed between patient pairs; however, no statistically significant differences could be observed.

As Fig. 1A and B highlight, a core fucosylated triantennary glycan was also observed (peak 5). The levels of this glycan in the 16 tissue pairs is shown in Fig. 1D and similar to the results observed in Fig. 1C, the levels of this core fucosylated species are not consistently altered in the HCC tissue as compared with the adjacent tissue. As summarized in Table 2, the core fucosylated triantennary peak had a mean value of 2.66% of the total glycan pool in the adjacent tissue, and 3.32% in the HCC tissue. This compares with an average of 2.9% in the control tissue (Table 3). These numbers were not statistically different (P > 0.05).

In contrast to core fucosylation, which was not consistently altered in the HCC tissue, as compared with the adjacent tissue, increased levels of tetra-antennary glycan (A4G4) were observed in the HCCs when compared with the adjacent tissue. Figure 2 shows a focus of the tetra-antennary N-linked glycan following treatment with neuraminidase (Arthrobacter ureafaciens) and α-(1-2,3,6)-jack bean mannosidase (Fig. 2A). Subsequently, the peak preliminarily identified as a A4G4 glycan was collected and digested individually with almond meal and bovine kidney fucosidase (for alpha 1-3,4,6-linked fucose; Fig. 2B) and bovine jack bean β-(1-4,6) galactosidase (Fig. 2C). As Fig. 2 shows, whereas treatment with the fucosidases did not alter this peak (Fig. 2B), indicating that this peak is not fucosylated, treatment with the beta-galactosidase shifted the peak to a level consistent with an A4G0 glycan (an agalactosylated tetra-antennary glycan). Digestion of this structure with hexosaminidase (Glyko; β-N-Acetylhexosaminidase/HEXase I) digested this structure to the chitobiose core (M3N2), confirming that this peak consisted of a tetra-antennary N-linked glycan. The levels of the tetra-antennary N-linked glycan in the HCC and adjacent tissue are shown in Fig. 2D. As this figure shows, the level of the tetra-antennary glycan was increased in 14 of 16 HCC samples, as compared with the adjacent tissue. The levels of the tetra-antennary glycan in normal tissue were similar to what was observed in the adjacent tissue (see Tables 2 and 3). The complete profile of all 16 tissue pairs, with the A4G4 peak indicated are shown in Supplementary Fig. S1.

The mean level of the A4G4 glycan in the HCC tissue was 2.87% of the total glycan pool and 1.55% in the adjacent tissue. Although this is less than a 2-fold difference, 14 of 16 of the HCC tissues were elevated for the glycan relative to the matched nontumor samples, and the mean difference was statistically different (P = 0.0003). The level of this glycan in normal liver was 1.59, very similar to the level observed in the nontumor tissue (Table 3).

In an effort to further confirm the results obtained by N-linked glycan sequencing, we conducted a lectin blot of total pooled protein from either the HCC or adjacent tissue. In Fig. 3B, the fucose binding Aleuria aurantia lectin (AAL) was used to determine the level of fucosylation in the HCC and the adjacent tissue. Consistent with the results obtained via N-linked glycan sequencing, the AAL lectin blot showed a similar banding pattern in the HCC tissue and in the adjacent tissue, indicating no major change in fucosylation in HCC tissue.

Changes in branching were analyzed using the Datura stramonium lectin (DSL), which has high affinity toward tri- and tetra-antennary complex-type oligosaccharides. As Fig. 3C shows, consistent with the results obtained in Fig. 2, there was substantially more binding of the DSL lectin with the HCC tissue, as compared with the adjacent tissue, suggesting increased levels of branching in the HCC tissue.

In an effort to determine whether the increased level of branched N-linked observed in the tissue of patients with HCC could be seen in the serum of patients, we have conducted glycan analysis on serum from 9 HCC and 8 cirrhotic patients. The N-linked glycan profile, with a focus on the A4G4 glycan, for 4 representative HCC and 4 cirrhotic, patients is shown. As Fig. 4A shows, there is an increase in the level of the A4G4 glycan in the HCC samples as compared with the cirrhotic samples. Figure 4B shows the level of the A4G4 glycan in all 9 HCC and 8 cirrhotic samples. As this figure shows, the level of A4G4 glycan in the HCC samples ranged from 1.33% of the total glycan pool to 4.8% of the total glycan pool with a media value of 3.00 (±1.13). In the patients with cirrhosis the level of A4G4 glycan ranged from 1.0% of the total glycan pool to 3.0% of the total glycan pool with a median value of 1.98 (±0.653). Although the sample size is small, the difference was statistically significant (P = 0.032).

Changes in N-linked glycosylation on serum proteins have been associated by us and by others with liver cancer. The most prominent change has been the increased levels of α-1,6- and α-1,3–linked fucosylation (13–15, 21, 22, 29–35). In an effort to identify the source of this increase we have conducted N-linked glycosylation analysis of HCC tissue, surrounding tissue and liver tissue from nondiseased livers. Surprisingly, the normal, HCC, and tumor-adjacent tissue had similar levels of fucosylation, suggesting that the increase in liver-derived fucosylated proteins observed in the circulation is not the result of increased production from either the HCC tissue or the surrounding tissue. Previous reports have indicated that the level of the fucosyltransferases involved in core fucosylation were similar in cancer and cirrhotic tissue (36). Our glycan analysis is consistent with this finding and extends it by showing that the levels of core fucosylation are similar in both diseased and healthy livers.

One hypothesis is that it is not the production of fucosylated proteins that is greater in HCC but rather increased secretion into the circulation that is responsible for the increased level of fucosylated proteins into the serum (37). The results presented here would support this hypothesis. Indeed, the level of core fucosylated glycan that was observed in liver tissue was much greater than that observed in the circulation of all groups, including individuals with no known liver disease. Interestingly, the level of fucosylation observed in the tissue was similar to what was observed in the serum of patients with HCC (>8.0%), further supporting the hypothesis that altered secretion is the main reason for increased level of fucosylated glycan in the serum of patients with HCC. It is noted that fucosylation changes could be specific to a small set of proteins that are masked by the overall glycan analysis. In a rodent model, only certain glycoproteins were fucosylated in serum, whereas most all glycoproteins were fucosylated in bile (38). Preliminary data suggest that certain kinds of fucosylated proteins were selectively secreted into bile ducts but not into the conditioned medium in HepG2 cells (Nakagawa and colleagues, manuscript in preparation). These possibilities remain to be tested through the glycan analysis of specific proteins following resolution via 2-dimensional electrophoresis.

One change that was observed in HCC tissue was an increase in tetra-antennary N-linked glycan. Tetra-antennary N-linked glycans arise from the action of the enzyme GnT-V (16), which catalyzes the addition of β-1,6-GlcNAc to the growing N-linked glycan to form tri- and tetra-antenna–like oligosaccharides. Increased branching is associated with metastasis and has been associated with alterations in the hexosamine cycle and activation of the AKT pathway (19, 39–42). It has also been reported that expression of GnT-V in the liver was dramatically enhanced in the hepatocarcinogenesis of a rodent model. Increases in β-1,6-GlcNAc structure through upregulation of GnT-V have long been associated with cancers but this report represents the first oligosaccharide analyses with liver cancer tissue. The result was consistent with immunohistochemical analysis of GnT-V in HCC tissue (43) and is consistent with the hypothesis that the elevated expression of UDP-N-acetylglucosamine: α-mannoside β-1,6 N-acetylglucosaminyltransferase (GnT-V) is an early event in hepatocarcinogenesis.

There are reports in the literature that increased branching of N-glycans correlates with reduced intercellular adhesion of epithelial cells (44). These published observations and our findings reported here lead us to propose the following working model (Fig. 5). We speculate that HCC is associated with an increase in tetra-antennary glycan addition to proteins involved cell–cell adhesion and/or tight junction integrity. The resulting weaker cell–cell junctions permit “leaking” of core fucosylated glycoproteins from the biliary system, which are predominantly fucosylated (32), into the blood (and potentially other molecules that are bile specific). This model would account for the apparent discrepancy between core fucose levels present in the serum (and elevated in HCC) versus the tissue (nondifference between tumor and nontumor). Further characterization of the proteins that carry the tetra-antennary glycans will be required to test the model.

The identity of the proteins that contain tetra-antennary glycan are unclear but under investigation. It is understood that the level of tetra-antennary glycan was low in the HCC samples and represents only a minor species of the total glycan pool. This is not unexpected as conformationally, not all proteins will have the space to allow for a tetra-antennary glycan. Tetra-antennary glycans are usually observed on cell surface molecules, such as growth factor receptors and adhesion molecules. While these molecules are partially cleaved from the cell surface, it might be difficult to find them in circulation as they would represent only a minor fraction of serum glycoproteins. More advances in glycotechnology for oligosaccharide analysis may be required to fully characterize the N-glycan and to identify the proteins containing these alterations.

In conclusion, we have conducted N-linked glycan analysis of HCC and adjacent tissue in an effort to determine whether the increase in core fucosylation found on liver-derived serum proteins from patients with HCC was a result of increased fucosylation. Surprisingly, no increase in fucosylation was observed in HCC. In contrast, increased levels of branching, most notably, increased levels of tetra-antennary glycan were observed.

No potential conflicts of interest were disclosed.

Conception and design: A.S. Mehta, P. Norton, H. Liang, E. Miyoshi, T. Block

Development of methodology: A.S. Mehta, H. Liang, M.A. Comunale, M. Wang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Norton, H. Liang, M.A. Comunale, M. Wang, A. Koszycki

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.S. Mehta, P. Norton, H. Liang, L. Rodemich-Betesh, A. Koszycki, T. Block

Writing, review, and/or revision of the manuscript: A.S. Mehta, P. Norton, H. Liang, M.A. Comunale, M. Wang, T. Block

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.S. Mehta, H. Liang, L. Rodemich-Betesh, K. Noda, E. Miyoshi, T. Block

Study supervision: A.S. Mehta

This work was supported by grants R01 CA120206 and R01 CA136607 from the National Cancer Institute (NCI), the Hepatitis B Foundation, and an appropriation from The Commonwealth of Pennsylvania.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.