DNA methylation alterations of ADCY5, MICAL2, and PLEKHG2 during the developmental stage of cryptogenic hepatocellular carcinoma.

AIM: The aim of this study was to clarify the significance of DNA methylation alterations of cryptogenic hepatocellular carcinomas (HCCs). METHODS: Using the Infinium assay, we performed genome-wide DNA methylation analysis of 250 liver tissue samples, including noncancerous liver tissue (U-N) and corresponding cancerous tissue (U-T) from patients with cryptogenic HCC without a history of excessive alcohol use and hepatitis virus infection, and whose U-N samples showed no remarkable histological features (no microscopic evidence of simple steatosis, any form of hepatitis including nonalcoholic steatohepatitis, or liver cirrhosis). RESULTS: We identified 3272 probes that showed significant differences of DNA methylation levels between U-N and normal liver tissue samples from patients without HCC, indicating that a distinct DNA methylation profile had already been established at the precancerous U-N stage. U-Ns have a DNA methylation profile differing from that of noncancerous liver tissue of patients with nonalcoholic steatohepatitis-related, viral hepatitis-related, and alcoholic liver disease-related HCCs. Such DNA methylation alterations in U-Ns were inherited by U-Ts. The U-Ns showed DNA methylation alteration of ADCY5, resulting in alteration of its mRNA expression, whereas noncancerous liver tissue of patients with nonalcoholic steatohepatitis-, viral hepatitis-, or alcoholic liver disease-related HCCs did not. DNA methylation levels of MICAL2 and PLEKHG2 in U-Ts were correlated with larger tumor diameter and portal vein involvement, respectively. CONCLUSIONS: U-N-specific DNA hypermethylation of ADCY5 may have significance, even from the precancerous stage in liver showing no remarkable histological features. DNA hypomethylation of MICAL2 and PLEKHG2 may determine the clinicopathological features of cryptogenic HCC.

DNA methylation status of the SPHK1 and LTB genes underlies the clinicopathological diversity of non-alcoholic steatohepatitis-related hepatocellular carcinomas.

PURPOSE: This study was performed to identify the DNA methylation profiles underlying the clinicopathological diversity of non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs).  METHODS: Genome-wide DNA methylation analysis of 88 liver tissue samples was performed using the Infinium assay. RESULTS: Principal component analysis revealed that distinct DNA methylation profiles differing from such profiles in normal control liver tissue had already been established in non-cancerous liver tissue showing NASH, which is considered to be a precancerous condition. Hierarchical clustering separated 26 NASH-related HCCs into Cluster I (n = 8) and Cluster II (n = 18). Such epigenetic clustering was significantly correlated with histopathological diversity, i.e. poorer tumor differentiation, tumor steatosis and development of a scirrhous HCC component. Significant differences in DNA methylation levels between the two clusters were accumulated in molecular pathways participating in cell adhesion and cytoskeletal remodeling, as well as cell proliferation and apoptosis. Among tumor-related genes characterizing Clusters I and II, differences in the levels of DNA methylation and mRNA expression for the SPHK1, INHBA, LTB and PDE3B genes were correlated with poorer tumor differentiation. 5-Aza-2'-deoxycytidine treatment of HCC cells revealed epigenetic regulation of the SPHK1 and LTB genes. Knockdown experiments showed that SPHK1 promotes cell proliferation, represses apoptosis and enhances migration, whereas LTB enhances migration of HCC cells. DNA hypomethylation resulting in increased expression of SPHK1 and LTB in poorly differentiated HCCs may underlie the aggressive phenotype of such HCCs. CONCLUSION: These data indicate that DNA methylation profiles may determine the clinicopathological heterogeneity of NASH-related HCCs via alterations of tumor-related gene expression.

Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis.

BACKGROUND: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH. RESULTS: Normal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort. CONCLUSIONS: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

Prognostication of early-onset endometrioid endometrial cancer based on genome-wide DNA methylation profiles.

OBJECTIVE: The aim of this study was to establish criteria that would indicate whether fertility preservation therapy would likely be safe for patients aged 40 years or less with endometrioid endometrial cancer based on their DNA methylation profile. METHODS: Forty-nine fresh-frozen tissue samples from patients with endometrial cancer from an initial cohort and 31 formalin-fixed paraffin-embedded tissue samples from a second cohort were subjected to genome-wide DNA methylation analysis using the Infinium MethylationEPIC BeadChip. RESULTS: Epigenomic clustering of early-onset endometrial cancer was correlated with the widely used recurrence risk classification. Genes showing differences in DNA methylation levels between the low-recurrence-risk category and intermediate- and high-risk categories were accumulated in pathways related to fibroblast growth factor and nuclear factor-κB signaling. DNA hypomethylation and overexpression of ZBTB38 were frequently observed in the low-risk category. Eight hundred thirty-one marker CpG probes showed area under the curve values of >0.7 on the receiver operating characteristic curve for discrimination of patients belonging to the low-risk category. By combining marker CpG sites, seven panels for placing patients into the low-risk category with 91.3% or more sensitivity and specificity in both the initial and second cohorts were established. CONCLUSIONS: DNA methylation diagnostics criteria using up to 6 of 8 CpG sites for LPP, FOXO1, RNF4, EXOC6B, CCPG1, RREB1 and ZBTB38 may be applicable to recurrence risk estimation for patients aged 40 years or less with endometrial cancer, regardless of tumor cell content, even if formalin-fixed paraffin-embedded biopsy or curettage materials are used.

Clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma: prediction of early recurrence based on genome-wide DNA methylation profiling.

PURPOSE: The present study was conducted to clarify the clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma (PDAC). METHODS: Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analyzed fresh-frozen tissues from an initial cohort (17 samples of normal control pancreatic tissue [C] from 17 patients without PDAC, and 34 samples of non-cancerous pancreatic tissue [N] and 82 samples of cancerous tissue [T] both obtained from 82 PDAC patients) and formalin-fixed paraffin-embedded T samples from 34 patients in a validation cohort. RESULTS: The DNA methylation profiles of N samples tended to differ from those of C samples, and 91,907 probes showed significant differences in DNA methylation levels between C and T samples. Epigenetic clustering of T samples was significantly correlated with a larger tumor diameter and early recurrence (ER), defined as relapse within 6 months after surgery. Three marker CpG sites, applicable to formalin-fixed paraffin-embedded surgically resected materials regardless of their tumor cell content, were identified for prediction of ER. The sensitivity and specificity for detection of patients belonging to the ER group using a panel combining these three marker CpG sites, including a CpG site in the CDK14 gene, were 81.8% and 71.7% and 88.9% and 70.4% in the initial and validation cohorts, respectively. CONCLUSION: These findings indicate that DNA methylation alterations may have a clinicopathological impact on PDAC. Application of our criteria will ultimately allow prediction of ER after surgery to improve the outcome of PDAC patients.

DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of 'pan-negative'-type lung adenocarcinomas.

Although some previous studies have examined epigenomic alterations in lung adenocarcinomas, correlations between epigenomic events and genomic driver mutations have not been fully elucidated. Single-CpG resolution genome-wide DNA methylation analysis with the Infinium HumanMethylation27 BeadChip was performed using 162 paired samples of adjacent normal lung tissue (N) and the corresponding tumorous tissue (T) from patients with lung adenocarcinomas. Correlations between DNA methylation data on the one hand and clinicopathological parameters and genomic driver mutations, i.e. mutations of EGFR, KRAS, BRAF and HER2 and fusions involving ALK, RET and ROS1, were examined. DNA methylation levels in 12 629 probes from N samples were significantly correlated with recurrence-free survival. Principal component analysis revealed that distinct DNA methylation profiles at the precancerous N stage tended not to induce specific genomic driver aberrations. Most of the genes showing significant DNA methylation alterations during transition from N to T were shared by two or more driver aberration groups. After small interfering RNA knockdown of ZNF132, which showed DNA hypermethylation only in the pan-negative group and was correlated with vascular invasion, the proliferation, apoptosis and migration of cancer cell lines were examined. ZNF132 knockdown led to increased cell migration ability, rather than increased cell growth or reduced apoptosis. We concluded that DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of 'pan-negative' lung adenocarcinomas. In addition, DNA methylation alterations at the precancerous stage may determine tumor aggressiveness, and such alterations that accumulate after driver mutation may additionally modify clinicopathological features through alterations of gene expression.

Impact of reduced ozone concentration on the mountain forests of Mt. Tateyama, Japan.

The central mountainous area of Japan is affected by air pollutant emissions from nearby countries such as China and Korea. Sharp increases in the consumption of fossil fuels in the early 21st century, associated with rapid industrialization in China, resulted in long-range transport of pollutants from East Asia and increases in the harmful effects of pollution. However, the air pollutants emissions have decreased since 2006, when air pollution countermeasures were implemented in China. Furthermore, climatic patterns during 2008-2013 reduced tropospheric ozone concentrations around Japan. Such major changes in the social and climatic environment may have had a significant impact on forests. To investigate this, long-term forest monitoring data obtained at Buna-daira (1190 m a.s.l.), Buna-zaka (1090 m a.s.l.) and Arimine (1350 m a.s.l.) were analyzed. Buna-daira and Buna-zaka forests face the continental side of Mt. Tateyama in Toyama Prefecture. In both stands, the girth growth rate of Fagus crenata was found to have increased after 2008; however, such a tendency was not detected at Arimine, which is surrounded by mountains. The growth rates of Cryptomeria japonica, a conifer resistant to air pollution, were found to remain unchanged or decrease. Here, regional long-range transport of air pollution (including ozone and sulfur oxide) has been demonstrated to influence mountain forests in Japan. In particular, recent decreases in regional air pollution may be an important factor controlling increases in F. crenata, likely through changes in interspecific relationships between species sensitive to and tolerant of air pollution.

Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.

The aim of this study was to develop a less invasive and accurate diagnostic system for upper urinary tract urothelial carcinoma (UTUC) based on genome-wide DNA methylation profiling. Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analysed 26 samples of normal control urothelial tissue (C), an initial cohort of 62 samples (31 samples of non-cancerous urothelium [N] from UTUC patients and 31 samples of the corresponding UTUCs), a validation cohort of 82 samples (41 N and 41 UTUC samples), and 14 samples of urinary bladder urothelial carcinoma (BUC). In the initial cohort, we identified 2,448 CpG sites showing significant differences in DNA methylation levels between both C and UTUC and N and UTUC, but not showing differences between C and N. Among these CpG sites, 10 were located within CpG islands or their shores and shelves included in genomic domains where DNA methylation levels are stably controlled, allowing discrimination of UTUC even from BUC. Receiver operating characteristic curve analysis for discrimination of UTUC from N in these 10 CpG and neighbouring sites (37 diagnostic panels in total) yielded area under the curve values of 0.959-1.000, with a sensitivity and specificity of 86.6-100% and 93.5-100%, respectively. The diagnostic impact was successfully confirmed in the validation cohort. Our criteria were useful for diagnosis of UTUC, regardless of its clinicopathological features. Application of our criteria to voided urine samples will ultimately allow non-invasive DNA methylation diagnosis of UTUC.

Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides.

Cell-penetrating peptides (CPPs) including arginine-rich peptides are attracting a lot of attention due to their potential as a novel intracellular drug delivery tool without substantial toxicity. On the other hand, disease-associated arginine-rich CPPs, such as poly-PR and poly-GR translated from C9orf72 gene, also efficiently enter neuronal cells and then kill them. Although both non-harmful CPPs and harmful poly-PR/GR penetrate the plasma membrane using same arginine residues, little is known about the factors which determine the toxicity of the pathogenic CPPs. Here, we show that poly-PR and poly-GR, but not other Arg-rich CPPs, specifically distributed to nucleolus via interaction with RNA. Importantly, C9orf72-dipeptides, but not other Arg-rich CPPs, caused inhibition of protein translation and cell death. Raising extracellular pH enhanced the cell penetration of poly-PR. The repeat number of (PR) affected the secondary structure and determined the intracellular delivery rate and neurotoxicity, and enforced intracellular delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity.