Abstract
Background: Recently, it has been reported that oncostatin M receptor-β (OSMR) is frequently methylated in primary colon cancer tissues, but not in normal tissues. We examined the methylation status of the OSMR gene in primary carcinomas and the corresponding normal tissues derived from 56 patients with colorectal cancer. Patients and Methods: The methylation status of the OSMR gene was examined in primary carcinomas and corresponding normal tissues derived from 56 patients with colorectal cancer using quantitative methylation-specific PCR (qMSP), and the correlation between the methylation status and the clinicopathological findings was evaluated. Results: Methylation of the OSMR gene was detected in 18 out of the 56 (32%) primary colon carcinomas. The clinicopathological data were then compared with the methylation results. A significant difference was observed in regard to the extent of tumour (p=0.0442). These results indicated that OSMR was more frequently methylated in non-invasive colorectal carcinomas. Conclusion: OSMR may act as a tumour suppressor in colorectal carcinoma and OSMR methylation may play an important role in non-invasive colorectal cancer.
There is now firm evidence that a series of genetic alterations in both dominant oncogenes and tumour suppressor genes are involved in the pathogenesis of human colorectal cancer. The activation of oncogenes such as the RAS gene, and the inactivation of tumour suppressor genes such as APC (adenomatous polyposis coli) and p53, have been identified in colorectal cancer (1-3). In addition, it has also been found that several other genes are related to the pathogenesis of colorectal cancer (4, 5). An investigation of genetic changes is important in clarifying the tumourigenic pathway of colorectal cancer (6).
Recently, it has been reported that oncostatin M receptor-β (OSMR) is frequently methylated in primary colon cancer tissues, but not in normal tissues (7). OSMR is a receptor of oncostatin M (OSM), an interleukin-6 (IL-6)-type cytokine identified as a potent suppressor of tumour cells. Promoter methylation of OSMR strongly correlates with its expression and resistance to growth inhibition by OSM with loss of OSMR in colorectal cancer cell lines (7). These results therefore prompted the present study in which the methylation status of the OSMR gene in surgically removed colorectal tumours was examined.
In the present study, the methylation status of the OSMR gene was examined in primary carcinomas and the corresponding normal tissues derived from 56 patients with colorectal cancer and a comparison between the methylation status and the clinicopathological findings was performed.
Patients and Methods
Sample collection and DNA preparation. Fifty-six primary tumour and corresponding normal tissue specimens were collected consecutively at Showa University Fujigaoka Hospital from colorectal cancer patients during colorectal surgery. The status of all tissue specimens were confirmed histologically. Written informed consent, as required by the Institutional Review Board, was obtained from all the patients. The samples were stored immediately at −80°C until analysis. The DNA was prepared as described elsewhere (8). The clinicopathological profiles of the patients enrolled in the study are shown in Table I.
Sodium bisulfite modification. One μg of the genomic DNA extracted from the tumour and the corresponding normal colorectal tissue specimens was subjected to bisulfite treatment using an Epitect Bisulfite Kit (Qiagen, Hilden, Germany).
Quantitative methylation-specific PCR (qMSP). The bisulfite-treated DNA was amplified with quantitative methylation-specific PCR (qMSP), using a Thermal Cycler Dice® Real-Time System TP800 (Takara Bio Inc., Otsu, Japan). Thermocycling was performed in a final volume of 25 μl containing 1.0 μl of the DNA sample, 100 nM of each of the OSMR or β-actin primers (forward and reverse), and 12.5 μl of SYBR Premix Ex Taq II (Takara Bio Inc., Otsu, Japan), which consisted of Taq DNA polymerase, reaction buffer and deoxynucleotide triphosphate mixture. The qMSP primer sequences for OSMR have been described elsewhere (7) and were: OSMR MS (sense), 5’-TTCGTGCGTTTTTGGTCG-3’, and OSMR MAS (antisense), 5’-CGAACTTTACGAACGAACG-3’. The PCR amplification consisted of 40 cycles (95°C for 5 s and 60°C for 30 s) after an initial denaturation step (95°C for 10 s). The bisulfite-treated DNA obtained from L132 cells that was fully methylated by SssI methylase was used as a positive control. To correct for differences in both quality and quantity between samples, β-actin was used as an internal control. The targets were obtained from the same bisulfite-treated DNA.
Clinicopathological features and OSMR methylation in colorectal cancer.
OSMR methylation scores. The relative amounts of OSMR methylated DNA in the colorectal carcinomas and the corresponding normal tissues that were normalised to the internal control β-actin were calculated. The OSMR methylation score in each tissue was defined as follows: relative amount of OSMR in tumour/relative amount of OSMR in corresponding normal tissue.
OSMR methylation was deemed to be positive when the methylation score was greater than 3.0.
Statistical analysis. The association between OSMR methylation and clinicopathological parameters was analysed using Chi-square tests or Student's t-tests. A p-value <0.05 indicated statistical significance.
Results
Methylation of the OSMR gene was detected in 18 out of the 56 (32%) primary colon carcinomas, suggesting that the aberrant methylation of OSMR was frequently observed in colorectal carcinomas.
The clinicopathological data was compared with the methylation results. No significant differences were observed between the presence of methylation in the colorectal carcinoma and patient gender or age, maximal tumour size, tumour site, histology, lymph node metastasis or Dukes' stage (Table I). A significant difference was observed in regard to the extent of tumour (p=0.0442) (Table I). Statistical analysis was performed by statistical software, JMP8. These results indicated that OSMR was more frequently methylated in non-invasive colorectal carcinomas.
Discussion
Colorectal cancer is one of the most aggressive malignancies and occurs at a high incidence in most countries (9). One treatment of this fatal cancer is surgery and subsequent chemotherapy and radiotherapy. For this purpose, it is important to identify the occurrence of genetic alterations as a new parameter to estimate the malignancy of the cancer. Previously, in order to define the role of checkpoint with forkhead and ring finger domains gene (CHFR) in the tumourigenic pathway of the colorectum, the methylation of CHFR was examined in tumours from colorectal cancer patients (10). An aberrant methylation of the CHFR gene was detected in 25 out of 98 (26%) primary colorectal cancers. Moreover, a significant difference was thus found in the extent of the tumour (p=0.035), thus indicating that in early colorectal cancer, the CHFR gene was more frequently methylated than in advanced cases. In the present study, we observed the frequent methylation of OSMR in colorectal cancer. We also compared the methylation status of OSMR in colorectal cancer patients with the clinicopathological features and demonstrated that OSMR in non-invasive colorectal cancer cases were more frequently methylated than in invasive cases. These findings suggested that OSMR and CHFR might act as tumour suppressors in at least some colorectal tumours and that methylation of these genes might therefore be a particular phenomenon of non-invasive colorectal cancer. Therefore, methylation of these genes could be used as a tumour marker in clinical samples such as serum and stool for the early detection of digestive tract cancer (11, 12).
This study provided firm evidence that can be used in further studies of the molecular mechanism of OSMR in colorectal cancer. This study also suggests that OSMR may play a role in the carcinogenic pathway in some patients with colorectal cancer. These observations indicate the possibility that tumour formation in the colorectum might thus be controlled by inducing the expression of silenced OSMR using demethylating reagents.
- Received January 25, 2011.
- Revision received February 21, 2011.
- Accepted March 10, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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