Gastric cancers arise through a multistep process characterized by the progressive accumulation of molecular alterations in which genetic and epigenetic mechanisms have been implicated. Gastric cancer is one of the human malignancies in which aberrant promoter CpG island hypermethylation is frequently found. Helicobacter pylori and Epstein-Barr virus, which are known carcinogens for gastric cancer, are closely associated with enhanced hypermethylation of CpG island loci in gastric non-neoplastic epithelial cells and cancer cells, respectively. Aberrant CpG island hypermethylation occurs early in the multistep cascade of gastric carcinogenesis and tends to increase with the step-wise progression of the lesion. Approximately 400 genes that are actively expressed in normal gastric epithelial cells are estimated to be inactivated in gastric cancers as a result of promoter CpG island hypermethylation. In this review, a variety of information is summarized regarding CpG island hypermethylation in gastric cancer.
CpG island; DNA methylation; Gastric cancer; Intestinal metaplasia
Several reports have described aberrant methylation in various types of human cancers. However, the interpretation of methylation frequency in various human cancers has some limitations because of the different materials and methods used for methylation analysis. To gain an insight into the role of DNA hypermethylation in human cancers and allow direct comparison of tissue specific methylation, we generated methylation profiles in 328 human cancers, including 24 breast, 48 colon, 61 stomach, 48 liver, 37 larynx, 24 lung, 40 prostate, and 46 uterine cervical cancer samples by analyzing CpG island hypermethylation of 13 genes using methylation-specific PCR. The mean numbers of methylated genes were 6.5, 4.4, 3.6, 3.4, 3.1, 3.1, 3.1, and 2.1 in gastric, liver, prostate, larynx, colon, lung, uterine cervix, and in breast cancer samples, respectively. The number of genes that were methylated at a frequency of more than 40% in each tumor type ranged from nine (stomach) to one (breast). Generally genes frequently methylated in a specific cancer type differed from those methylated in other cancer types. The findings indicate that aberrant CpG island hypermethylation is a frequent finding in human cancers of various tissue types, and each tissue type has its own distinct methylation pattern.
Carcinomas; CpG Island; DNA Methylations; Tumor Suppressor Gene
DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.
CpG Islands; DNA Methylation; Carcinoma, Hepatocellular; Microarray; Prognosis
We analyzed the clinical data of T3 colorectal cancer patients to assess whether T3 subdivision correlates with node (N) or metastasis (M) staging and stage-independent factors.
Five hundred fifty-five patients who underwent surgery for primary colorectal cancer from January 2003 to December 2009 were analyzed for T3 subdivision. T3 subdivision was determined by the depth of invasion beyond the outer border of the proper muscle (T3a, <1 mm; T3b, 1 to 5 mm; T3c, >5 to 15 mm; T3d, >15 mm). We investigated the correlation between T3 subdivision and N, M staging and stage-independent prognostic factors including angiolymphatic invasion (ALI), venous invasion (VI) and perineural invasion (PNI).
The tumors of the 555 patients were subclassified as T3a in 86 patients (15.5%), T3b in 209 patients (37.7%), T3c in 210 patients (37.8%) and T3d in 50 patients (9.0%). The nodal metastasis rates were 39.5% for T3a, 56.5% for T3b, 75.7% for T3c and 74.0% for T3d. The distant metastasis rates were 7.0% for T3a 9.1% for T3b, 27.1% for T3c and 40.0% for T3d. Both N and M staging correlated with T3 subdivision (Spearman's rho = 0.288, 0.276, respectively; P < 0.001). Other stage-independent prognostic factors correlated well with T3 subdivision (Spearman's rho = 0.250, P < 0.001 for ALI; rho = 0.146, P < 0.001 for VI; rho = 0.271, P < 0.001 for PNI).
Subdivision of T3 colorectal cancer correlates with nodal and metastasis staging. Moreover, it correlates with other prognostic factors for colorectal cancer.
T3 subdivision; Colorectal neoplasms; Neoplasm staging
It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine− and bromodeoxyuridine [BrdU]+) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.
Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes.
We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients) of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients). There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05). This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07), high body mass index (p-value = 0.06), and symptoms of gastroesophageal reflux (p-value = 0.07). Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage.
DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to epidemiological data and clinical outcome. Two new epigenetic subgroups of gastroesophageal adenocarcinomas were identified that differ to some extent in their survival rates, risk factors of exposure, and GATA5 DNA methylation.
Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.
Tufting enteropathy is a rare autosomal recessive disorder presenting with early-onset severe intractable diarrhea. The epithelial cell adhesion molecule gene (EpCAM) has recently been identified as the gene responsible for tufting enteropathy. Based on histology, a diagnosis of tufting enteropathy was made in two Korean siblings. They developed chronic diarrhea and failure to thrive. They had a broad nasal bridge and micrognathia. Duodenal and colonic biopsies showed villous atrophy, disorganization of surface enterocytes, and focal crowding resembling tufts. Protracted diarrhea continued and so cyclic parenteral nutrition was supplied. The sister had juvenile rheumatoid arthritis. Mutation analysis of EpCAM identified two compound heterozygous mutations in these siblings: 1) a donor splicing site mutation in intron 5 (c.491+1G>A) and 2) a novel nonsense mutation in exon 3 (c.316A>T, Lys106X). Analysis of EpCAM will be useful for genetic counseling and prenatal diagnosis of tufting enteropathy.
Tufting enteropathy; Diarrhea; Epithelial cell adhesion molecule; Mutation
Promoter CpG island hypermethylation has become recognized as an important mechanism for inactivating tumor suppressor genes or tumor-related genes in human cancers of various tissues. Gene inactivation in association with promoter CpG island hypermethylation has been reported to be four times more frequent than genetic changes in human colorectal cancers. Hepatocellular carcinoma is also one of the human cancer types in which aberrant promoter CpG island hypermethylation is frequently found. However, the number of genes identified to date as hypermethylated for hepatocellular carcinoma (HCC) is fewer than that for colorectal cancer or gastric cancer, which can be attributed to fewer attempts to perform genome-wide methylation profiling for HCC. In the present study, we used bead-array technology and coupled methylation-specific PCR to identify new genes showing cancer-specific methylation in HCC. Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner. Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression. Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.
Bead Array; CpG Islands; DNA Methylation; Carcinoma, Hepatocellular
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome caused by a defect in cholesterol biosynthesis. The incidence is very low in Asians and only one case has been reported in Korea thus far. Recently, we found an infant with neonatal cholestasis. He had microcephaly, ambiguous genitalia, cleft palate, syndactyly of toes, patent ductus arteriosus and hypertrophic pyloric stenosis. The serum cholesterol was decreased and serum 7-dehydrocholesterol was markedly elevated. Genetic analysis of the DHCR7 gene identified a novel missense mutation (Pro227Ser) as well as a known mutation (Gly303Arg) previously identified in a Japanese patient with SLOS. Although rare in Korea, SLOS should be considered in the differential diagnosis of neonatal cholestasis, especially in patients with multiple congenital anomalies and low serum cholesterol levels.
Smith-Lemli-Opitz Syndrome; Cholestasis; 7-dehydrocholesterol reductase; Mutation
Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAFV600E–a mutation found in ∼10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAFV600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAFV600E is not required for its maintenance.
Hypoxia-inducible factor 1α (HIF-1α) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1α is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1α protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1α stability. We found that STAT3 interacts with C-terminal domain of HIF-1α and stabilizes HIF-1α by inhibition of pVHL binding to HIF-1α. The binding between HIF-1α and pVHL, negative regulator of HIF-1α stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1α protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1α protein via inhibition of interaction between pVHL and HIF-1α. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1α through competition with pVHL for binding to HIF-1α, and then stabilizes HIF-1α protein levels.
anoxia; hypoxia-inducible factor1, α subunit; neoplasms; STAT3 transcription factor; ubiquitination; von Hippel-Lindau tumor suppressor protein
Transcriptional factor 4 (TCF4), encoding a basic helix-loop-helix transcriptional factor, has recently been demonstrated as a causative gene for Pitt-Hopkins syndrome, a neurodevelopmental disease. Examination of gastric cancers using the restriction landmark genomic scanning technique revealed methylation at a NotI enzyme site in TCF4 intron 8 and further identified CpG dinucleotide hypermethylation in TCF4 exon 1, strongly associated with gene silencing in gastric cancer cell lines. Treatment with 5-aza-2′-deoxycytidine and/or trichostatin A restored TCF4 expression in TCF4-silenced gastric cancer cell lines. Real-time reverse transcription–polymerase chain reaction analysis of 77 paired primary gastric tumor samples revealed that 38% of analyzed tumors had a >2-fold decrease in TCF4 expression compared with adjacent normal-appearing tissue, and the decrease significantly correlated with increased CpG methylation in TCF4 exon 1. Clinicopathologic data showed that decreased TCF4 expression occurred significantly more frequently in intestinal-type (22/37, 59%) than in diffuse-type (7/37, 19%) gastric cancers (P = 0.0004) and likewise more frequently in early (12/18, 67%) than in advanced (17/59, 29%) gastric cancers (P = 0.004). CpG methylation markedly increased with patient age among normal-appearing tissues, suggesting that CpG methylation in gastric mucosa may be one of the earliest events in carcinogenesis of intestinal-type gastric cancers. Furthermore, ectopic expression of TCF4 decreased cell growth in a gastric cancer cell line, and the knock down of TCF4 using small interfering RNA increased cell migration. Based on these results, we propose that the observed frequent epigenetic-mediated TCF4 silencing plays a role in tumor formation and progression.
A hepatic mesenchymal hamartoma is an uncommon benign tumor in children and little is known about the spectrum of its radiological features. The purpose of this study is to describe the spectrum of radiological features of a hepatic mesenchymal hamartoma in children.
Materials and Methods
Thirteen children with a pathologically confirmed hepatic mesenchymal hamartoma (M:F = 7:6; mean age, 3 years 2 months) were included in our study. Ultrasonography (US) was performed in nine patients including color and power Doppler US (n = 7). CT scans were performed in all patients. We evaluated the imaging findings of the hepatic mesenchymal hamartomas and the corresponding pathological features.
Each patient had a single tumor (mean diameter: 13 cm [1.8-20 cm]). On CT and/or US, four patients (31%) had a "multiseptated cystic tumor", five patients (38%) had a "mixed solid and cystic tumor", and four patients (31%) had a "solid tumor." The septa of the cystic portion were thin in the multiseptated cystic tumors and irregularly thick in the mixed solid and cystic tumors as seen on US. On a post-contrast CT scan, solid portions or thick septa of the tumors showed heterogeneous enhancement. The amount of hepatocytes was significantly different among the three tumor groups according to the imaging spectrum (p = 0.042).
A hepatic mesenchymal hamartoma in children can show a wide spectrum of radiological features, from a multiseptated cystic tumor to a mixed solid and cystic tumor, and even a solid tumor.
Children; Hepatic tumor; Mesenchymal hamartoma
We performed immunohistochemical staining against Hepatocyte (Hep) and CD10 antibodies in 75 hepatocellular carcinoma (HCC), 50 cholangiocarcinomas, 49 colorectal adenocarcinomas, and 308 gastric adenocarcinomas by tissue array method. We also evaluated the various non-neoplastic adult tissues and fetal digestive organs. Hep was expressed in 80% of HCCs, and HCCs without Hep expression were more likely to have a higher Edmondson & Steiner grade than HCCs with Hep expression (p=0.004). In non-HCCs, 16% of cholangiocarcinomas, 8.2% of colorectal carcinomas, and 44.2% of gastric carcinomas expressed Hep. Gastric carcinomas with Hep expression were significantly associated with early gastric carcinomas (p<0.001). In non-neoplastic tissues, Hep was found expressed in normal hepatocytes, small intestinal mucosa, and intestinal metaplasia of the stomach. Fetal hepatocytes expressed Hep after 19 weeks of gestation. CD10 was detected in 46.7% (35/75) of HCCs, and canalicular staining pattern was predominant in HCCs. In conclusion, the expression of Hep and CD10 may help to distinguish HCCs from non-HCCs.
Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.
In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.
Conclusion and Significance
The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.
Colorectal carcinoma (CRC) with CpG island methylator phenotype (CIMP) is recognized as a distinct subgroup of CRC, and CIMP status affects prognosis and response to chemotherapy. Identification of CIMP status in CRC is important for proper patient management. In Eastern countries, however, the clinicopathologic and molecular characteristics and prognosis of CRCs with CIMP are still unclear.
A total of 245 patients who underwent their first surgical resection for sporadic CRC were enrolled and CIMP status of the CRCs was determined using the quantitative MethyLight assay. The clinicopathologic and molecular characteristics were reviewed and compared according to CIMP status. In addition, the three-year recurrence-free survival (RFS) of 124 patients with stage II or stage III CRC was analyzed in order to assess the effectiveness of fluoropyrimidine-based adjuvant chemotherapy with respect to CIMP status.
CIMP-high CRCs were identified in 34 cases (13.9%), and were significantly associated with proximal tumor location, poorly differentiated carcinoma, mucinous histology, and high frequencies of BRAF mutation, MGMT methylation, and MSI-high compared to CIMP-low/negative carcinomas. For patients with stage II or III CIMP-low/negative CRCs, no significant difference was found in RFS between those undergoing surgery alone and those receiving surgery with fluoropyrimidine-based adjuvant chemotherapy. However, for patients with CIMP-high CRCs, patients undergoing surgery with fluoropyrimidine-based adjuvant chemotherapy (n = 17; three-year RFS: 100%) showed significantly better RFS than patients treated with surgery alone (n = 7; three-year RFS: 71.4%) (P = 0.022).
Our results suggest that selected patients with CIMP-high CRC may benefit from fluoropyrimidine-based adjuvant chemotherapy with longer RFS. Further large scale-studies are required to confirm our results.
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy.
Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells.
Low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased anticancer drug activity in vitro. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC.