Evaluation of Methylation of MGMT ( O 6 -Methylguanine-DNA Methyltransferase) Gene Promoter in Sporadic Colorectal Cancer

Abstract

The DNA repair gene O⁶ -methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer (CRC). The aim of this study was to demonstrate that MGMT methylation may be one of the candidate mediators of field cancerization in the colon mucosa. Therefore, quantitative methylation-specific polymerase chain reaction was performed on tumor itself and additional samples of 5 and 10 cm away from the tumor in 40 CRC patients. Moreover, colon mucosa was examined from 30 cases with no evidence of cancer as a control. MGMT promoter methylation was present in 27.5% of colorectal tumor specimens. Tumors that showed MGMT promoter methylation had substantial MGMT promoter methylation in their normal adjacent mucosa. The methylation was also observed in 36.36% (4/11) of normal samples with MGMT promoter methylation in the adjacent tumors, in 20.79% (6/29) of samples without MGMT methylation in the adjacent tumors, and in 6.66% (2/30) of control samples (p<0.006 and p<0.001 respectively). Finally, the mean of MGMT methylation levels was significantly higher in the cancerous group than in the control group (6.25±1.702 vs. 0.086±0.036, p<0.001). Some CRCs arise from a field defect defined by epigenetic inactivation of MGMT. Detection of such abnormality may ultimately be useful in risk assessment for CRCs.

Introduction

C olorectal cancer (CRC) is one of the most common neoplasias worldwide that typically starts in either the colon or rectum (Kamimae et al., 2011). These types of cancers may also be referred separately as a rectal cancer or colon cancer that has many features in common (Prakash et al., 2012). The molecular aspects underlying the sporadic CRC are not well understood; however, it has been shown that CRC may be caused by DNA mutations (defects) which turn on oncogenes or inactivate tumor suppressor genes (Schuebel et al., 2007). Genetic and epigenetic changes in CRC, such as regional DNA hypermethylation and global DNA hypomethylation, have been previously studied, and DNA methylation has been proposed as a candidate mediator of this field defect (van Engeland et al., 2003). The DNA repair protein encoded by the O⁶ -methylguanine-DNA methyltransferase (MGMT) gene is involved in defending cells against alkylating agents (Pegg, 2000; Kaina et al., 2001). Several studies have reported that transcriptional silencing of MGMT gene in multiple tumor types is strongly associated with hypermethylation of the CpG islands on its promoter (Costello et al., 1994; Qian et al., 1995). MGMT is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O⁶ -guanine in DNA (Pegg, 1990). Alkylation of DNA at the O⁶ position of guanine is an important step in the formation of mutations in malignancies, primarily because of the tendency of O⁶ -methylguanine to pair with thymine during replication. Subsequently, this will result in a conversion of guanine–cytosine to adenine–thymine pairs in DNA (Coulondre and Miller, 1977; Erickson et al., 1980). Loss of MGMT expression is not common because of deletion or rearrangement of the gene (Day et al., 1980; Fornace et al., 1990; Pieper et al., 1990), but rather methylation of CpG islands in the MGMT promoter region is associated with silencing of the MGMT gene in different cell lines (Costello et al., 1994; Qian and Brent, 1997; Watts et al., 1997). This phenomenon is, therefore, associated with loss of protein expression in primary human neoplasia (Esteller et al., 1999). Furthermore, in vitro treatment with demethylating agents restores the expression of the MGMT gene of such cell lines (Qian and Brent, 1997; Esteller et al., 1999). It was demonstrated that MGMT was hypermethylated in primary colon cancer tumors, indicating that hypermethylation of the MGMT promoter has a pivotal role in this neoplasm (Esteller et al., 1999). It has been noted that initially in the colon, normal-appearing tissues have aberrant methylation of promoter-associated CpG islands, albeit at low levels (Issa et al., 1994; Issa, 2002). In addition, alterations in the MGMT gene impair the ability of the MGMT protein to remove alkyl groups from the O⁶ -position of guanine, thereby increasing the mutation rate and the risk of cancer (Povey et al., 2002). In our study, it was, therefore, hypothesized that MGMT methylation will be one of the mediators of field cancerization in the colon mucosa.

Materials and Methods

Patients and samples

Samples of primary colorectal tumors and specimens from the corresponding normal-appearing tissue (5 and 10 cm away from the tumor) were collected from 40 patients who had undergone surgery at the Ghaem University Hospital (n=25), the Mehr Hospital (n=5), and the Razavi Hospital (n=15), Mashhad, Iran, in accordance with institutional policies. Ethics approval was obtained from the Human Research Ethics Committee of Mashhad University of Medical Sciences. All patients provided written informed consent.

Tumors were selected solely on the basis of their availability. Additional two samples of normal appearing colonic mucosa were obtained from patients. The two samples originated from tissue located 5 and 10 cm away from the original tumor. Clinicopathologic data were available from all of the 40 CRC patients. In addition, colonic biopsy specimens from 30 individuals who had no family history of CRC and had no colonic lesions at screening colonoscopy were obtained. These healthy control specimens were also selected on the basis of availability. All tissue samples were fresh frozen and stored at −80°C.

DNA extraction and bisulfite conversion

Genomic DNA was extracted from patient tissue biopsies using standard phenol/chloroform method. Bisulfite treatment and purification of genomic DNA was performed using the Epitect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. For each conversion reaction, 1 μg of genomic DNA was employed and after conversion and purification, DNA was eluted in 70 μL of the provided elution buffer.

Quantitative methylation–specific polymerase chain reaction

Quantitative real-time polymerase chain reaction (PCR) was performed for MGMT using the following primer/probe set (Metabion) (Hoque et al., 2006): forward 5′-CGAATATAC TAAAACAACCCGCG-3′, reverse 5′-GTATTTTTTCGGGAGCGAGGC-3′; probe 5′-FAMAATCCTCGCGATACGCACCGT TTACGTAMRA-3′, yielding a122 bp amplicon. A primer/probe set specific for the unmethylated promoter region of the ACTB gene was used as reference gene (Hoque et al., 2006): forward 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′; reverse 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′; probe 5′-FAMACCACCACCCAACACACA ATAACAAACACATA-MRA-3′, yielding an amplicon size of 133 bp. Leukocyte DNA from a healthy blood donor was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Inc.) to generate completely methylated DNA. Serial dilutions (pure up to 1:10,000) of this DNA were used to construct calibration curves for MGMT and ACTB genes.

For each 20 μL PCR, 4 μL eluate containing the bisulfite converted DNA, 10 μL Perfect real-time premix Ex Taq (RR039; Takara, Kyoto, Japan,), 0.5 μL Rox Refrence dyeiiiiIIii II (Takara), 1.5 μL each of forward and reverse primer (concentration10 pmol/μL resulting in final concentration of 0.0375 μM), and 2.5 μL probe (concentration2.5 pmol/μL resulting in final concentration of 0.0156 μM) were used. PCR conditions were as follows: one step at 95°C for 30 s, 50 cycles at 95°C for 4 s, and 60°C for 30 s.

PCRs were performed in 48-well plates in a StepOne Real-Time PCR System (Applied Biosystems). Each plate included calibration curves for the ACTB and MGMT genes, patient DNA samples, positive control (CG Genome Universal Methylated DNA [mDNA],S7821; Millipore), negative control (CG Genome Universal Unmethylated DNA [uDNA],S7822; Millipore), and multiple water blanks. The relative level of methylated DNA was determined as a ratio of MGMT to ACTB and then multiplied by 1000 for easier tabulation (average value of triplicates of gene of interest/average value of triplicates of ACTB×1000). For each sample, quantitative methylation-specific PCR (QMSP) analysis was repeated on three separate plates, and median values were considered for statistical analyses.

Statistical analysis

All statistical analyses were carried out by SPSS 16.0 (SPSS, Inc.). To compare differences in methylation levels or other continuous values between groups, t tests or ANOVA with a post hoc Tukey test were carried out. Fisher's exact test was used for the analysis of categorical data. p<0.05 (two-sided) was considered significant.

Results

In this study, MGMT promoter methylation has been analyzed in CRC. Specimens of colorectal tumors and biopsies of 5 and 10 cm away from the tumor in 40 patients were measured using a sensitive and specific method of real-time QMSP. By using the quantitative QMSP assay, we found that the mean level of MGMT promoter methylation in the CRC samples was higher than in the samples of 5 cm away from the tumors (6.254% vs. 0.6495%: difference=5.6074%, 95% CI=0.0694% to 11.2789%; p=0.05). However, the difference between the mean level of MGMT promoter methylation in the colorectal neoplastic tissue and in the samples of 10 cm away from the tumors was not statistically significant (p=0.221). Among those 40 samples of the ternary, the MGMT promoter was also methylated (i.e., methylation level of at least 3%) in 27.5% colorectal tumor samples, in 12.5% samples of 5 cm, and in 12.5% samples of 10 cm away from the tumors. Subsequently, we examined the association between MGMT promoter methylation and clinicopathologic features of patients with primary CRC. Initially, we analyzed the data using methylation status as a categorical variable; samples with detectable methylation by QMSP (i.e., those with a methylation level ≥3%) were considered positive for MGMT promoter methylation (Table 1). We found that the MGMT promoter methylation status of samples was not associated with patient age at diagnosis, with the patient sex, with the grade, with the location of the tumor in the colon, or with tumor–node–metastasis stage.

Table 1.

Clinicopathologic Features of Patients by Tumor MGMT Promoter Methylation Status

Characteristic	No. without promoter methylation	No. with promoter methylation	p-Value ^a
Sex
Female	21	5
Male	8	6	0.17
Age
<60	13	5
≥60	16	6	0.19
Tumor location in colon
Proximal	12	2
Distal	17	9	0.563
Tumor stage
1	7	2
2	8	3
3	7	2
4	7	4	0.397
Tumor grade
1	13	4
2	12	4
3	4	3	0.696

Methylation of tumor samples was measured by real time after bisulfite conversion, and a sample was considered positive for methylation if the methylation level was ≥3%.

Fisher's exact test (two sided).

MGMT, O ⁶-methylguanine-DNA methyltransferase.

We also analyzed MGMT methylation level as a continuous quantitative variable, limiting the analysis to 11 tumors that were MGMT promoter methylation positive by the categorical classification. In this group of 11 patients, there was no association between the degree of methylation and patient age, patient sex, grade, tumor stage, or the location of the tumor in the colon (data not shown). We used similar methodologies to examine the degree of methylation of the MGMT promoter in the samples of 5 and 10 cm away from tumors in the same patients. As shown in Tables 2 and 3, MGMT promoter methylation (≥3%) was detectable in five samples of 5 cm and in five samples of 10 cm away from the tumors out of 40 patients studied.

Table 2.

Clinicopathologic Features of Patients by MGMT Methylation Status of Normal-Appearing Mucosa 5 cm Away from Tumor

Characteristic	No. without promoter methylation	No. with promoter methylation	p-Value ^a
Sex
Female	23	3
Male	12	2	0.259
Age
<60	15	3
≥60	20	2	0.042
Tumor location in colon
Proximal	13	1
Distal	22	4	0.146
Tumor stage
1	9	0
2	8	3
3	8	1
4	10	1	0.494
Tumor grade
1	15	2
2	15	1
3	5	2	0.614

Methylation of normal-appearing mucosa samples was measured by real time after bisulfite conversion, and a sample was considered positive for methylation if the result was ≥3%.

Fisher's exact test (two sided).

Table 3.

Clinicopathologic Features of Patients by MGMT Methylation Status of Normal-Appearing Mucosa 10 cm Away from Tumor

Characteristic	No. without promoter methylation	No. with promoter methylation	p-Value ^a
Sex
Female	25	1
Male	10	4	0.133
Age
<60	14	4
≥60	21	1	0.024
Tumor location in colon
Proximal	12	2
Distal	23	3	0.215
Tumor stage
1	9	0
2	10	1
3	7	2
4	9	2	0.508
Tumor grade
1	14	3
2	14	2
3	7	0	0.465

Methylation of normal-appearing mucosa samples was measured by real time after bisulfite conversion, and a sample was considered positive for methylation if the result was ≥3%.

Fisher's exact test (two sided).

No association was found between the presence of MGMT promoter methylation and any of the clinicopathologic variables examined. Results of a quantitative analysis of methylation level in five samples of 5 and10 cm away from tumors that were positive for MGMT promoter methylation are shown in Table 3. Mean MGMT promoter methylation level in the samples of 5 cm away from tumors was lower in patients who were 60 years or older at surgery than in patients who were younger than 60 years (0.1245% vs. 1.2911% p<0.042 by the independent sample test [t-test]). In addition, this level in the samples of 10 cm away from tumors was lower in patients who were 60 years or older at surgery than in patients who were younger than 60 years (0.0459% vs. 4.5278% p<0.024 by the independent sample test [t-test]). We considered samples of 5 and 10 cm together as a group of samples adjacent to the tumor and used similar method to analyze the MGMT promoter methylation. Again, this level in the samples of adjacent mucosa was lower in patients who were 60 years or older at surgery than in patients who were younger than 60 years (p=0.94 by the independent sample test [t-test]). Patients who had MGMT promoter methylation-positive cancers also had detectable methylation of the MGMT promoter in the samples of 5 or 10 cm away from cancer. When analyzed as a categorical variable, methylation was positive in 2 (18.18%) of 11 samples of 5 cm away from methylation-positive tumors compared with only 3 (10.34%) of 29 samples of 5 cm away from methylation-negative tumors. However, the difference was not statistically significant (p=0.08).We, therefore, used a similar method to analyze the MGMT promoter methylation in the samples of 10 cm away from the cancer. As a whole, we have detected the MGMT promoter methylation in four tissue samples of surrounding tumor in 11 cancer patients whose tumor samples were MGMT methylation positive (36.36%; 95% CI=21% to 51%). We have also observed that there is a methylation in 6 tissue samples around the tumor in 29 cancer patients whose tumor was without MGMT methylation (20.79%; 95% CI=3% to 19%). In the current research, we have examined the methylation status of the MGMT promoter in colonic biopcy specimens obtained at screening colonoscopy from 30 patients who had no family history of CRC and no colonic lesions (mean age was 42.1 years). It was found that two specimens (6.66%; 95% CI=1% to 25%) had detectable MGMT promoter methylation (p<0.006 and p<0.001 for a comparison between each of the latter two groups and the first group, respectively). The mean MGMT methylation level was significantly higher in the tumoral group than in the control group (6.25±1.702 vs. 0.086±0.036, p<0.001) (Table 4).

Table 4.

Comparison Demographic and Clinicopathologic Features in Tumoral and Control Group

Characteristic	Tumoral group	Control group	p-Value (t-test)
Sex
Female	14	13
Male	26	17	0.46
Age (mean±SD)	61.05±14.06	42.1±16.81	0.524
Methylation percent (mean±SEM)	6.2542±2.72006	0.0863±0.06727	<0.001

SD, standard deviation; SEM, standard error of the mean.

Discussion

Recently, it was reported that the methylation status of the MGMT promoter is strongly associated with clinical outcome in human cancers such as malignant gliomas (Villalva et al., 2012). It has also been demonstrated that in glioblastoma patients, MGMT gene promoter is a strong favorable prognostic factor (Havik et al., 2012). Esteller et al. (2000) have shown that inactivation of the MGMT gene by promoter methylation is a predictor of overall survival and response to alkylating agents with additional supporting data which promise that methylated markers are prognostic tools. In primary colon cancers, it was also demonstrated that MGMT is hypermethylated in CRC tumors (Esteller et al., 2001). Candiloro et al. have detected MGMT promoter methylation in peripheral blood mononuclear cell DNA from normal individuals. It was shown that this methylation has a strong correlation with the presence of T allele in the MGMT promoter (Candiloro and Dobrovic, 2009). In the current research, we have examined whether MGMT promoter methylation will be characterized in normal-appearing cells (samples of 5 and 10 cm away from tumors) that are precursors to the neoplasm. In principle, the molecular abnormalities responsible for a field defect may be detectable at a high frequency in patients with cancer but at a low frequency in patients without neoplasia. The abnormalities mentioned earlier will even be traced in healthy individuals who are at a risk of cancer or during the early stages of the neoplastic process (Shen et al., 2005). Real-time QMSP has been used to detect promoter hypermethylation of several genes, including MGMT, in primary tumors and bodily fluid from cancer patients (Lo et al., 1999; Eads et al., 2000; Jeronimo et al., 2001; Harden et al., 2003; Fackler et al., 2004; Reesink-Peters et al., 2004; Schmiemann et al., 2005; Belinsky et al., 2006; Kahn et al., 2008; Vlassenbroeck et al., 2008). In our study, we have used QMSP method to analyze MGMT promoter methylation. A number of different methods for assessing promoter methylation status have been reported (Singer-Sam et al., 1990; Frommer et al., 1992; Herman et al., 1996; Huang et al., 1999; An et al., 2002; Colella et al., 2003; Ogino et al., 2006; Mikeska et al., 2007), but the most widely used is methylation-specific PCR (MSP) analysis after bisulphite treatment (Cankovic et al., 2007). However, this method is not quantitative and bears a significant risk of false-positive and false-negative results (Herman et al., 1996; Esteller et al., 1999; Derks et al., 2004). There are also various techniques for standardization of the analytical process during the assessment of MGMT promoter hypermethylation. Mellai et al. (2009) have analyzed the products of methylation-specific PCR with capillary electrophoresis, and Esteller et al. (1999) have used a qualitative method to analyze inactivation of the DNA repair gene of MGMT. In this study, we had a limited number of tumor samples available. We have endeavored to minimize the risk of contamination of normal tissues with cancerous cell via the following approaches: First, normal tissue was confirmed histologically. Second, promoter methylation was present in the tissue located 10 cm away from the tumor, and, finally, promoter methylation was considered detectable in some patients who did not have tumors. Therefore, it was attempted to utilize QMSP to detect methylation, and the percentage of cases with detectable MGMT promoter methylation in samples of each of 5 and 10 cm (as two separate groups) was 18.18% (2/11). The difference between MGMT promoter methylation in normal-appearing colonic mucosa (samples of 5 cm away from tumors) and MGMT promoter methylation in adjacent tumors was statistically significant. However, contrary to our expectations, the difference between MGMT promoter methylation in the normal tissue of 10 cm away from tumors and MGMT promoter methylation in adjacent tumors was not statistically significant.

Hawkins et al. (2009) have reported that the low level of methylation is detected by quantitative methylation-specific PCR in the normal colonic mucosa of cases. It has also been reported that the MGMT was hypermethylated in 44 of 95 (46%) primary CRC. It was shown that 50% patients whose cancer had MGMT promoter methylation also had substantial MGMT promoter methylation in the normal adjacent mucosa (Shen et al., 2005). In addition to these findings, we have found that MGMT promoter methylation is detectable in the normal mucosa of patients without neoplasia. This research demonstrated that the mean MGMT methylation level was significantly higher in the tumoral group than in the control group. We considered samples of 5 and 10 cm away from tumors together as a group of samples of adjacent to tumors. This methylation was seen in 36.36% (4/11; 95% CI=21% to 51%) of normal samples with MGMT promoter methylation in the adjacent tumors, 20.79% (6/29; 95% CI=3% to 19%) of samples without MGMT methylation in adjacent tumors, and 6.66% (2/30; 95% CI=1% to 25%) of control samples (p<0.006 and p<0.001 for a comparison between each of the latter two groups and the first group, respectively). These findings showed that MGMT promoter methylation has frequently occurred in the normal-appearing colonic mucosa of CRC patients whose tumors had MGMT promoter methylation, but it was less frequent if the tumor did not have MGMT methylation. Shen et al. (2005) have demonstrated that gene methylation increases with age that accompanies sporadic colorectal tumorigenesis. Contrary to this finding, among patients who had MGMT promoter methylation in their normal-appearing colonic mucosa, we have observed that methylation density had a declining correlation with age.

Conclusion

In conclusion, our data demonstrated that some CRCs arising from a field defect defined by epigenetic inactivation of MGMT detection of this abnormality might ultimately be useful in risk assessment for CRC. Further work may include the investigation of MGMT promoter methylation in relation to MGMT mRNA or MGMT protein expression, and also the detection of DNA methylation profiling in other types of clinical samples such as stool or plasma. These findings suggest that quantification of MGMT promoter hypermethylation will have a key role in identifying CRC patients at high risk during the early disease. On the basis of the results presented, additional studies or clinical trials will be useful to investigate the prognostic value of MGMT methylation in CRC and other types of gastrointestinal tract cancers.

Footnotes

Acknowledgments

The authors are thankful for the financial support of the Mashhad University of Medical Sciences, Mashhad, Iran. They are grateful to the volunteers involved in the study, laboratory staff, and all those who contributed in terms of time and effort.

Disclosure Statement

No competing financial interests exist.

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