Elsevier

Gene

Volume 644, 20 February 2018, Pages 66-73
Gene

Research paper
Promoter methylation and expression of DNA repair genes MGMT and ERCC1 in tissue and blood of rectal cancer patients

https://doi.org/10.1016/j.gene.2017.10.056Get rights and content

Highlights

  • Blood and tumor tissue MGMT & ERCC1 methylation were associated with cancer rectum.

  • Blood MGMT & ERCC1 methylation status may represent the rectal tissue methylation.

  • MGMT and ERCC1 methylation were not associated with clinicopathological features.

  • MGMT and ERCC1 methylation were correlated with response to therapy.

  • MGMT and ERCC1 genes methylation was associated with down-regulation of their mRNA expression.

Abstract

Rectal cancer involves one-third of colorectal cancers (CRCs). Recently, data supported that DNA methylation have a role in CRC pathogenesis. In the present study we aimed to analyze the methylation status of MGMT and ERCC1 promoter regions in blood and tissue of patients with benign and malignant rectal tumors. We also studied the methylated MGMT and ERCC1 genes and their relations with clinicopathological features. Furthermore, we suggested that methylation may play a critical function in the regulation of MGMT and ERCC1 expression.

Fifty patients with non-metastatic cancer rectum and 43 patients with benign rectal lesions were involved in the study. DNA extraction from blood and rectal specimens was done to analyze the methylation status of MGMT and ERCC1 genes by methylation-specific PCR method. RNA was extracted also to determine the expression levels of these genes by real time-PCR.

The frequency of MGMT and ERCC1 methylation was significantly higher in rectum cancers than in benign tumors both for the tissue and the blood (p < 0.001). There was no relation between MGMT or ERCC1 methylation and clinicopathological features; while they were correlated with the response to therapy. An interesting finding that the agreement of the methylation levels in the blood and rectal tissue was classified as good (κ = 0.78) for MGMT gene and as very good (κ = 0.85) for ERCC1. Lastly, the MGMT and ERCC1 genes methylation was associated with down-regulation of their mRNA expression when compared with the non-methylated status.

Our findings provided evidence that both blood and tumor tissue MGMT and ERCC1 methylation were associated with cancer rectum. MGMT or ERCC1 methylation in blood could be suitable non-invasive biomarkers differentiating benign and malignant rectal tumors. Furthermore, the methylation of the MGMT and ERCC1 promoter regions was associated with down-regulation of their mRNA expression.

Introduction

Rectal cancer constitutes one-third of colorectal cancers (CRCs). It has a specific anatomic position in the small pelvis which makes the operative resection with clear margins more difficult but permits for chemoradiotherapy as an efficient neoadjuvant treatment (Pramateftakis et al., 2010).

Recently, researches revealed that both genetic and epigenetic agents have a part in CRC pathogenesis. DNA methylation is considered as an epigenetic post-replication DNA modification by a methyl group addition to carbon 5 of the cytosine pyrimidine ring, mainly in cytosine-phospho-guanine (CpG) dinucleotide (Korkmaz et al., 2011). The CpG dinucleotides clusters are presented in promoter areas of genes and are termed CpG islands. A CpG island is defined as a stretch of DNA extended at least 500 bp and ≥ 55% of which consists of GC, moreover; a ratio of observed and expected CpG is more than 0.65 (Korkmaz et al., 2011). DNA methylation of promoter regions of these CpG islands represses their genes by either decreasing the DNA availability for transcription factors (TFs) or enrolling extra silencing associated proteins (Jones and Liang, 2009). DNA hypermethylation results in transcriptional silencing of specific tumor suppressor genes (TSGs) (Ng and Yu, 2015). One class of these TSGs is the genes that their proteins shared in the repair of damaged DNA. Notably, DNA repair mechanism dysfunction allows DNA damage to escape repair and could result in genomic instability; one of important characteristic of tumorigenesis (Farkas et al., 2014).

Direct reversal of DNA damage is the simplest form of DNA repair. Methylation of guanine at the O6-position can be excised by O6-methylguanine DNA methyltransferase (MGMT) enzyme (Mishina et al., 2006). MGMT is also able to take off other alkylations at the O6-position of guanine (Mishina et al., 2006). Unrepaired O6-methylguanine sites can lead to mutagenesis as distorted pairing of guanine with cytosine or thymidine leads to G: C to A: T transitions on replication (Singer and Dosanjh, 1999).

Nucleotide excision repair (NER) is another mechanism of DNA repair, which removes helix-disrupted DNA damages as thymine dimers. ERCC1/xeroderma pigmentosum (Xp-f) complex is a DNA endonuclease necessary for one of the two incision steps of NER as ERCC1 endonuclease incises the DNA on the 5′ side of the damaged site. The ERCC1/Xp-f also supports repair of DNA double-strand breaks (De Dosso et al., 2013) and inter-strand crosslinks (Clauson et al., 2013).

Up to date, majority of published reports were done randomly on patients with either colon or rectal cancer; despite both tumors have completely different biological and clinical entities (Iacopetta, 2002). Few studies only have studied the association of the DNA methylation status with rectal cancer. A set of five methylated TSGs was shown in early-stage rectal cancer (Leong et al., 2011). Vymetalkova et al. have been investigated the methylated DNA signatures as potential biomarkers in rectal cancer (Vymetalkova et al., 2016). CDKN2A promoter methylation has been proposed to be indicator of more aggressive tumors with worse prognosis (Kohonen-Corish et al., 2014). CpG island methylator phenotype (CIMP) positivity was correlated with poor radio-chemotherapy's response and significantly reduced disease-free survival (DFS) in locally advanced rectal cancer (Jo et al., 2012). However, Molinari et al. found that methylation of selected TSGs can't detect rectal cancer patients at high risk of relapse (Molinari et al., 2013).

In the present study we analyzed the methylation status of MGMT and ERCC1 promoter regions in blood and tissue of benign and malignant rectal tumors patients. Also, the methylated MGMT and ERCC1 genes and their relations with clinicopathological features as well as response to therapy have been investigated. Furthermore, we proposed that methylation may have a critical role in the regulation of MGMT and ERCC1 repression.

Section snippets

Sample collection

This work included 93 patients diagnosed with rectal tumor. Fifty patients with non-metastatic cancer rectum (stage I, II, III) and 43 benign tumor patients were collected following approval from the Ethics Committees of Faculty of Medicine, Zagazig University. We obtained informed consent from all patients. Blood samples were collected prior to the beginning of treatment. Pretreatment rectal tumor biopsy specimens were divided into two parts. One part was sent for routine pathology

Clinicopathological features results

Our findings demonstrated non statistical significant differences between rectal cancer patients and cases with benign lesions regarding age and sex (p > 0.05). Data listed in Table 1 summarized the clinicopathological characteristics in rectal cancer patients. There was a statistically significant difference between patients < 65 years and patients ≥ 65, site of tumor, pathology, grading and staging. On the other side, there was no significant difference in the terms of sex, CEA levels, lymph nodes

Discussion

The DNA repair genes are subtypes of TSGs that are largely methylated in the development of carcinogenesis. In the present study, we analyzed the methylation status of two DNA repair genes; MGMT and ERCC1 in benign and malignant rectal tumors. Results displayed that the frequencies of MGMT and ERCC1 methylation in rectum cancers and benign tumors was statistically significant different in both tissue and blood. A methylation-positive pattern of MGMT was seen for 80% of tumor tissues and for 58%

Conflict of interest

None.

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