CpG island hypermethylation of SOCS-1 gene is inversely associated with HBV infection in hepatocellular carcinoma

The CpG island hypermethylation of the suppressor of cytokine signaling-1 (SOCS-1) gene is frequently methylated in hepatocellular carcinoma (HCC), but its clinicopathological significance and the potential risk factors for the epigenetic change in HCC remain poorly understood. The methylation status of SOCS-1 CpG island was evaluated in fresh-frozen tissues from 284 HCC patients using the methylation-specific polymerase chain reaction. The expression of SOCS-1 protein was analyzed by immunohistochemical staining. SOCS-1 methylation was found in 163 (57%) of 284 HCCs. SOCS-1 methylation was positively associated with patient age (P = 0.002) and HCV infection status (P = 0.004), and was inversely associated with HBV infection (P = 0.0002). In the multivariate logistic regression analysis, the HB_sAg-negative HCCs showed a 2.78 (95% CI = 1.31–5.89, P = 0.007) times greater risk of SOCS-1 methylation than the HB_sAg-positive HCCs. SOCS-1 methylation also occurred at a 4.34 times (95% CI = 1.24–14.25, P = 0.02) higher prevalence in antiHCV-positive cases than in antiHCV-negative cases. No prognostic effect of SOCS-1 methylation was observed in the HCCs. In conclusion, the present study suggests that SOCS-1 methylation in HCC may be negatively associated with HB_sAg status.     

Aberrant TIG1 methylation associated with its decreased expression and clinicopathological significance in hepatocellular carcinoma

Recently, it has been reported that tazarotene-induced gene 1 (TIG1) methylation was frequently detected in a variety of human cancers. However, the relationship between the TIG1 methylation and the characteristics of hepatocellular carcinoma (HCC) remains unknown. The aim of present study was to observe the promoter methylation of TIG1 in HCC tissues and assess its prognostic significance for HCC. Real-time quantitative polymerase chain reaction and methylation-specific polymerase chain reaction were used, respectively, to examine the mRNA expression and methylation status of TIG1 in 91 pairs of HCC and adjacent noncancerous tissues. The mRNA expression level of TIG1 was significantly lower in HCC tissues than in adjacent noncancerous tissues. The rate of TIG1 promoter methylation was significantly higher in HCC tissues than in adjacent noncancerous tissues (P?<?0.001). A strong correlation between downregulation and promoter methylation was found in these tumors (P?<?0.001). More importantly, TIG1 methylation status was related to tumor size (P?=?0.015), histological differentiation (P?=?0.004), and tumor stage (P?<?0.001). Kaplan–Meier survival analysis showed that TIG1 promoter hypermethylation was associated with a worse outcome in patients with HCC. Further, Cox multivariate analysis indicated that TIG1 methylation status was an independent prognostic factor for the overall survival rate of HCC patients. In conclusion, our data suggested that epigenetic silencing of TIG1 gene expression by promoter hypermethylation may play an important role in HCC.     

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.     

Reduction of T-Box 15 gene expression in tumor tissue is a prognostic biomarker for patients with hepatocellular carcinoma

Genome-wide analysis is widely applied to detect molecular alterations during oncogenesis and tumor progression. We analyzed DNA methylation profiles of hepatocellular carcinoma (HCC), and investigated the clinical role of most heypermethylated of tumor, encodes T-box 15 (TBX15), which was originally involved in mesodermal differentiation. We conducted a genome-wide analysis of DNA methylation of tumor and non-tumor tissue of 15 patients with HCC, and revealed TBX15 was the most hypermethylated gene of tumor (Beta-value in tumor tissue = 0.52 compared with non-tumor tissue). Another validation set, which comprised 58 HCC with radical resection, was analyzed to investigate the relationships between tumor phenotype and TBX15 mRNA expression. TBX15 mRNA levels in tumor tissues were significantly lower compared with those of nontumor tissues (p < 0.0001). When we assigned a cutoff value = 0.5-fold, the overall survival 5-year survival rates of the low-expression group (n = 17) were significantly shorter compared with those of the high-expression group (n = 41) (43.3% vs. 86.2%, p = 0.001). Multivariate analysis identified low TBX15 expression as an independent prognostic factor for overall and disease-free survival. Therefore, genome-wide DNA methylation profiling indicates that hypermethylation and reduced expression of TBX15 in tumor tissue represents a potential biomarker for predicting poor survival of patients with HCC.     

Clinical significance of expression of Hint1 and potential epigenetic mechanism in gastric cancer

HINT1, a member of the evolutionary highly conserved HIT protein superfamily, is ubiquitously expressed in diverse species including mammalian tissues. Accumulating evidence shows that HINT1 is a haploinsufficient tumor suppressor. In the present study, we explored possible correlations between the expression level of HINT1 and clinicopathological features in tissues from gastric cancer (GC) patients. Decreased expression of HINT1 detected by qPCR and Western blotting in tumor tissues was found in 58.82 and 39.2% of the patients, respectively. Significantly reduced expression of HINT1 was found in poorly differentiated tumor tissues (p = 0.027). Environmental interference (either H. pylori or EBV infection) (p = 0.005) was associated with the expression of HINT1. Moreover, compared with the GC tissues, the level of Hint1 detected by qPCR was significantly higher in the adjacent non-cancerous tissues (p = 0.03). We treated AGS cells, a GC cell line with low expression level of Hint1, with 5 and 10 μmol/l 5-Aza-dC for 72 h and found that HINT1 could be induced by 5-Aza-dC in a dose-dependent manner. These results suggested that Hint1 expression was lower in GC tissues and some etiological factors, such as H. pylori/EB infection or promoter hypermethylation may play a role in gastric tumorigenesis.     

Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP− primary glioblastoma

To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP− primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ?18 months) and 20 short-term survivors (STS; overall survival ?9 months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP− samples. Methylation levels of 11 CpG loci (10 genes) were statistically significantly lower, and 43 CpG loci (40 genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP− samples (P < 0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP− samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP− primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP− primary GBMs.     

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age ⩾12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8–30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6–9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.     

Repeated tumor pO2 measurements by multi-site EPR oximetry as a prognostic marker for enhanced therapeutic efficacy of fractionated radiotherapy

Purpose

To investigate the temporal effects of single or fractionated radiotherapy on subcutaneous RIF-1 tumor pO₂ and to determine the therapeutic outcomes when the timing of fractionations is guided by tumor pO₂.

Methods

The time-course of the tumor pO₂ changes was followed by multi-site electron paramagnetic resonance (EPR) oximetry. The tumors were treated with single 10, 20, and 10 Gy × 2 doses, and the tumor pO₂ was measured repeatedly for six consecutive days. In the 10 Gy × 2 group, the second dose of 10 Gy was delivered at a time when the tumors were either relatively oxygenated or hypoxic. The changes in tumor volumes were followed for nine days to determine the therapeutic outcomes.

Results

A significant increase in tumor pO₂ was observed at 24 h post 10 Gy, while 20 Gy resulted in a significant increase in tumor pO₂ at 72-120 h post irradiation. The tumors irradiated with a second dose of 10 Gy at 24 h, when the tumors were oxygenated, had a significant increase in tumor doubling times (DTs), as compared to tumors treated at 48 h when they were hypoxic (p < 0.01).

Conclusion

Results indicate that the time of tumor oxygenation depends on the irradiation doses, and radiotherapeutic efficacy could be optimized if irradiations are scheduled at times of increased tumor oxygenation. In vivo multi-site EPR oximetry could be potentially used to monitor tumor pO₂ repeatedly during fractionated schemes to optimize radiotherapeutic outcome. This technique could also be used to identify responsive and non-responsive tumors, which will facilitate the design of other therapeutic approaches for non-responsive tumors at early time points during the course of therapy.     

Decursin chemosensitizes human multiple myeloma cells through inhibition of STAT3 signaling pathway

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease’s high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31-3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients.     

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection

Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.     

Role of ZIC1 methylation in hepatocellular carcinoma and its clinical significance

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in humans, and its prognosis is generally poor even after surgery. The zinc finger of the cerebellum (ZIC1) gene is a novel tumor suppressor gene that plays a crucial role in vertebrate development. Altered expression of ZIC1 is observed in various types of human cancers. The aims of the present study were to investigate the methylation status of ZIC1 in HCC and evaluate its clinical implication. The methylation status of ZIC1 was analyzed in 132 pairs of HCC and corresponding noncancerous tissues by methylation-specific polymerase chain reaction (PCR) (MSP). The expression of ZIC1 messenger RNA (mRNA) in HCC tissues was examined by real-time PCR. Methylation frequency of ZIC1 in HCC was significantly higher than that in the corresponding noncancerous tissues (P?P?=?0.022), histological differentiation (P?=?0.033), and tumor stage (P?=?0.009). The downregulation of the ZIC1 mRNA expression in HCC was correlated with the ZIC1 methylation (P?P?mRNA and associated with poor survival in HCC. Overall, aberrant methylation is an important mechanism for ZIC1 inactivation in HCC, and ZIC1 methylation may be a promising biomarker for the diagnosis and prognosis of HCC.     

Identification of the bleomycin hydrolase gene as a methylated tumor suppressor gene in hepatocellular carcinoma using a novel triple-combination array method

In the present study, we sought to identify novel suppressor genes of hepatocellular carcinoma (HCC) using our newly designed triple-combination array. Using this method, the bleomycin hydrolase gene (BLMH) was detected as a candidate suppressor gene. We found that 28 of 48 (58.3%) tumor tissues showed BLMH promoter hypermethylation, and its expression level was significantly reduced in tumor tissues (P = 0.001). The present study suggests that our new method can detect novel genes of interest and that BLMH is a suppressor gene in HCC.     

Association of GSTP1 expression with resistance to docetaxel and paclitaxel in human breast cancers

Aims

It has been reported that glutathione S-transferase P1 (GSTP1) expression is implicated in resistance to taxanes (docetaxel and paclitaxel) in human breast cancer cells in vitro. In the study presented here, we examine whether GSTP1 expression is associated with resistance to docetaxel or paclitaxel in human breast cancers. We also investigated the relationship between GSTP1 methylation status and response to these taxanes.

Material and methods

Sixty two primary breast cancer patients were treated with docetaxel or paclitaxel as primary systemic treatment (PST). GSTP1 expression was detected immunohistochemically and the hypermethylation status GSTP1 gene was identified with a methylation specific primer assay.

Results

The mean tumor reduction rate for all patients (n = 62) was significantly (p < 0.001) higher in GSTP1 negative (0.73 ± 0.04; mean ± standard error) than GSTP1 positive (0.31 ± 0.09) tumors. The subset analysis showed that the mean reduction rate was significantly (p = 0.005) higher in GSTP1 negative (0.59 ± 0.06) than GSTP1 positive (0.11 ± 0.13) tumors in the docetaxel group as well as in the paclitaxel group (p = 0.006; GSTP1 negative tumors: 0.84 ± 0.05; GSTP1 positive tumors: 0.56 ± 0.08). On the other hand, GSTP1 methylation showed no significant association with the reduction rate.

Conclusion

Our present study has suggested that GSTP1 protein expression, but not GSTP1 methylation status, might be associated with response to docetaxel and paclitaxel. This suggests that GSTP1 immunohistochemical expression might be a potentially clinically useful predictive factor for response to docetaxel and paclitaxel.