Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Identification of odontogenic ameloblast associated as a novel target gene of the Wnt/ß-catenin signaling pathway.

The Wnt/ß-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant ß-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant ß-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic, ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the ß-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.

Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.

Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient.

Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.

Efficacy of liquid-based genetic diagnosis of endometrial cancer.

Although liquid-based cytology (LBC) has increased the sensitivity of cytological diagnosis of endometrial cancer (EC) compared with conventional smear cytology, the sensitivity of LBC for the detection of EC is between 70% and 96% and remains unsatisfactory. In the present study, we compared the efficacy of LBC with liquid-based genetic diagnosis (LBGDx) by amplicon sequencing of five genes including PTEN, PIK3CA, CTNNB1, KRAS, and TP53 in 48 LBC subjects who underwent endometrial screening. Consequently, LBC classified 15 samples as "positive or suspicious for malignancy" and the 15 were later confirmed as EC. However, LBC failed to identify five cases who were diagnosed as EC by additional transvaginal ultrasound and endometrial curettage, indicating that the sensitivity of cytology alone was 75% (15/20). LBGDx identified 11 pathogenic PTEN variants in 10 subjects, six PIK3CA variants in nine, three CTNNB1 variants in five, two KRAS variants in four, and three TP53 variants in three. Collectively, at least one pathogenic variant was identified in 19 subjects, which included 17 EC (15 endometrioid carcinoma and 2 endometrial carcinosarcomas), and one cervical adenocarcinoma. However, LBGDx did not identify any pathogenic mutations in three of the 20 EC, indicating that the sensitivity of LBGDx alone was 85% (17/20). Although five EC were negative for malignancy by LBC and three were negative for pathogenic mutations by LBGDx, the combination of LBC and LBGDx would successfully diagnose all 20 EC. These data suggested that LBGDx is a useful strategy to improve the sensitivity of screening of EC by LBC.

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway.

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high-methylation CRC. The methylation levels of six Group-1 and 14 Group-2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group-1 and Group-2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high-methylation CRC. But TSA showed low-methylation levels of Group-1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high-methylation CRC. High-methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1-methylated SSA/P showed lower methylation level of MLH1 compared with high-methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1-methylated and MLH1-unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF-ß and BMP signaling (but not in TP53 signaling) were significantly involved in high-methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.

TP53 mutation at early stage of colorectal cancer progression from two types of laterally spreading tumors.

Although most sporadic colorectal cancers (CRC) are thought to develop from protruded adenomas through the adenoma-carcinoma sequence, some CRC develop through flat lesions, so-called laterally spreading tumors (LST). We previously analyzed epigenetic aberrations in LST and found that LST are clearly classified into two molecular subtypes: intermediate-methylation with KRAS mutation and low-methylation with absence of oncogene mutation. Intermediate-methylation LST were mostly granular type LST (LST-G) and low-methylation LST were mostly non-granular LST (LST-NG). In the present study, we conducted a targeted exon sequencing study including 38 candidate CRC driver genes to gain insight into how these genes modulate the development of LST. We identified a mean of 11.5 suspected nonpolymorphic variants per sample, including indels and non-synonymous mutations, although there was no significant difference in the frequency of total mutations between LST-G and LST-NG. Genes associated with RTK/RAS signaling pathway were mutated more frequently in LST-G than LST-NG (P = 0.004), especially KRAS mutation occurring at 70% (30/43) of LST-G but 26% (13/50) of LST-NG (P < 0.0001). Both LST showed high frequency of APC mutation, even at adenoma stage, suggesting its involvement in the initiation stage of LST, as it is involved at early stage of colorectal carcinogenesis via adenoma-carcinoma sequence. TP53 mutation was never observed in adenomas, but was specifically detected in cancer samples. TP53 mutation occurred during development of intramucosal cancer in LST-NG, but during development of cancer with submucosal invasion in LST-G. It is suggested that TP53 mutation occurs in the early stages of cancer development from adenoma in both LST-G and LST-NG, but is involved at an earlier stage in LST-NG.

Wnt/ß-catenin signaling regulates amino acid metabolism through the suppression of CEBPA and FOXA1 in liver cancer cells.

Deregulation of the Wnt/ß-catenin pathway is associated with the development of human cancer including colorectal and liver cancer. Although we previously showed that histidine ammonia lyase (HAL) was transcriptionally reduced by the ß-catenin/TCF complex in liver cancer cells, the mechanism(s) of its down-regulation by the complex remain to be clarified. In this study, we search for the transcription factor(s) regulating HAL, and identify CEBPA and FOXA1, two factors whose expression is suppressed by the knockdown of ß-catenin or TCF7L2. In addition, RNA-seq analysis coupled with genome-wide mapping of CEBPA- and FOXA1-binding regions reveals that these two factors also increase the expression of arginase 1 (ARG1) that catalyzes the hydrolysis of arginine. Metabolome analysis discloses that activated Wnt signaling augments intracellular concentrations of histidine and arginine, and that the signal also increases the level of lactic acid suggesting the induction of the Warburg effect in liver cancer cells. Further analysis reveals that the levels of metabolites of the urea cycle and genes coding its related enzymes are also modulated by the Wnt signaling. These findings shed light on the altered cellular metabolism in the liver by the Wnt/ß-catenin pathway through the suppression of liver-enriched transcription factors including CEBPA and FOXA1.

Expression of PVRL4, a molecular target for cancer treatment, is transcriptionally regulated by FOS.

PVRL4 (or nectin­4) is a promising therapeutic target since its upregulated expression is found in a wide range of human cancer types. Enfortumab vedotin, an antibody­drug conjugate targeting PVRL4, is clinically used for the treatment of urothelial bladder cancer. In addition, rMV­SLAMblind, a genetically engineered oncolytic measles virus, can infect cancer cells and induce apoptosis through interaction with PVRL4. Although PVRL4 transcript levels are elevated in breast, lung and ovarian cancer, the mechanisms of its upregulation have not yet been uncovered. To clarify the regulatory mechanisms of elevated PVRL4 expression in breast cancer cells, Assay for Transposase­Accessible Chromatin­sequencing and chromatin immunoprecipitation­sequencing (ChIP­seq) data were used to search for its regulatory regions. Using breast cancer cells, an enhancer region was ultimately identified. Additional analyses, including ChIP and reporter assays, demonstrated that FOS interacted with the PVRL4 enhancer region, and that alterations of the FOS­binding motifs in the enhancer region decreased reporter activity. Consistent with these data, exogenous expression of FOS enhanced the reporter activity and PVRL4 expression in breast cancer cells. Furthermore, RNA­seq analysis using breast cancer cells treated with PVRL4 small interfering RNA revealed its possible involvement in the cytokine response and immune system. These data suggested that FOS was involved, at least partly, in the regulation of PVRL4 expression in breast cancer cells, and that elevated PVRL4 expression may regulate the response of cancer cells to cytokines and the immune system.

Visinin-like 1, a novel target gene of the Wnt/ß-catenin signaling pathway, is involved in apoptosis resistance in colorectal cancer.

BACKGROUND: Abnormal activation of Wnt/ß-catenin signaling is associated with various aspects of cancer development. This study explored the roles of novel target genes of the Wnt/ß-catenin signaling pathway in cancer cells. METHODS: Using the haploid chronic myelogenous leukemia cell line HAP1, RNA sequencing (RNA-seq) was performed to identify genes whose expression was increased by APC disruption and reversed by ß-catenin knockdown (KD). The regulatory mechanism and function of one of the candidate genes was investigated in colorectal cancer (CRC) cells. RESULTS: In total, 64 candidate genes whose expression was regulated by Wnt/ß-catenin signaling were identified. Of these candidate genes, the expression levels of six were reduced by ß-catenin KD in HCT116 CRC cells in our previous microarray. One of these genes was Visinin-like 1 (VSNL1), which belongs to the neuronal calcium-sensor gene family. The expression of VSNL1 was regulated by the ß-catenin/TCF7L2 complex via two TCF7L2-binding elements in intron 1. VSNL1 KD-induced apoptosis in VSNL1-positive CRC cells. Additionally, forced expression of wild-type VSNL1, but not a myristoylation, Ca2+ -binding, or dimerization-defective mutant, suppressed the apoptosis induced by camptothecin and doxorubicin in VSNL1-negative CRC cells. CONCLUSION: Our findings suggest that VSNL1, a novel target gene of the Wnt/ß-catenin signaling pathway, is associated with apoptosis resistance in CRC cells.

Bromodomain protein BRD8 regulates cell cycle progression in colorectal cancer cells through a TIP60-independent regulation of the pre-RC complex.

Bromodomain-containing protein 8 (BRD8) is a subunit of the NuA4/TIP60-histone acetyltransferase complex. Although BRD8 has been considered to act as a co-activator of the complex, its biological role remains to be elucidated. Here, we uncovered that BRD8 accumulates in colorectal cancer cells through the inhibition of ubiquitin-dependent protein degradation by the interaction with MRG domain binding protein. Transcriptome analysis coupled with genome-wide mapping of BRD8-binding sites disclosed that BRD8 transactivates a set of genes independently of TIP60, and that BRD8 regulates the expression of multiple subunits of the pre-replicative complex in concert with the activator protein-1. Depletion of BRD8 induced cell-cycle arrest at the G1 phase and suppressed cell proliferation. We have also shown that the bromodomain of BRD8 is indispensable for not only the interaction with histone H4 or transcriptional regulation but also its own protein stability. These findings highlight the importance of bromodomain as a therapeutic target.

Motile sperm domain containing 1 is upregulated by the Wnt/ß-catenin signaling pathway in colorectal cancer.

Aberrant activation of the Wnt/ß-catenin signaling pathway plays a crucial role in the development and progression of colorectal cancer. Previously, we identified a set of candidate genes that were regulated by this signaling pathway, and the present study focused on motile sperm domain containing 1 (MOSPD1). Immunohistochemical staining revealed that the expression of MOSPD1 was elevated in tumor cells from colorectal cancer tissues compared with in non-tumor cells. Using ChIP-seq data and the JASPAR database, the regulatory region(s) in the MOSPD1 gene as a target of the Wnt/ß-catenin signaling pathway were searched, and a region containing three putative TCF-binding motifs in the 3'-flanking region was identified. Additional analyses using reporter assay and ChIP-quantitative PCR suggested that this region harbors enhancer activity through an interaction with transcription factor 7 like 2 (TCF7L2) and ß-catenin. In addition, chromatin conformation capture assay revealed that the 3'-flanking region interacts with the MOSPD1 promoter. These data suggested that MOSPD1 was regulated by the ß-catenin/TCF7L2 complex through the enhancer element located in the 3'-flanking region. These findings may be helpful for future studies regarding the precise regulatory mechanisms of MOSPD1.

Cancer-associated IDH mutations induce Glut1 expression and glucose metabolic disorders through a PI3K/Akt/mTORC1-Hif1α axis.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations and their key effector 2-hydroxyglutarate (2-HG) have been reported to promote oncogenesis in various human cancers. To elucidate molecular mechanism(s) associated with IDH1/2 mutations, we established mouse embryonic fibroblasts (MEF) cells and human colorectal cancer cells stably expressing cancer-associated IDH1R132C or IDH2R172S, and analyzed the change in metabolic characteristics of the these cells. We found that IDH1/2 mutants induced intracellular 2-HG accumulation and inhibited cell proliferation. Expression profile analysis by RNA-seq unveiled that glucose transporter 1 (Glut1) was induced by the IDH1/2 mutants or treatment with 2-HG in the MEF cells. Consistently, glucose uptake and lactate production were increased by the mutants, suggesting the deregulation of glucose metabolism. Furthermore, PI3K/Akt/mTOR pathway and Hif1α expression were involved in the up-regulation of Glut1. Together, these results suggest that Glut1 is a potential target regulated by cancer-associated IDH1/2 mutations.

Anti-apoptotic effect by the suppression of IRF1 as a downstream of Wnt/ß-catenin signaling in colorectal cancer cells.

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the ß-catenin/TCF7L2 complex. Although genes upregulated by Wnt/ß-catenin signaling have been intensively studied, the roles of downregulated genes are poorly understood. Previously, we reported that interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) was downregulated by the Wnt/ß-catenin signaling, and that the suppressed expression of IFIT2 conferred antiapoptotic property to colorectal cancer (CRC) cells. However, the mechanisms underlying how Wnt/ß-catenin signaling regulates IFIT2 remain to be elucidated. In this study, we have uncovered that the expression of IFIT2 is induced by IRF1, which is negatively regulated by the Wnt/ß-catenin signaling. In addition, we found that downregulation of IRF1 is mediated by its degradation through the ubiquitination-proteasome pathway, and that decreased activity of a deubiquitinase complex containing USP1 and UAF1 is involved in the degradation of IRF1 by Wnt/ß-catenin signaling. These data should provide better understanding of the Wnt signaling pathway and human carcinogenesis.

The frequency of promoter DNA hypermethylation is decreased in colorectal neoplasms of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an inherited disorder characterized by numerous colorectal adenomatous polyps with predisposition to the development of colorectal cancer (CRC). Here, we conducted genome-wide DNA methylation analysis of FAP neoplasms, including seven cancer samples and 16 adenoma samples, using an Infinium 450K BeadArray. As controls for sporadic colorectal neoplasms and mucosae, we used Infinium 450k data from 297 CRC samples, 45 colorectal adenoma samples, and 37 normal mucosa samples with reference to The Cancer Genome Atlas and other databases. Unsupervised two-way hierarchical clustering analysis of FAP and sporadic CRC/adenoma revealed that CRC was classified into four DNA methylation epigenotypes (MEs): high-ME (HME), intermediate-ME (IME), low-ME (LME), and normal-like ME (NME). Five FAP neoplasms (two cancer and three adenoma) were clustered with IME, whereas 18 FAP neoplasms (five cancer and 13 adenoma) were clustered into NME. IME FAP neoplasms significantly correlated with KRAS mutations, similar to sporadic CRC. Within IME cases, however, aberrant DNA methylation was significantly less frequent in FAP neoplasms than sporadic neoplasms, and these unmethylated genes included WNT family genes and several types of oncogenes. In summary, FAP neoplasms were classified into at least two molecular subtypes, i.e., NME in the majority of cases showing mostly no aberrant methylation and IME in some cases accompanied by KRAS mutations but less frequent aberrant DNA methylation than sporadic neoplasms, suggesting that FAP may follow a tumorigenesis pathway different from that of sporadic CRC.

DNA methylation epigenotype and clinical features of NRAS-mutation(+) colorectal cancer.

Sporadic colorectal cancer (CRC) is classified into several molecular subtypes. We previously established two groups of DNA methylation markers through genome-wide DNA methylation analysis to classify CRC into distinct subgroups: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively). HME CRC, also called CpG island methylator phenotype (CIMP)-high CRC, shows methylation of both Group 1 markers (CIMP markers) and Group 2 markers, while IME/CIMP-low CRC shows methylation of Group 2, but not of Group 1 markers, and LME CRC shows no methylation of either Group 1 or Group 2 markers. While BRAF- and KRAS-mutation(+) CRC strongly correlated with HME and IME, respectively, clinicopathological features of NRAS-mutation(+) CRC, including association with DNA methylation, remain unclear. To characterize NRAS-mutation(+) CRC, the methylation levels of 19 methylation marker genes (6 Group 1 and 13 Group 2) were analyzed in 61 NRAS-mutation(+) and 144 NRAS-mutation(-) CRC cases by pyrosequencing, and their correlation with clinicopathological features was investigated. Different from KRAS-mutation(+) CRC, NRAS-mutation(+) CRC significantly correlated with LME. NRAS-mutation(+) CRC showed significantly better prognosis than KRAS-mutation(+) CRC (P = 3 × 10-4 ). NRAS-mutation(+) CRC preferentially occurred in elder patients (P = 0.02) and at the distal colon (P = 0.006), showed significantly less lymph vessel invasion (P = 0.002), and correlated with LME (P = 8 × 10-5 ). DNA methylation significantly accumulated at the proximal colon. NRAS-mutation(+) CRC may constitute a different subgroup from KRAS-mutation(+) CRC, showing significant correlation with LME, older age, distal colon, and relatively better prognosis.

Frequent promoter hypermethylation associated with human papillomavirus infection in pharyngeal cancer.

Oropharyngeal squamous cell carcinoma (OPSCC) incidence has increased dramatically due to human papillomavirus (HPV); however, associated epigenetic alterations are not well studied. We performed genome-wide DNA methylation analysis using an Infinium 450k BeadArray for clinical OPSCC and non-cancerous samples and cancer cell lines with/without 5-aza-2'-deoxycytidine and/or trichostatin A treatment. Frequent promoter hypermethylation and methylation-associated silencing were detected in 144 genes, which included those involved in cell-cell signaling and neuron differentiation. The methylation of nine genes (GHSR, ITGA4, RXRG, UTF1, CDH8, FAN19A4, CTNNA2, NEFH, and CASR) was quantitatively validated in 70 pharyngeal SCC cases by pyrosequencing. Hypermethylation significantly correlated with HPV-L1 positivity, but not with age or smoking status. p16INK4A was generally activated in HPV-L1(+) tumors, and p16-positive cases significantly associated with better prognosis. RXRG hypermethylation strongly correlated with positivity of HPV-L1 and p16 (P = 3 × 10-5 and P = 5 × 10-4, respectively). RXRG-methylation(+) significantly associated with better prognosis when analyzing all tumor cases (P = 0.04), and when analyzing the p16-negative poorer-outcome group (P = 0.03). Thus, aberrant DNA methylation might be involved in HPV-associated OPSCC; in addition, DNA methylation could serve as a marker to classify subgroups based on outcome.

Inhibition of DNA Methylation at the MLH1 Promoter Region Using Pyrrole-Imidazole Polyamide.

Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2'-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)-imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_-16 (Py-Im-ß-Im-Im-Py-γ-Im-Py-ß-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human MLH1 gene. MLH1 is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_-16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the MLH1 promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_-16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the MLH1 promoter under hypoxic conditions (1% O2), methylation of the MLH1 promoter was inhibited in the region surrounding the compound binding site. Silencing of the MLH1 expression was also inhibited. Promoter methylation and silencing of MLH1 were not inhibited when MLH1_non was added. These results indicate that Py-Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells.

Two subtypes of colorectal tumor with distinct molecular features in familial adenomatous polyposis.

While sporadic colorectal cancer (CRC) is classified into several molecular subtypes, stratification of familial colorectal tumors is yet to be well investigated. We previously established two groups of methylation markers through genome-wide DNA methylation analysis, which classified sporadic CRC and adenoma into three distinct subgroups: high-, intermediate-, and low-methylation epigenotypes. Here, we investigated familial adenomatous polyposis (FAP), through quantitative methylation analysis of 127 samples (16 cancers, 96 adenomas, and 15 benign mucosa from 14 patients with FAP) using six Group-1 and 14 Group-2 methylation markers, APC, BRAF, and KRAS mutation analysis, and CTNNB1 and TP53 immunohistochemical analysis. All the 14 patients presented with APC germline mutation. Three were from the same family and presented the same APC mutation. FAP tumors lacked BRAF-mutation(+) high-methylation epigenotype and were classified into two methylation epigenotypes. While 24 of 112 tumor samples showed intermediate-methylation epigenotype significantly correlating with KRAS-mutation(+) (P=3×10-4), 88 tumor samples showed low-methylation epigenotype correlating with the absence of KRAS- and BRAF-mutations. Similar to sporadic CRC, CTNNB1 was frequently activated at the adenoma stage, and TP53 mutation occurred during cancer development from adenoma. Whereas some patients showed a single epigenotype in all tumors throughout the colon, tumors with two distinct epigenotypes developed within a family with the same APC mutation or even within one patient. Methylation accumulation significantly correlated with proximal location and older age. These results indicate that there are at least two distinct molecular subtypes of FAP tumors, resembling sporadic CRC and independent from the APC germline mutation status.

Aberrant promoter methylation of PPP1R3C and EFHD1 in plasma of colorectal cancer patients.

Aberrant DNA methylation is a common epigenetic alteration involved in colorectal cancer (CRC). In our previous study, we performed methylated DNA immunoprecipitation-on-chip analysis combined with gene re-expression analysis by 5-aza-2'-deoxycytidine treatment, to identify methylation genes in CRC genome widely. Among these genes, 12 genes showed aberrant hypermethylation frequently in >75% of 149 CRC samples but did not in normal samples. In this study, we aim to find out any of these methylation genes to be utilized for CRC detection using plasma DNA samples. Primers for methylation-specific PCR and pyrosequencing were designed for seven of the 12 genes. Among them, PPP1R3C and EFHD1 were rarely hypermethylated in peripheral blood cells, but frequently hypermethylated in 24 CRC tissue samples and their corresponding plasma samples. In plasma samples, PPP1R3C was methylated in 81% (97/120) of CRC patients, but only in 19% (18/96) of noncancer patients (P = 6 × 10(-20) , Fisher's exact test). In combined analysis with EFHD1, both genes were methylated in 53% (64/120) of CRC patients, but only in 4% (4/96) of noncancer patients (P = 2 × 10(-16) ), giving high specificity of 96%. At least one of the two genes was methylated in 90% (108/120) of CRC patients, and 36% (35/96) of control patients, giving high sensitivity of 90%. Compared with low sensitivity of carcinoembryonic antigen (17% at stage I, 40% at stage II) and CA19-9 (0% at stage I, 13% at stage II) for early-stage CRCs, sensitivity of aberrant methylation was significantly higher: PPP1R3C methylation at 92% (11/12) for stage I and 77% (23/30) for stage II, and methylation of at least one gene at 100% (12/12) for stage I and 87% (26/30) for stage II. PPP1R3C methylation or its combined use of EFHD1 methylation was highly positive in CRC plasma samples, and they might be useful in detection of CRC, especially for early-stage CRCs.