Abstract
Background: Recent studies suggest that altered patterns of stanniocalcin 1 (STC1) gene expression have a role in human carcinogenesis. This study examined the relationship between the relative expression of the STC1 gene and clinicopathological factors in patients with colorectal cancer. Patients and Methods: Surgical specimens of cancer tissue and adjacent normal mucosa were obtained from 202 patients with colorectal carcinomas. The relative expression levels of STC1 mRNA in the cancer and the normal adjacent mucosa were measured by quantitative real-time, reverse-transcriptase polymerase chain reaction. Results: The relative expression levels of the STC1 gene were higher in the cancer tissue than in the normal adjacent mucosa and high expression of STC1 correlated with poor postoperative survival. Conclusion: High expression of the STC1 gene might be a useful predictor of poor postoperative outcome in patients with colorectal cancer.
Stanniocalcin (STC) is a glycoprotein hormone that regulates calcium and phosphate levels produced in bony fish by the corpuscle of Stannius, which is located near the kidney (1, 2). A human ortholog of fish STC, STC1, has been identified by molecular biological techniques (3, 4). Human STC1 complementary DNA (cDNA) encodes a 247 amino-acid protein. The gene resides on the short arm of chromosome 8 (8p11.2-p21) and contains four exons (5, 6). In contrast to fish STC, mammalian STC1 is expressed in various tissues, including the kidney, ovary, prostate, thyroid, colon, bone and spleen (3, 4, 6), and appears to be involved in not only calcium/phosphate regulation (7-9), but also in diverse biological processes. Modulation of STC1 expression has been demonstrated in numerous developmental, physiological, and pathological processes including pregnancy (10), lactation (10), angiogenesis (11-14), organogenesis (15-18), cerebral ischemia (19), hypertonic stress (20), oxidative stress (21), and apoptosis (22, 23). STC1 was originally cloned as part of a search for cancer-related genes, and recent studies have indicated that altered STC1 expression patterns may have a role in carcinogenesis. Increased STC1 gene expression has been found in hepatocellular (24, 25), colorectal (12, 25, 26), and medullary thyroid carcinomas (27), increased STC1 protein expression has been found in ovarian caner (28), and aberrant STC1 gene expression has been found in breast carcinomas (29-32). The mechanisms involved remain poorly understood and it remains unclear whether the expression of SCT1 is associated with the malignant potential of cancer. In agreement with other studies, the present results showed that STC1 gene expression levels were higher in the cancer tissue than in the normal adjacent mucosa. A paralog of STC1 (STC2) was identified by searching expressed sequence tag databases for sequences related to STC1 (2). STC2 cDNA has 34% homology with both STC1 and eel STC (2). STC2 is less strongly related to fish STC than to STC1. Similar to STC1, STC2 is expressed in various tissues and is associated with several types of cancer, including breast cancer (29), renal cell carcinoma (33), and colorectal cancer (34), but these proteins are thought to have different biological roles (2).
In this study, the expression levels of the STC1 gene were measured in cancer tissue and adjacent normal mucosa obtained from patients with colorectal cancer. To evaluate the clinical significance of STC1, the correlation between the relative expression of this gene and clinicopathological features was examined. Additinally, the influence of STC1 gene expression on the outcomes of patients with colorectal cancer was assessed.
PCR primers and conditions.
Patients and Methods
Patients and samples. Surgical specimens of cancer tissue and adjacent normal mucosa were obtained from 202 patients with colorectal cancer who underwent surgery at Kanagawa Cancer Center and at the Gastroenterological Center of Yokohama City University Medical Center between 2002 and 2006. Informed consent was obtained from each patient. The ethics committees of Kanagawa Cancer Center and Yokohama City University Medical Center approved the protocol before initiation of the study. All the tissue samples were embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and were immediately stored at −80°C until use. No patient had any other malignancies. The specimens were stained with hematoxylin and eosin and examined histopathologically. Sections that consisted of >80% carcinoma cells were used to prepare the total RNA.
Quantitative real-time, reverse-transcriptase polymerase chain reaction (PCR). The total RNA isolated from the colorectal cancer and adjacent normal mucosa was prepared with the use of Trizol (Gibco Life Tech, Gaithersburg, MD, USA). cDNA was synthesized from 2 μg of total RNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). After synthesis, the cDNA was diluted 1:4 with water and stored at −20°C until use. Quantitative real-time PCR was performed with an iQ SYBR-Green Supermix (Bio-Rad Laboratories). The PCR was carried out in a total volume of 15 μl containing cDNA derived from 75 ng of RNA, 0.27 μM of each primer, 7.5 μl of iQ SYBR-Green Supermix containing dATP, dCTP, dGTP and dTTP at concentrations of 400 μM each and 50 units/ml of iTaq DNA polymerase. The PCR consisted of 10 min at 94°C, followed by 50 cycles of denaturation of the cDNA for 30 s at 94°C, annealing for 30 s at 60°C, and a primer extension for 1 min at 72°C followed by 72°C for 10 min. The PCR primer sequences of STC1 and β-actin, used as an internal control, are shown in Table I.
Statistical analysis. The gene expression levels in colorectal cancer were compared with those in normal adjacent mucosa with the use of the Wilcoxon test. The relationships between the gene expression levels and potential explanatory variables, including age, gender, tumor size, histological type, depth of invasion, lymph node metastasis, location, lymphatic invasion, venous invasion and liver metastasis, were evaluated with the chi-square test. The postoperative survival rate was analyzed with the Kaplan-Meier method, and differences in survival rates were assessed with the log-rank test. A Cox proportional-hazards model was used for multivariate analysis. All the statistical analyses were performed using Dr.SPSS II, version 11.0.1 J for Windows software (SPSS Inc., Chicago, IL, USA). Two-sided p-values were calculated, and differences were considered significant at p-values of <0.05.
Results
STC1 mRNA expression. STC1 mRNA expression levels were significantly higher in the cancer tissues than in the normal adjacent mucosa (p=0.004; Figure. 1).
Relationship of STC1 gene expression level to clinicopathological features. The expression levels of the STC1 gene were categorized as low or high in relation to the median value. The STC1 gene expression level was not related to age, gender, tumor size, histological type, depth of invasion, lymph node metastasis, tumor location, lymphatic invasion, or venous invasion. However, the STC1 gene expression level correlated with liver metastasis (low expression: 24/101 [23.8%], high expression: 38/101 [37.6%], p=0.047; Table II).
STC1 expression and postoperative survival. Overall survival curves were plotted according to STC1 mRNA expression level by the Kaplan-Meier method. The median follow-up period was 1178 days. In the study group as a whole (202 patients), the overall survival rate was significantly lower in the patients with high STC1 mRNA expression than in those with low expression (p=0.016; Figure 2).
Univariate analysis with Cox proportional-hazards model identified seven prognostic factors: histological type, tumor size, depth of invasion, lymph node invasion, lymphatic invasion, liver metastasis and STC1 expression. The other clinicopathological features, such as age, gender, location, and venous invasion, were not statistically significant prognosis factors (Table III). A multivariate analysis of the prognosis factors with a Cox proportional-hazards model confirmed that high STC1 expression was a significant independent predictor of poor survival in colorectal cancer (Table IV).
Discussion
Wascher et al. (32) reported that in early-stage breast cancer, the detection of STC1 mRNA in bone marrow and blood significantly correlated with multiple histopathological prognostic factors, including primary tumor size, number of positive lymph nodes, TMN stage, and overall American Joint Committee on Cancer (AJCC) stage. Gerritsen et al. (12) proposed that increased STC1 expression was related to tumor vasculature in colon cancer. Ieta et al. (34) reported that high STC2 expression positively correlated with lymph node metastasis, lymphatic invasion, tumor depth, tumor size and AJCC stage in colorectal cancer and was associated with significantly poorer overall survival than low STC2 expression. However, the clinical significance of STC1 gene expression in colorectal cancer remains unclear. To our knowledge, no previous study has examined the relationship between STC1 expression and patient outcome. In the present study, STC1 expression was associated with liver metastasis.and high STC1 expression correlated with poor postoperative survival. Fujiwara et al. (25) and Wascher et al. (32) have suggested that STC1 mRNA might be a useful molecular marker for the detection of tumor cells in blood. Tumor cells expressing high levels of STC1 probably exist in the circulation and metastasize via the bloodstream, which would be consistent with the finding that a high STC1 expression level was associated with liver metastasis. In conclusion, high STC1 gene expression might be a useful predictor of poor postoperative survival in patients with colorectal cancer.
Comparison of STC1 mRNA expression levels between colorectal cancer tissue (C) and adjacent normal mucosa (N). Box boundaries, the 25th and 75th percentiles of the observed values; capped bars, the 10th and 90th percentiles; solid line, median. p-Value was calculated by the Wilcoxon test. STC1 gene expression levels were higher in cancer tissue than in normal adjacent mucosa (p=0.004).
Postoperative survival of patients with colorectal cancer.
Relationship of STC1 gene expression level to clinicopathological features.
Univariate analysis of clinicopathological factors for overall survival.
Multivariate analysis of clinicopathological factors for overall survival.
- Received August 3, 2010.
- Revision received December 2, 2010.
- Accepted December 3, 2010.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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