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. 2023 Aug 25;102(34):e34860. doi: 10.1097/MD.0000000000034860

The deficiency of 5-methylcytosine (5mC) and its ramification in the occurrence and prognosis of colon cancer

Xin-Xin Yan a,*, Na Guo a, Song-Wei Ru a, Zhi-Yuan Wang b, Hai-Juan Sui c, Yin-Shi Xu d, Zhen-Dan Yao e
PMCID: PMC10470721  PMID: 37653838

Abstract

The incidence and mortality of colon cancer are increasing, and effective biomarkers for its diagnosis are limited. 5-methylcytosine (5mC), a vital DNA methylation marker, plays important roles in gene expression, genomic imprinting, and transposon inhibition. This study aimed to identify the predictors of colon cancer prognosis and lay the foundation for research on therapeutic targets by detecting the levels of 5mC, 5-hydroxymethylcytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxylcytosine (5caC) in colon cancer and adjacent non-tumor tissues. A tissue microarray including 100 colon cancer tissue samples and 60 adjacent non-tumor tissue samples was used. The expression levels of 5mC and its ramifications were assessed by immunohistochemistry. According to the expression levels, patients were divided into moderately positive and strongly positive groups, and the correlation between clinicopathological characteristics and methylation marks was assessed using 2-sided chi-square tests. The prognostic values of 5mC, 5hmC, 5fC, and 5caC were tested using Kaplan–Meier analyses. Compared with adjacent non-tumor tissues, the overall levels of DNA methylation were lower in colon carcinoma lesions. However, the clinical parameters were not significantly associated with these methylation markers, except for 5hmC, which was associated with the age of cancer patients (P value = .043). Kaplan–Meier analysis disclosed that moderate positive group had a significantly shorter disease specific survival than strong positive group for patients with different levels of 5mC (65.2 vs 95.2 months, P = .014) and 5hmC (71.2 vs 97.5 months, P = .045). 5mC and its ramifications (5hmC, 5fC, and 5caC) can serve as biomarkers for colon cancer. 5mC and 5hmC are stable predictors and therapeutic targets in colon cancer. However, further understanding of its function will help to reveal the complex tumorigenic process and identify new therapeutic strategies.

Keywords: 5hmC, 5mC, biomarkers, colon cancer, DNA methylation

1. Introduction

Colorectal cancer is one of the most common malignancies of the gastrointestinal tract. It is the second most common tumor in women and third most common tumor in men.[1] Although the global incidence of colorectal adenocarcinoma is generally stable or declining, the incidence of colorectal cancer in people over 50 years of age is increasing.[2] China is one of the countries with a high incidence of colorectal cancer. Cancer statistics show that the incidence and mortality of colorectal cancer have been increasing in China from 2000 to 2020.[3,4] There are estimated 2.2 million new colon cancer cases and 1.1 million deaths by 2030, especially in developing countries.[5]Colon cancer patients have no obvious early symptoms, which increases the difficulty of early diagnosis.[6,7] In addition, colon cancer has a high metastasis rate; more than 50% of colon cancers present with or develop liver metastasis[8] which causes a poor prognosis. Recently, Colon cancer has recently become the third leading cause of death worldwide.[9,10] With the development of gene detection technology, chemotherapy, molecular targeted therapy, and immunotherapy, the diagnosis and treatment of colon cancer have greatly improved. However, existing therapeutic targets cannot achieve the expected therapeutic effects and the 5-year survival rate of patients with advanced colon cancer is low.[11] Therefore, further research on novel markers and therapeutic targets is warranted. Tumorigenesis is caused by oncogene expression and inactivation of tumor suppressor genes,[12] and epigenetic changes are involved in the pathogenesis, molecular heterogeneity, and progression of colorectal cancer.[13] DNA methylation is the most common epigenetic modification observed. 5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and transposon inhibition.[14] It regulates gene activity without changing the main DNA sequence, and is involved in many biological processes. The 10 to 11 translocation protein converts 5mC to 5-hydroxymethylcytosine (5hmC) and produces 5-formyl cytosine (5fC) and 5-carboxyl cytosine (5caC) from 5mC in an enzyme activity dependent manner.[14] In this study, we investigated the expression and clinical significance of these 4 DNA methylation markers in colon cancer and adjacent non-tumor tissues. Our findings may provide potential predictors of colon cancer prognosis and lay the foundation for the identification of therapeutic targets.

2. Materials and methods

2.1. Clinical sample

A tissue microarray (TMA) consisting of 100 colon cancer tissue samples and 60 adjacent non-tumor tissue samples (Lot No. HColA160Su02) was purchased from Outdo Biotech (Shanghai, China). Immunohistochemistry detection of MLH1, MSH2, MSH6, and PMS2 in colon cancer TMA was performed according to the manufacturer’s instructions. The negative expression of any of these 4 markers was defined as microsatellite instability (MSI). The study was approved by the Ethics Committee of the OutdoBiotech Company (No. SHYJS-CP-1704001).

2.2. Immunohistochemistry staining

Immunohistochemical staining for 5mC, 5hmc, 5fC, and 5caC was performed on the 4 TMAs. Slicing and heating were performed manually. After dewaxing the TMA sections (4μm), endogenous peroxidase was blocked with TBS/H2O2. The primary antibodies against 5mC (cat. no. 629765; GeneTex, 1:1000 dilution) and 5hmc (cat. no. 39769; Active Motif, 1:100 dilution), 5fC (cat. no. 61223; Active Motif, 1:100 dilution) and 5caC (cat. no. 61225; Active Motif, 1:100 dilution), and 5hmc, 5fC, and 5caC were stained with horseradish peroxidase conjugated with rabbit secondary antibody (Envision, cat. no. K4003, Dako) and horseradish peroxidase-conjugated mouse secondary antibodies (cat. no. K4001; Dako) was used to detect 5mC. Colorimetric signals were detected using diaminobenzidine, and the sections were counterstained with hematoxylin and examined under a microscope.

The mean integrated optical density (IOD) of nuclei in cancerous and adjacent non-tumor tissues was determined using image analysis. Immunoreactivities of 5mC, 5hmc, 5fC, and 5caC were assessed by 2 independent observers using Pannoramic Viewer software (3DHISTECH, Hungary, No. PANNORAMIC DESK/MIDI/250/1000). The captured images of 500 × 500 pixels were saved in the JPG format. Each image was analyzed using AperioImageScope (version 10). The Aperio Positive Pixel Count Algorithm was used to quantify the number of specific stains present in a slide image. These inputs have been preconfigured for brown quantification in 3 intensity ranges: weak positive, positive, and strong positive. Three images were obtained for each segment and the IOD values were averaged.

2.3. Statistical analysis

All statistical analyses were performed using SPSS 20.0 (IBM, Armonk, NY). The Student t test was used to assess the statistical significance of the differences between the 2 groups. Associations between methylation marks and clinicopathological characteristics were evaluated using 2-sided chi-square tests. Kaplan–Meier analyses were used to test the prognostic values of 5mC, 5hmC, 5fC, and 5caC.

3. Results

3.1. Overall expression of DNA methylation and its correlation with clinical parameters

The relevant clinical parameters, including age, sex, site, gross pattern, stage, and MSI, are summarized in Table 1. The mean age ± SD of the patients was 62.4 ± 11.6, and the male to female ratio was 1.3:1. Nearly half of the patients (48%) were diagnosed with right colon cancer and 61% of the patients were diagnosed with UC. 57% of the patients had good performance status (stage II). It was found that 31% of patients had MSI. Considering the different levels of DNA methylation markers, cases were divided into moderately positive and strongly positive groups. The clinicopathological parameters of the different groups are summarized in Table 1. The level of 5hmC showed a significant difference in mean age (P value = .043). The strongly positive 5hmC group had a more advanced age than the moderately positive group.

Table 1.

Associations of 5mC, 5hmC, 5fC, and 5caC with the clinicopathological parameters of colon carcinomas (*P < .05).

Parameters Sample Levels of 5mC Levels of 5hmC Levels of 5fC Levels of 5caC
Groups Total tissues
(n = 100)		 Colon
cancer
(n = 100)		 Adjacent
non-tumor
(n = 60)		 Moderate
positive
(n = 72)		 Strong
positive
(n = 18)		 Moderate
positive
(n = 81)		 Strong
positive
(n = 15)		 Moderate
positive
(n = 77)		 Strong
positive
(n = 17)		 Moderate
positive
(n = 47)		 Strong
positive
(n = 11)
Age (yr) 62.4 ± 11.64 62.4 ± 11.64 62.15 ± 12.46 63.61 ± 11.26 58.94 ± 11.54 61.84 ± 12.09 65.60 ± 8.22 61.9 ± 12.29 63.94 ± 8.98 61.45 ± 12.47 64.73 ± 12.63
P = .898 P = .979 P = .043* P = .075 P = .687
Gender
 Male (%) 58 58 58.3 59.7 55.6 56.5 66.7 57.1 58.8 59.6 54.5
 Female (%) 42 42 41.7 40.3 44.4 43.5 33.3 42.9 41.2 40.4 45.5
P = .967 P = .748 P = .461 P = .899 P = .760
Site
 Right colon (%) 48 50 45 44.4 61.1 48.2 46.7 44.2 58.8 40.4 45.5
 Left colon (%) 24 24 26.7 25 16.7 24,7 20 28.6 5.9 27.7 27.3
 Rectosigmoid colon (%) 28 26 28.3 30.6 22.2 27.1 33.3 27.3 35.3 31.9 27.3
P = .828 P = .447 P = .859 P = .088 P = .943
Gross pattern
 Lumpy (%) 22 21 16.7 20.8 27.8 23.5 13.3 19.5 41.2 14.9 36.4
 Ulcerous (%) 61 61 66.7 61.1 50 58.8 73.3 66.2 35.5 70.2 54.5
 Invasive (%) 17 18 16.7 18.1 22.2 17.6 13.3 14.3 23.5 14.9 9.1
P = .744 P = .688 P = .535 P = .057 P = .258
Stage
 II (%) 57 55 55 54.2 55.6 54.1 73.3 58.4 64.7 57.4 63.6
 III (%) 38 40 40 41.7 33.3 40 26.7 37.7 29.4 38.3 27.3
 IV (%) 5 5 5 4.2 11.1 5.9 0 3.9 5.9 4.3 9.1
P = 1.000 P = .471 P = .218 P = .785 P = .684
Microsatellite instability (MSI) (%) 31 31 25 26.4 38.9 30.6 33.3 32.5 23.5 25.5 27.3
P = .417 P = .295 P = .832 P = .470 P = .906
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5caC = 5-carboxylcytosine, 5fC = 5-formylcytosine, 5hmC = 5-hydroxymethylcytosine, 5mC = 5-methylcytosine.

3.2. The immunohistochemical staining of 5mC, 5hmC, 5fC, and 5caC

Epithelial dysplasia is a histopathological marker of a premalignant disorder of the colon, and is characterized by a reduction in the number and size of mature cells. Small transparent mucus vacuoles were observed in the cytoplasm of a few of the cancer cells. The nuclei were round, half-moon, or irregular with obvious deformities. The chromatin was thickened and showed a thick mass or coarse reticular shape divided into uneven sections. Nucleoli were obvious and thickened. The cytoplasmic boundary of the cell was not clear, and the nucleus was located at the edge of the cell mass, causing the marginal cells to bulge and form a morula (Fig. 1A). At the same time, it was found that the abnormal development of colon cancer cells is related to DNA methylation markers. Taking 5hmc as an example, 5hmC is rarely expressed in the heterogeneous nucleus of cancer cells (Fig. 1C), but is highly expressed in the homogeneous nucleus of normal tissues (Fig. 1B). The same is true of other markers.

Figure 1.

Figure 1.

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HE and immunohistochemistry staining of colon cancer. A represent HE staining (×20), and B represent immunohistochemical staining of 5hmC in cancer tissue (×40), C represent immunohistochemical staining of 5hmC in non-tumor tissue (×40). 5hmC = 5-hydroxymethylcytosine.

The study found that the levels of DNA methylation markers 5mC, 5hmC, 5fC, and 5caC were lower in colorectal carcinoma lesions than in adjacent non-tumor tissues (Fig. 2). The mean IOD values of 5mC immunohistochemical staining was 0.39 ± 0.03 in cancer and 0.41 ± 0.02 in adjacent non-tumor tissues. The mean IOD values of 5hmC immunohistochemical staining was 0.37 ± 0.05 in cancer and 0.42 ± 0.04 in adjacent non-tumor tissues. The mean IOD values of 5fC immunohistochemical staining was 0.29 ± 0.05 in cancer and 0.33 ± 0.03 in adjacent non-tumor tissues. The mean IOD values of 5caC immunohistochemical staining was 0.31 ± 0.04 in cancer and 0.34 ± 0.04 in adjacent non-tumor tissues.

Figure 2.

Figure 2.

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Immunohistochemical staining of 5mC, 5hmC, 5fC and 5caC in colon cancer tissues and adjacent non-tumor tissues. A/C/E/G represent cancer tissues, and B/D/F/H represent adjacent non-tumor tissues (**P < .01). 5caC = 5-carboxylcytosine, 5fC = 5-formylcytosine, 5hmC = 5-hydroxymethylcytosine, 5mC = 5-methylcytosine.

3.3. The correlation of DNA methylation with survival of patients

Most colon carcinoma cases were stage II (57%) and the overall survival time was 98 months. Kaplan–Meier analysis revealed that the moderate positive group had a significantly shorter overall survival than the strong positive group for 5mC (65.2 vs 95.2 months, P = .014) and 5hmC (71.2 vs 97.5 months, P = .045), as shown in Figure 3A and B. However, there were no significant correlations between differentiated levels of 5fC (78.6 vs 85.6 months, P = .519) or 5caC (74.3 vs 70 months, P = .392) and the overall survival of colon carcinoma patients (Fig. 3C and D).

Figure 3.

Figure 3.

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Overall survival of colon carcinoma patients with different levels of 5mC, 5hmC, 5fC, and 5caC. (A) The relationship of different 5mc levels with survival time of colorectal carcinoma patients (P = .014). (B) The relationship of different 5hmC levels with survival time of colon carcinoma patients (P = .045). (C) The relationship of different 5fC levels with survival time of colon carcinoma patients (P = .519). (D) The relationship of different 5caC levels with survival time of colon carcinoma patients (P = .392). 5caC = 5-carboxylcytosine, 5fC = 5-formylcytosine, 5hmC = 5-hydroxymethylcytosine, 5mC = 5-methylcytosine.

4. Discussion

Epigenetics refers to changes in gene expression levels due to non-gene sequence changes in cancer development and progression, which can be exploited as clinically relevant disease biomarkers for the diagnosis, prognostication, and prediction of treatment response.[15] DNA methylation, one of the earliest and most deeply studied mechanisms, controls gene expression by changing the structure of chromatin, DNA conformation, DNA stability, and DNA-protein interactions. Epigenetic mechanisms are believed to play vital roles in cancer development.[16] 5mC, a normal epigenetic modification of CpG-rich regions of DNA, is a product of cytosine produced by the catalysis of DNA methyltransferase. In mammals, active demethylation is mediated by the 10 to 11 translocation family of dioxygenases, which oxidize 5mC to 5hmC, 5fC, and 5caC.[17] Typically, tumor suppressor genes are hypermethylated whereas tumor genes are hypomethylated. Hypermethylation often causes transcriptional silencing of downstream genes. 5mC has been shown to be associated with repression of the chromatin state and transcriptional silencing, serving as a sensitive marker of oxidative damage-induced tumorigenesis and a typical hallmark of cancer cells.[18] The epigenetic marker, 5hmC, has recently been identified as a novel prognostic biomarker for cancers of the colon, liver, stomach, thyroid, and pancreas.[19]

In this study, we comprehensively investigated 5mC, 5hmC, 5fC, and 5caC levels in colon cancer and adjacent non-tumor tissues using immunohistochemical analysis. The expression of 5mC, 5hmC, 5fC, and 5caC in colon cancer tissues was significantly lower than that in adjacent tissues, and was mainly distributed in the nucleoli of tumor cells. DNA hypomethylation is characteristic of tumor cells and may disrupt normal gene structure and function, resulting in chromosomal instability by reactivating previously silenced retrotransposons[20]and This may be a cause of cancer development. Studies have shown that Global DNA hypomethylation is a potential biomarker for colorectal cancer risk[21]and breast cancer risk[22] in peripheral blood leukocytes.

Previous studies have found that downregulation of 5hmC is associated with the progression of cervical intraepithelial neoplasia, as detected by immunohistochemistry analysis.[23] 5hmC expression is reduced in high-grade brain tumors compared to low-grade tumors and normal brain tissues.[24]5mC and 5hmC presented different expression patterns in cervical squamous cell carcinoma. In cervical cancer, 5mC is upregulated, whereas 5hmC is downregulated in cervical cancer.[25] 5hmC, 5fC, and 5caC are the derivatives of 5mC. 5mC is present at a frequency approximately 10 to 100 folds higher than that of 5hmC and 40 to 1000 folds higher than that of 5fC and 5caC.[26] Therefore, 5mC is considered to be a more stable indicator. 5hmC is an independent epigenetic marker, with a weak positive correlation with 5mC. 5fC and 5caC are probably short-lived intermediates of active demethylation processes. In contrast to 5mC, the distribution of 5hmC is nonrandom in tissues and cell lines.[27,28] Moreover, many studies have demonstrated that 5hmC expression is significantly decreased in various tumors.[29,30]

In conclusion, 5mc, 5hmC, 5fC, and 5caC expression levels were significantly decreased in colon carcinoma, indicating that these were associated with tumor formation and development. It has been found that a variety of tumor suppressor genes are the targets of DNA hypermethylation in cancers, and abnormal DNA methylation promotes cell tumorigenesis through tumor suppressor genes silencing.[31] In this study, the lower methylation in colon carcinoma may be due to lower methylation of tumor suppressor genes. Further research is needed to explore the types and levels of methylation of specific tumor suppressor genes, which can help to develop antitumor therapies using tumor suppressor gene demethylation drugs.

By analyzing the association of 5mC and its derivatives with the clinicopathological parameters of colon carcinoma, we found that only age had a slightly significant association with 5hmC. Other clinicopathological parameters, including sex, site, gross pattern, stage, and MSI were not significantly associated with the expression levels of 5mC and its derivatives. In tumor tissues, lower methylation expression can promote tumor development; for instance, methyl island hypomethylation can cause oncogene overexpression, promote tumor development, and result in poor prognosis in cancer patients.[32] This mechanism might be caused by abnormal methylation of tumor suppressor genes; for example, hypomethylation leads to dysregulation of septin 9, which might eventually lead to cancer development.[33,34] Zhang et al[25] indicated that a decreased 5hmC level predicts poor prognosis in patients with cervical squamous cell carcinoma. The mechanism may be that decreased expression levels of the 5hmC protein affect proper gene transcription, cause abnormal proliferation and growth of tumor cells, promote tumor recurrence and metastasis, and reduce the survival of patients with cervical cancer. A lower level or loss of 5hmC might predict poor prognosis in non-small cell lung cancer, intrahepatic cholangiocarcinoma, and small hepatocellular carcinoma.[35–37] In addition to 5hmC, we found that a lower level of 5mc was also connected with a poor prognosis of colon cancer. Therefore, 5mC was deemed a stable indicator for predicting the prognosis of patients with colon cancer.

5mC and its ramifications (5hmC, 5fC, and 5caC) are epigenetic markers. Compared with adjacent noncancerous tissues of colon cancer, tumor tissues had lower 5mC, 5hmC, 5fC, and 5caC levels, and lower methylation levels of 5hmC and 5mC predicted a poor prognosis. Epigenetic modifications have been identified as vital factors in the occurrence and development.[38] Within cancer cells, DNA methylation levels are reduced in regions of low CpG density compared to those in normal cells, whereas a subset of CpG islands is hypermethylated in a cancer cell-specific manner.[39] Simultaneously, many genes involved in DNA methylation can undergo hypermethylation in specific cancer cells.[40] We found that 5mC and its ramifications, 5hmC, 5fC, and 5caC, can serve as colon cancer biomarkers, whereas 5mC and 5hmC can be used as markers for the prognosis of colon cancer. The sample size of this study is limited, so it is necessary to further expand the sample size for further research and further understanding of its function will help reveal the complex tumorigenic process and identify new therapeutic strategies.

5. Conclusion

The levels of 5mC and its ramifications are significantly decreased in colon cancer. The 5mC and 5hmC levels have been identified as valuable prognostic predictors of colon cancer. It is necessary to further explore the methylation types and levels of specific tumor suppressor genes, which can help develop antitumor therapies using demethylating drugs.

Acknowledgements

The authors received financial support for the research, authorship, and/or publication of this article from the Foundation of Aerospace Center Hospital (YN202107), Foundation of Aerospace Group Medical Technology Group (2021YK02), and Foundation of Haidian District Health Development Research Cultivation Program (HP2023-02-507004).

Author contributions

Data curation: Songwei Ru, Haijuan Sui.

Funding acquisition: Xinxin Yan.

Project administration: Xinxin Yan, Yinshi Xu, Zhendan Yao.

Software: Zhiyuan Wang.

Supervision: Zhiyuan Wang.

Visualization: Haijuan Sui.

Writing – original draft: Xinxin Yan, Na Guo.

Writing – review & editing: Xinxin Yan, Na Guo.

Abbreviations:

5caC
5-carboxylcytosine
5fC
5-formylcytosine
5hmC
5-hydroxymethylcytosine
5mC
5-methylcytosine
IOD
integrated optical density
MSI
microsatellite instability
TMA
tissue microarray

The authors have no conflicts of interest to disclose.

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

How to cite this article: Yan X-X, Guo N, Ru S-W, Wang Z-Y, Sui H-J, Xu Y-S, Yao Z-D. The deficiency of 5-methylcytosine (5mC) and its ramification in the occurrence and prognosis of colon cancer. Medicine 2023;102:34(e34860).

Contributor Information

Na Guo, Email: guona721gn@163.com.

Song-Wei Ru, Email: rusongwei@asch.net.cn.

Zhi-Yuan Wang, Email: zhiyuan8552@126.com.

Hai-Juan Sui, Email: suihaijuan@jzmu.edu.cn.

Yin-Shi Xu, Email: 721ywb@sina.com.

Zhen-Dan Yao, Email: yaozd1984@sina.com.

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