Clinical significance of altered S100A2 expression in gastric cancer
- Authors:
- Published online on: January 15, 2013 https://doi.org/10.3892/or.2013.2236
- Pages: 1556-1562
Abstract
Introduction
Although there has been a sharp decline in both the worldwide incidence and mortality due to gastric cancer during the second half of the 20th century, gastric cancer continues to be a major health issue (1). To date, surgery is the most common treatment method, whereas chemotherapy to treat gastric cancer has a limited success rate as this cancer is not particularly sensitive to chemotherapeutic drugs. Thus, early detection is the key to successful treatment to ensure the long-term survival of patients since it is impossible to completely surgically resect gastric cancer in the advanced stage. The risk factors of gastric cancer include Helicobacter pylori infection, consumption of smoked foods, salted fish and meat and pickled vegetables, as well as obesity, tobacco smoke, chronic gastritis and blood type A. These risk factors alter the expression and function of critical cell growth-related genes. Therefore, it is important to develop novel strategies for the prevention, treatment, and prediction of prognosis of gastric cancer. In addition, understanding the molecular mechanisms responsible for gastric cancer development and progression will help to identify useful biomarkers to predict disease progression or provide a means to prevent or delay this disease from occurring.
To this end, the family of S100 proteins is involved in the regulation of various cellular processes, such as cell cycle progression and cell differentiation. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and they include ~25 members clustering on human chromosome 1q21, whose encoding proteins contain two EF-hand calcium-binding motifs. Each member contains two EF-hands connected by a central hinge, which are S100 family-specific in the N-terminus and canonical in the C-terminus. Among these proteins, 11-kDa acidic S100A2 is directly involved in protein phosphorylation, cell cycle regulation, growth, motility, differentiation, survival and chemoattraction (2). The S100A2 promoter has been shown to be transcriptionally activated by wild-type p53, but not by mutated p53, suggesting that S100A2 is a p53 target gene (3,4). Previous studies have demonstrated the loss of S100A2 expression in breast cancer and several other types of cancer (2). Indeed, ectopic overexpression of S100A2 suppressed oral squamous cancer cells to grow, migrate, invade in Matrigel, and form colonies in soft agar or grow tumors in nude mice (5). In contrast, knockdown of S100A2 expression restrained head and neck cancer cell migration (6). However, other studies have shown the opposite effect; for example, S100A2 overexpression has been detected in pancreatic carcinoma (7), non-small cell lung carcinomas (8) and thyroid carcinoma (9). Therefore, the aim of this study was to further clarify the association between S100A2 expression and clinicopathological data. We determined the expression of S100A2 mRNA and protein in a large number of gastric cancer and precancerous lesions and then associated its expression with clinical significance in gastric cancer.
Materials and methods
Study population and pathological diagnosis
In the present study, we collected gastric cancer tissues from gastrectomy, gastric biopsies from gastritis, and gastric intestinal metaplasia (IM) and adenoma from endoscopy from patients at Department of Surgery, Shengjing Hospital of China Medical University between January 1995 and January 2005. None of the patients underwent chemotherapy, radiotherapy, or adjuvant treatment before surgery. The tissue specimens were routinely fixed in 10% neutral formalin, embedded in paraffin, and cut into 4-μm section. These sections were then stained with hematoxylin and eosin (H&E) to carry out the pathological diagnosis. In addition, part of the tissue samples was snap-frozen in liquid nitrogen and then stored at −80°C for later protein extraction and RNA isolation. The tumor-node-metastasis (TNM) stage for each gastric cancer specimen was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumor spread (10). Histological architecture of gastric cancer was expressed in terms of Lauren's classification (11,12). In this study, we also included additional clinicopathological data, such as tumor size, depth of invasion, and lymphatic and venous invasion. In addition, lymphatic and venous invasion of gastric cancer cells was diagnosed using H&E staining and D2-40 immunostaining and EvG staining, respectively. The Ethics Committee of China Medical University approved our research protocol, and each patient or their guardian provided a consent form for participation in this study. The patients were followed up through their medical records and telephone conversations.
Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from tissue specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. These RNA samples were reversely transcribed into cDNA using RevertAid™ reverse transcriptase (Takara, Dalian, China). Real-time PCR was then performed using these cDNA samples in an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, Foster City, CA, USA), and the qPCR conditions were 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C for 15 sec, and 60°C for 1 min. The primers for GAPDH (135 bp, 201–335, NM_002046.3) were 5′-CAATGACCCCTTCATTGACC-3′ (sense) and 5′-TG GAAGATGGTGATGGGATT-3′ (antisense). The primers for S100A2 (140 bp, 440–579, NM_005978.3) were 5′-GAAGGA ACTTCTGCACAAGG-3′ (sense) and 5′-GTGCCAGGAAA ACAGCATAC-3′ (antisense). GAPDH mRNA was used as an internal control for loading and handling of samples. Each assay was performed in triplicate, the average was then calculated, and the level of S100A2 mRNA was expressed as 2−ΔΔCt, where ΔCt = Ct (S100A2) - Ct (GAPDH) and ΔΔCt = ΔCt (carcinoma) - ΔCt (adjacent non-neoplastic mucosa).
Protein extraction and western blot analysis
Protein was extracted from tissue samples using a homogenizer in RIPA lysis buffer. These protein samples were concentrated using the BAC method (Bio-Rad Laboratories, Hercules, CA, USA). After denaturation, the protein samples were separated by electrophoresis on an SDS-polyacrylamide gel (15% acrylamide) and then transferred onto Hybond membranes (Amersham, Freiburg, Germany). The membranes were then incubated overnight at 4°C in 5% skim milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween 20). For immunoblotting, the membrane was then incubated for 1 h with a rabbit antibody against S100A2 (Abcam, Cambridge, UK; 1:500). After rinsing with TBS-T, the membrane was further incubated with an anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Dako, Carpinteria, CA, USA) at a dilution of 1:1,000 for 1 h. Then, positive protein bands were visualized by incubating the membrane with ECL-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and exposed to X-ray film (Fuji, Japan). Next, the membrane was washed with WB Stripping Solution (pH 2.0–3.0; Nacalai, Tokyo, Japan) for 1 h, and then the western blot procedures were repeated for detection of GAPDH protein expression using an anti-GADPH antibody from Sigma (St. Louis, MO, USA) at a dilution of 1:10,000 as an internal control antibody. Densitometric quantification of S100A2 protein expression in gastric samples was performed using Scion Image software (Scion Corp., Frederick, MD, USA) and was compared to GADPH levels.
Tissue microarray (TMA) and immunohistochemistry
Representative areas of gastric lesions were first identified in H&E-stained sections, and a 2-mm diameter tissue core per donor block was punched out and transferred into a recipient block, with a maximum of 48 cores per block using a manual arraying device (MTA-1; Beecher Instruments Inc., Sun Prairie, WI, USA). After being re-embedded into the paraffin blocks, 4-μm consecutive sections were incised from the recipient blocks and mounted onto polylysine-coated glass slides. After confirmation with H&E staining, these TMA sections were used for immunohistochemistry experiments.
For the immunohistochemistry experiments, the sections were first deparaffinized in xylene and rehydrated through graded concentrations of alcohol. The sections were then subjected to blockage of endogenous peroxidase activity in 1.5% hydrogen peroxide/methanol at room temperature for 10 min and subjected to antigen retrieval using a microwave oven (Oriental Rotor Ltd., Co., Tokyo, Japan) and retrieval solution (Target Retrieval Solution; Dako, Carpinteria, CA, USA) for 15 min. Next, the sections were incubated with a rabbit polyclonal S100A2 antibody at a dilution of 1:200 for 1 h at room temperature. After washing three times with TBS-T, the sections were further incubated with an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Dako) at a dilution of 1:1,000 for 1 h. The final color was visualized by exposing the sections to 0.5 mg/ml 3,3′-diaminobenzidine and 0.005% hydrogen peroxide for ~5 min. After counterstaining with Mayer's hematoxylin, the sections were dehydrated, cleared and mounted. Negative control sections were incubated with TBS-T instead of the primary antibody.
The stained TMA sections were subsequently reviewed and scored by two experienced pathologists without any knowledge of the clinicopathological data. For each tissue core, the number of cells positively staining for S100A2 was counted in five fields at a magnification of ×200. The percentage of positively stained cells was calculated and scored as follows: 0–5%, negative (−); 6–25%, weakly positive (+); 26–50%, moderately positive (++); and >50%, strongly positive (+++). Finally, these two scores were combined to indicate the semi-quantitative expression of S100A2 protein, i.e., −, +, ++ and +++.
Statistical analysis
Spearman's correlation test was performed to analyze the rank data, and the Student's t-test was used to compare the means of different groups. Kaplan-Meier survival plots were generated, and comparisons between survival curves were tested using log-rank analysis. Cox's proportional hazards model was employed for multivariate analysis. SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) was used to generate all statistical data, and P<0.05 was considered to indicate a statistically significant result.
Results
In the present study, we first assessed S100A2 mRNA levels using qRT-PCR in a subset of gastric tissue specimens. Reduced expression of S100A2 mRNA was observed from gastritis, intestinal metaplasia and dysplasia to cancer tissue samples (P<0.001, Fig. 1). Loss of S100A2 mRNA expression occurred more frequently in gastric cancer with a larger tumor size, deeper invasive depth and lymph node metastasis and in intestinal-type carcinoma (P<0.05). Similar data were also observed for S100A2 protein expression in these samples analyzed by western blot analysis (Fig. 2).
We immunostained the tissue samples for S100A2 expression and found that S100A2 protein was positively expressed in the cytoplasm of the gastric epithelial cells and in the intestinal metaplasia, adenomatous dysplasia, and carcinoma tissues (Fig. 3). Specifically, S100A2 protein was expressed in all 73 cases of gastritis, 90.7% (78/86) of intestinal metaplasia, 73.0% (46/63) of adenomatous dysplasia, and 48.9% (170/348) of gastric cancer tissues. There was a statistically significant difference in the level of the S100A2 protein between gastritis, intestinal metaplasia, and dysplasia samples and cancer tissues (P<0.001, Table I). In gastric cancer, the expression of S100A2 protein was frequently absent in younger patients compared to that of older patients (P<0.05), and S100A2 expression was inversely associated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis and TNM stage (P<0.05). S100A2 protein was also more highly expressed in the intestinal type of gastric cancer than in the diffuse type (P<0.05). However, S100A2 expression was not associated with patient gender (P>0.05; Table II).
Table IIAssociation of S100A2 protein expression with the clinicopathological features of the gastric cancer patients. |
Next, we assessed the association of S100A2 protein expression with the survival of the gastric cancer patients. Survival data were available for all 348 gastric cancer patients with a follow-up period ranging from 1 month to 10.1 years (median, 65.1 months). Fig. 4 indicates that loss of S100A2 expression contributed to poorer survival of the gastric cancer patients (P<0.05). Kaplan-Meier analysis demonstrated that the cumulative survival rate of patients with weak and moderate S100A2 expression was obviously higher than those with loss of S100A2 expression. Moreover, multivariate analyses revealed that S100A2 expression, depth of invasion, lymphatic invasion, venous invasion, lymph node metastasis, distant metastasis, and TNM stage were independent predictors for gastric cancer patient survival.
Discussion
In the present study, we analyzed the expression of S100A2 mRNA and protein in gastritis, intestinal metaplasia, adenomatous dysplasia, and gastric cancer tissue specimens to determine whether the expression levels were associated with clinical features. We found that S100A2 protein was observed in the cytoplasm of gastric tissue specimens, which confirmed previous data (13), whereas we did not find nuclear localization of S100A2 protein, which was previously observed in oral and esophageal mucosa (5,14). Our data demonstrated that expression of S100A2 mRNA and protein gradually decreased from gastritis, intestinal metaplasia and adenomatous dysplasia to gastric cancer.
A previous study showed that S100A2 was expressed in tissue specimens of patients with benign prostate hyperplasia, while it was reduced in prostate cancer (15). Another study reported that expression of S100A2 mRNA was positive in 77.5% of esophageal squamous cell carcinoma tissues, which was lower than that in normal mucosa (100%) detected by in situ hybridization (16). However, normal esophageal mucosa expressed S100A2 in the cell nuclei, whereas two-thirds of Barrett's dysplasia and adenocarcinoma samples with S100A2 expression had stronger cytosolic staining of S100A2 protein (14). An additional study demonstrated that 2 out of 8 (25%) esophageal cancer cell lines and 14 out of 30 (47%) primary esophageal squamous cell carcinomas exhibited S100A2 expression compared to paired normal tissues (17), suggesting that S100A2 maybe related to the progression of esophageal squamous cell carcinoma. In the present study, we found gradually reduced expression of S100A2 from gastritis, intestinal metaplasia and dysplasia to carcinoma. We believe that our tissues covered a series of lesions throughout gastric cancer development, and a gradual loss of S100A2 expression in these specimens indicates that S100A2 is associated with carcinogenesis. Indeed, a previous study revealed that intestinal metaplasia is an adaptive condition for an injured gastric epithelium, and inflammation can develop into globoid dysplasia and then to gastric signet ring cell carcinoma, as shown by morphological appearance and biological characteristics (18). Moreover, pathological and genetic observations of intestinal dysplasia have demonstrated that gastric dysplasia is a premalignant lesion that has a high probability of undergoing malignant transformation (19). Thus, our data, for the first time, demonstrated that loss of S100A2 expression may contribute to gastric carcinogenesis. Other studies have shown that loss of S100A2 expression is only due to promoter hypermethylation (20–22).
Furthermore, the present data revealed that expression of S100A2 mRNA and protein was inversely linked to tumor size, depth of tumor invasion, lymphatic and venous invasion, lymph node metastasis, and TNM stage; these findings are supported by other studies in various types of cancer (23–25). These findings suggest that S100A2 plays a role in the suppression of growth, invasion and metastasis of gastric cancer. However, the function of S100A2 may be tissue-specific. For example, in esophageal cancer, the level of S100A2 mRNA expression has been shown to be closely associated with de-differentiation and lymph node metastasis (16), while positive S100A2 expression has been significantly associated with lymphatic invasion in lung cancer (26). In contrast, Nagy et al(6) showed that S100A2 has a clear inhibitory effect on cell motility by antisense oligonucleotides and extracellular treatments. Furthermore, Tsai et al reported that ectopic expression of S100A2 in the human malignant squamous cell carcinoma cell line KB resulted in significant inhibition of proliferation, migration and invasion. Moreover, S100A2 significantly reduced the number of colonies formed in semi-solid agar and decreased tumor growth and burden in nude mice with oral squamous cancer (5). Taken altogether, we speculate that downregulation of S100A2 expression is involved in the development and progression of gastric cancer.
Although gastric cancer originates from the same gastric epithelium, its morphological features vary substantially for individual patients. According to Lauren's classification, intestinal-type gastric cancer is characterized by cohesive carcinoma cells forming gland-like tubular structures with an expanding or infiltrative growth pattern, which includes well and moderately differentiated adenocarcinoma (11,12). In contrast, diffuse-type gastric cancer displays less apparent adenocarcinoma or lacks cell adhesion and contains poorly differentiated and signet ring cell carcinoma (11,12). The present data demonstrated that the expression of S100A2 mRNA and protein was higher in intestinal-type gastric cancer than in diffuse-type, suggesting that S100A2 may contribute to the development of diffuse-type gastric cancer but does not play a role in intestinal-type. A previous study showed that S100A2 is more highly expressed in well and moderately differentiated cancer than in poorly differentiated gastric cancer (13), which supports the present data.
To date, there have been no reports describing the prognostic significance of S100A2 expression in gastric cancer. Our present study showed that S100A2 expression was associated with a more favorable prognosis of gastric cancer patients. Multivariate analysis using Cox's proportional risk analysis indicated that depth of invasion, lymphatic or venous invasion, lymph node metastasis, distal metastasis, TNM stage, and S100A2 expression were independent factors for the prognosis of carcinoma patients. These findings suggest that S100A2 is an independent prognostic factor for gastric cancer. Others have reported that reduced or increased expression of S100A2 is an independent predictive factor, depending on the type of human cancer (27–30). Nevertheless, the data from our present study using a large number of tissue samples, demonstrated the clinical significance of S100A2 expression. Further studies will investigate how the S100A2 gene contributes to suppression of gastric cancer development and progression.
Acknowledgements
This study was supported, in part, by a grant from the Natural Science Foundation of Liaoning Province (#201202279).
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