Epigenetic biomarkers of promoter DNA methylation in the new era of cancer treatment

Abstract

Promoter DNA methylation, which occurs on cytosine nucleotides across CpG islands, results in gene silencing and represents a major epigenetic alteration in human cancer. Methylation-specific PCR can amplify these modifications as markers in cancer cells. In the present work, we rigorously review the published literatures describing DNA methylation in the promoters of critical tumor suppressor genes; detection of promoter DNA methylation in various body fluids permits early detection of cancer cells during perioperative courses of clinical treatment. The latest whole-genome comprehensive explorations identified excellent epigenetic biomarkers that could be detected at high frequency with high specificity; these biomarkers, which are designated highly relevant methylation genes (HRMG), permit the discrimination of tumor tissues from the corresponding normal tissues; these markers are also associated with unique cancer phenotypes, including dismal prognosis. In humans, HRMG include the CDO1, GSHR, RASSF1 and SFRP1 genes, with these markers permitting discrimination depending on the organs tested. The combination of several HRMG increased the early detection of cancer and exhibited reliable surveillance potential in human body fluids. Cancer clinics using such epigenetic biomarkers are entering a new era of enhanced decision-making with the potential for improved cancer prognosis.

Keywords: CDO1; body fluid; cancer; methylation; tumor suppressor gene.

Figure 1

A, Methylated cytosines (green boxes) can bind to MeCP2 protein, which is itself a complex composed of proteins with SWI/SNF and HDAC activities, leading, in turn, to alterations of chromatin remodeling and histone acetylation (respectively). These activities regulate the state (open or closed) of chromatin. B, Open chromatin facilitates access by the various transcription factors, permitting transcription to generate RNA. Heterochromatin protein 1 (HP1) functions include gene repression by heterochromatin formation

Figure 2

A, Direct sequencing revealed cytosine methylation (at the blue location) in primary tumor tissues (upper panel), while no cytosine methylation was detected (at the red locations) in the corresponding normal tissues. These cytosine methylation sites were consistent with CpG islands in the promoter DNA of the HOPX gene. B, Cloned sequencing after PCR amplification of products revealed finer‐scale status of the methylated cytosines of the CpG portions of the HOPX gene promoter. Black circles represent methylated residues, while white circles indicate residues that lacked methylation (as assessed by cloned sequencing). TaqMeth values by Q‐MSP (HOPX value/beta‐actin value × 100) are indicated within parentheses (right corners of each panel). C, Q‐MSP for CDO1 gene methylation was performed in DLD1 cells, revealing almost full methylation (93.3% of the CpG sites were methylated as judged by cloned sequencing) at various dilutions. PCR amplification detected signals at dilutions of 10‐, 100‐ and 1000‐fold, but not at 10 000‐ and 100 000‐fold dilutions

Figure 3

A, Methylation of the CDO1 gene is shown in order of the frequencies in various cancers of esophageal adenocarcinoma (100%), endometrial adenocarcinoma (98%), pancreatic adenocarcinoma (94%), large intestine adenocarcinoma (CRC; 91%), stomach (gastric) adenocarcinoma (87%), esophageal squamous cell carcinoma (SCC) (83%), lung (non‐small cell) cancer (82%), breast adenocarcinoma (72%), bladder cancer (78%), biliary tract cancer (73%), gallbladder cancer (72%) and kidney cancer (38%). B, Area under curve (AUC) of the receiver operating characteristic (ROC) curve to discriminate primary tumor tissues from the corresponding normal tissues is shown for pancreatic cancer. Quantitative methylation‐specific PCR (Q‐MSP) showed significant difference (P < .0001) in CDO1 methylation between the primary tumor tissues and the corresponding normal tissues

Figure 4

Methylation value increased as the tumor progressed. A, In colorectal carcinogenesis, the CDO1 methylation level increased significantly during the course of Vogelstein adenoma carcinoma sequence (P < .0001). B, In gallbladder carcinogenesis, the CDO1 methylation level increased significantly as the disease progressed from stage I to stage II

Figure 5

A, In gastric cancer, DNA cytology test using CDO1 methylation showed an approximately 2‐fold higher detection rate of minimal residual disease in the peritoneum, among cases that were positive for CY1 (n = 104). B, In esophageal squamous cell carcinoma (ESCC) with neoadjuvant chemotherapy showing histological grade 2/3, CDO1 methylation level was significantly lower than in other cases (n = 41)