Introduction
Recently, sugar chains have drawn extensive attention from sugar biologists due to their increasing role in tumor growth and invasion and metastasis of cancer. Each step of the sugar chain synthesis requires a specific glycosyltransferase to catalyze the transfer of a monosaccharide moiety from a glycosyl donor to an acceptor substrate; therefore, the abnormal sugar chain is controlled by the transfer of enzymes from sugar chain synthesis catalyzed glycosylation in the final analysis. Polypeptide N-acetylgalactosaminyltransferase (pp GalNAc-T, EC2.4.1.4.1) is an O-sugar chain synthesis-starting glycosyltransferase, which catalyzes the transfer of the GalNAc group of UDP-GalNAc to the polypeptide chain Thr or Ser hydroxyl in a specific sequence in order to synthesize GalNAcα-O-Ser/Thr peptide; the peptide chain gradually extends under the action of other glycosyltransferases (1). A growing number of studies have demonstrated that pp GalNAc-Ts is closely connected with tumor occurrence and development. Ishikawa et al(2) identified that pp GalNAc-T3 expression is a useful indicator of tumor differentiation in early gastric cancer and its expression is positively correlated with the depth of tumor invasion and lymph node metastasis. Wu et al(3) revealed that the expression of pp GalNAc-T14 is associated with clinicopathological characteristics of breast carcinoma. Berois et al(4) reported that pp GalNAc-T6 is expressed in the majority of human breast carcinomas, but not in normal breast epithelium and is sporadically found in non-malignant breast diseases. Gu et al(5) hypothesised that low expression of pp GalNAc-T3 may be a useful indicator of early tumor recurrence in stage I lung adenocarcinoma. pp GalNAc-T10 is a member of the pp GalNAc family; however, limited study has been conducted. In this study, an immunohistochemical method was applied to detect pp GalNAc-T10 protein expression in different gastric mucosal lesions. Expression of pp GalNAc-T10 in gastric cancer tissues was identified to be higher than that in adjacent non-tumor gastric tissue. pp GalNAc-T10 protein expression in gastric cancer was significantly positively correlated with histological type and degree of differentiation of the gastric cancer. Therefore, pp GalNAc-T10 may be a specific biomarker for gastric cancer.
Materials and methods
Collection of tissue samples and histological classification
In this study, 96 gastric adenocarcinoma surgical samples and 88 5-cm adjacent cancer non-tumor gastric mucosa control samples were used. All specimens were collected from the two Affiliated Hospitals of Suzhou University. The study was approved by the ethics committee of Southern Medical University, Guangzhou, China. The patients, ranging in age from 38 to 83 years (median age, 62 years; mean age, 63.4 years), had received no other treatment prior to surgery. All patients provided informed consent to participate in this study. The clinicopathological parameters included gender, age, TNM stage, Laurén’s type, histological grade, depth of tumor invasion and lymph node metastasis (Table II) in which TNM staging system is according to the 2004 AJCC (American Joint Committee on Cancer)/UICC (Union Internationale Contre le Cancer) gastric cancer staging.
Tissue microarray production
Cores (diameter 1 mm) were taken from the marked region of individual formalin-fixed paraffin-embedded gastric adenocarcinoma specimens and the 5-cm adjacent cancer non-tumor gastric mucosa control samples. In 8 cases, a non-tumor gastric mucosa control sample was missing. Two cores from each tissue were assembled adjacently into a single recipient TMA block (Outdo Biotech, Shanghai, China).
Immunohistochemistry
For pp GalNAc-T10 immunostaining, quenching of endogenous peroxidase activity was performed with 3% H2O2 in PBS for 20 min, which was then blocked with normal goat serum (Gibco, Carlsbad, CA, USA) for 20 min. Anti-pp GalNAc-T10 (Abcam, Cambridge, MA, USA) was incubated overnight at 4°C and peroxidase-conjugated goat anti-mouse polyvalent antibody (Maixin-Bio, Jinan, China) was incubated for 60 min at room temperature. Reactions were revealed with diaminobenzidine, washed in water, counterstained in Mayer’s hematoxylin and dehydrated in ethanol and xylene and were then mounted. Between each step, sections were washed in PBS. For every assay, negative controls using PBS without primary antibody were included.
Immunocytochemical analysis
The immunostaining frequency for each tumor was scored as follows: 0 for negative samples or ≤5% stained tumor tissue; 1 for staining between 6 and 25% of the tumor tissue; 2 for staining between 26 and 50% of the tumor tissue; 3 for staining between 51 and 75% of the tumor tissue; and 4 for staining >75% of the tumor tissue. Signal intensity was scored as strong (3), moderate (2), weak (1) and null (0). Total immunostaining score resulted from the addition of both parameters. Scores were established jointly by four observers under a multi-head microscope. Clinicopathological information was masked to the observers.
Statistical analysis
SPSS 13.0 statistical software was used for statistical analysis. Gastric cancer tissues and adjacent non-tumor gastric tissue protein expression differing in intensity were tested with the two sample rate of the matched pair, designed to compare the Chi-square. The Chi-square test was applied to analyze the correlation between intensity and clinicopathological parameters of the protein expression. P<0.05 was considered to indicate a statistically significant difference.
Discussion
It is widely acknowledged that glycosyltransferases affect the development of tumors by altering the structure of sugar chains. The changes in cell surface sugar chains have an impact on the interaction between cancer cells, and cancer cells and the extracellular matrix. This in turn has a major impact on the behavior of cancer cells, including tumor growth, invasion and metastasis. The T-antigen O-glycosylation originates in the peptide: the enzyme family of pp GalNAc-Ts. Certain members of the pp GalNAc-T family have reported abnormal expression in squamous, colon, stomach and lung cancer, leukemia and other types of cancer. pp GalNAc-T10 is a member of the pp GalNAc-T family, which is highly expressed in human gastrointestinal tissues (6), but at present there is no study relating pp GalNAc-T10 expression to cancer. In this study, pp GalNac-T10 protein expression was immunohistochemically analyzed, in 96 gastric adenocarcinoma samples and 88 5-cm adjacent cancer non-tumor gastric mucosa control samples. Results demonstrated that pp GalNAc-T10 protein expression in gastric cancer tissues was higher than that in adjacent non-tumor gastric tissues, and the expression had a significant positive correlation with the degree of tumor differentiation (P<0.05). Expression in poorly differentiated gastric carcinoma was significantly higher than that in cases of high and mid degree in differentiation, and the expression intensity in the diffuse-type gastric cancer was significantly higher than in the intestinal-type gastric cancer. pp GalNAc-T10 protein expression was not significantly positively correlated with clinical stage, depth of invasion or lymph node metastasis (P>0.05). Therefore, abnormal expression of pp GalNAc-T10 may be a significant event which determines whether normal cells tranform into malignant poorly differentiated gastric cancer cells. This may also be used as a diagnostic indicator of malignant gastric cancer cells. Through analysis of the protein expression in gastric cancer cells, we may be able to provide suggestions for the diagnosis of gastric cancer development and treatment.
We speculate that the impact of pp GalNAc-T10 on the proliferation and differentiation of gastric cancer results from its action on certain cell signaling pathways, particularly the transforming growth factor-β (TGF-β) signal-regulated pathway. The TGF-β superfamily is a newly discovered family of proteins that regulate cell growth and differentiation. Studies have reported that disruption of TGF-β signaling in diffuse-type gastric carcinoma models may accelerate tumor growth (7) and TGF-β decreases cancer-initiating cell proliferation within diffuse-type gastric carcinoma cells (8). Otherwise, N-acetyl aminotransferase is likely to play an important role in diffuse-type gastric cancer cells (9) in a TGF-β signal-regulated pathway. TGF-β1 mRNA level is significantly decreased in the overexpression of pp GalNAc-T2 (10). Therefore, we hypothesize that the pp GalNAc-T family may be correlated with the TGF-β family. Further studies are required to study the impact of pp GalNAc-T10 on the proliferation and differentiation of gastric cancer, and its correlation with TGF-β signaling pathway.
In the occurrence and development of gastric cancer, changes in sugar chains originate from the transferring of the glycosyltransferase. Through in-depth study of the pp GalNAc-T family we may be able to obtain a deeper knowledge of the pathogenesis of gastric cancer and provide a more effective reference for existing diagnosis and treatment options.