Metformin regulates expression of DNA methyltransferases through the miR-148/-152 family in non-small lung cancer cells.

BACKGROUND: To understand the molecular mechanisms involved in regulation of DNA methyltransferases (DNMTs) by metformin in non-small cell lung cancer (NSCLC) cells. METHODS: Expression levels of DNMTs in response to metformin were analyzed in NSCLC cells. MicroRNAs regulating expression of DNMTs at the post-transcriptional level were searched using miRNA-target databases (miRDB and miRTarBase), TCGA RNASeqV2 lung cancer data, and miRNA-seq. RESULTS: Metformin dose-dependently downregulated expression of DNMT1 and DNMT3a at the post-transcriptional level and expression of DNMT3b at the transcriptional level in A549 lung cancer cells. Activity of DNMTs was reduced by about 2.6-fold in A549 cells treated with 10 mM metformin for 72 h. miR-148/-152 family members (miR-148a, miR-148b, and miR-152) targeting the 3'UTR of DNMTs were associated with post-transcriptional regulation of DNMTs by metformin. Metformin upregulated expression of miR-148a, miR-148b, and miR-152 in A549 and H1650 cells. Transfection with an miR-148b plasmid or a mimic suppressed expression of DNMT1 and DNMT3b in A549 cells. Transfection with the miR-148a mimic in A549 and H1650 cells decreased the luciferase activity of DNMT1 3'UTR. A combination of metformin and cisplatin synergistically increased expression levels of miR-148/-152 family members but decreased expression of DNMTs in A549 cells. Low expression of miR-148b was associated with poor overall survival (HR = 2.56, 95% CI 1.09-6.47; P = 0.04) but not with recurrence-free survival. CONCLUSIONS: The present study suggests that metformin inhibits expression of DNMTs by upregulating miR-148/-152 family members in NSCLC cells.

Aberrant Methylation of SLIT2 Gene in Plasma Cell-Free DNA of Non-Small Cell Lung Cancer Patients.

This study aimed to understand aberrant methylation of SLITs genes as a biomarker for the early detection and prognosis prediction of non-small cell lung cancer (NSCLC). Methylation levels of SLITs were determined using the Infinium HumanMethylation450 BeadChip or pyrosequencing. Five CpGs at the CpG island of SLIT1, SLIT2 or SLIT3 genes were significantly (Bonferroni corrected p < 0.05) hypermethylated in tumor tissues obtained from 42 NSCLC patients than in matched normal tissues. Methylation levels of these CpGs did not differ significantly between bronchial washings obtained from 76 NSCLC patients and 60 cancer-free patients. However, methylation levels of SLIT2 gene were significantly higher in plasma cell-free DNA of 72 NSCLC patients than in that of 61 cancer-free patients (p = 0.001, Wilcoxon rank sum test). Prediction of NSCLC using SLIT2 methylation was achieved with a sensitivity of 73.7% and a specificity of 61.9% in a plasma test dataset (N = 40). A Cox proportional hazards model showed that SLIT2 hypermethylation in plasma cell-free DNA was significantly associated with poor recurrence-free survival (hazards ratio = 2.19, 95% confidence interval = 1.21-4.36, p = 0.01). The present study suggests that aberrant methylation of SLIT2 in plasma cell-free DNA is a valuable biomarker for the early detection of NSCLC and prediction of recurrence-free survival. However, further research is needed with larger sample size to confirm results.

Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells.

This study aimed at understanding the effect of metformin on histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin significantly reduced H3K4me3 level at the promoters of positive cell cycle regulatory genes such as CCNB2, CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. Metformin suppressed the expression of H3K4 methyltransferases MLL1, MLL2, and WDR82. The siRNA-mediated knockdown of MLL2 significantly downregulated global H3K4me3 level and inhibited lung cancer cell proliferation. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, and a Cox proportional hazards analysis showed that recurrence-free survival of lung adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08-4.72; p = 0.02) times poorer than in those without it. Metformin showed little effect on DNA methylation and chromatin accessibility at the promoter regions of cell cycle regulatory genes. The present study suggests that metformin reduces H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. Additionally, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.

Negative Effect of Reduced NME1 Expression on Recurrence-Free Survival in Early Stage Non-Small Cell Lung Cancer.

This study aimed to understand whether the effect of non-metastatic cells 1 (NME1) on recurrence-free survival (RFS) in early stage non-small cell lung cancer (NSCLC) can be modified by ß-catenin overexpression and cisplatin-based adjuvant chemotherapy. Expression levels of NME1 and ß-catenin were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 425 early stage NSCLC patients. Reduced NME1 expression was found in 39% of samples. The median duration of follow-up was 56 months, and recurrence was found in 186 (44%) of 425 patients. The negative effect of reduced NME1 expression on RFS was worsened by cisplatin-based adjuvant chemotherapy (adjusted hazard ratio = 3.26, 95% CI = 1.16-9.17, p = 0.03). ß-catenin overexpression exacerbated the effect of reduced NME1 expression on RFS and the negative effect was greater when receiving cisplatin-based adjuvant chemotherapy: among patients treated with cisplatin-based adjuvant chemotherapy, hazard ratios of patients with reduced NME1 expression increased from 5.59 (95% confidence interval (CI) = 0.62-50.91, p = 0.13) to 15.52 (95% CI = 2.94-82.38, p = 0.001) by ß-catenin overexpression, after adjusting for confounding factors. In conclusion, the present study suggests that cisplatin-based adjuvant chemotherapy needs to be carefully applied to early stage NSCLC patients with overexpressed ß-catenin in combination with reduced NME1 expression.

Clinicopathological Significance of RUNX1 in Non-Small Cell Lung Cancer.

This study aimed to understand the clinicopathological significance of runt-related transcription factor 1 (RUNX1) in non-small cell lung cancer (NSCLC). The methylation and mRNA levels of RUNX1 in NSCLC were determined using the Infinium HumanMethylation450 BeadChip and the HumanHT-12 expression BeadChip. RUNX1 protein levels were analyzed using immunohistochemistry of formalin-fixed paraffin-embedded tissues from 409 NSCLC patients. Three CpGs (cg04228935, cg11498607, and cg05000748) in the CpG island of RUNX1 showed significantly different methylation levels (Bonferroni corrected p < 0.05) between tumor and matched normal tissues obtained from 42 NSCLC patients. Methylation levels of the CpGs in the tumor tissues were inversely related to mRNA levels of RUNX1. A logistic regression model based on cg04228935 showed the best performance in predicting NSCLCs in a test dataset (N = 28) with the area under the receiver operating characteristic (ROC) curve (AUC) of 0.96 (95% confidence interval (CI) = 0.81-0.99). The expression of RUNX1 was reduced in 125 (31%) of 409 patients. Adenocarcinoma patients with reduced RUNX1 expression showed 1.97-fold (95% confidence interval = 1.16-3.44, p = 0.01) higher hazard ratio for death than those without. In conclusion, the present study suggests that abnormal methylation of RUNX1 may be a valuable biomarker for detection of NSCLC regardless of race. And, reduced RUNX1 expression may be a prognostic indicator of poor overall survival in lung adenocarcinoma.

Metformin and tenovin-6 synergistically induces apoptosis through LKB1-independent SIRT1 down-regulation in non-small cell lung cancer cells.

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non-histone proteins; however, antitumour effects by suppressing SIRT1 activity in non-small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin-6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin-fixed paraffin-embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence-free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin-6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin-6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin-6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1-deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up-regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase-3-dependent apoptosis. The study concluded that metformin with tenovin-6 may enhance antitumour effects through LKB1-independent SIRT1 down-regulation in NSCLC cells.

Genome-wide analysis of DNA methylation in bronchial washings.

Background: The objective of this study was to discover DNA methylation biomarkers for detecting non-small lung cancer (NSCLC) in bronchial washings and understanding the association between DNA methylation and smoking cessation. Methods: DNA methylation was analyzed in bronchial washing samples from 70 NSCLCs and 53 hospital-based controls using Illumina HumanMethylation450K BeadChip. Methylation levels in these bronchial washings were compared to those in 897 primary lung tissues of The Cancer Genome Atlas (TCGA) data. Results: Twenty-four CpGs (p < 1.03E-07) were significantly methylated in bronchial washings from 70 NSCLC patients compared to those from 53 controls. The CpGs also had significant methylation in the TCGA cohort. The 123 participants were divided into a training set (N = 82) and a test set (N = 41) to build a classification model. Logistic regression model showed the best performance for classification of lung cancer in bronchial washing samples: the sensitivity and specificity of a marker panel consisting of seven CpGs in TFAP2A, TBX15, PHF11, TOX2, PRR15, PDGFRA, and HOXA11 genes were 87.0 and 83.3% in the test set, respectively. The area under the curve (AUC) was equal to 0.87 (95% confidence interval = 0.73-0.96, p < 0.001). Methylation levels of two CpGs in RUNX3 and MIR196A1 genes were inversely associated with duration of smoking cessation in the controls, but not in NSCLCs, after adjusting for pack-years of smoking. Conclusions: The present study suggests that NSCLC may be detected by analyzing methylation changes of seven CpGs in bronchial washings. Furthermore, smoking cessation may lead to decreased DNA methylation in nonmalignant bronchial epithelial cells in a gene-specific manner.

Metformin induces cell cycle arrest at the G1 phase through E2F8 suppression in lung cancer cells.

A target molecule responsible for cell cycle arrest by metformin was discovered using a gene chip array in lung cancer cells and the effect of metformin on E2F8 was assessed. The siRNA-mediated knockdown of E2F8 significantly suppressed G1-S progression while ectopic expression of E2F8 relieved metformin-induced G1 arrest. The mRNA levels of p21 were found to be inversely related to those of E2F8 in lung cancer cells while siRNA-mediated knockdown of p21 partly rescued siE2F8-induced arrest of the cell cycle. Metformin had no effect on degradation of E2F8 mRNA. Activation and inhibition of AMPK by AICAR and Dorsomorphin, respectively, did not affect E2F8 suppression by metformin. The clinical significance of E2F8 was analyzed in The Cancer Genome Atlas (TCGA) data. One hundred six (13%) of 848 TCGA lung cancers overexpressed E2F8 mRNA. The overexpression of E2F8 was associated with poor overall survival (adjusted hazard ratio = 1.58, 95% confidence interval = 1.13-2.22; P = 0.008). The present study suggests that metformin may induce cell cycle arrest at the G1 phase by suppressing E2F8 expression in lung cancer cells. In addition, E2F8 may be associated with poor overall survival in lung cancer patients irrespective of histology.

Bronchial biopsy specimen as a surrogate for DNA methylation analysis in inoperable lung cancer.

Background: This study was aimed at understanding whether bronchial biopsy specimen can be used as a surrogate for DNA methylation analysis in surgically resected lung cancer. Methods: A genome-wide methylation was analyzed in 42 surgically resected tumor tissues, 136 bronchial washing, 12 sputum, and 8 bronchial biopsy specimens using the Infinium HumanMethylation450 BeadChip, and models for prediction of lung cancer were evaluated using TCGA lung cancer data. Results: Four thousand seven hundred and twenty-six CpGs (P < 1.0E-07) that were highly methylated in tumor tissues were identified from 42 lung cancer patients. Ten CpGs were selected for prediction of lung cancer. Genes including the 10 CpGs were classified into three categories: (i) transcription (HOXA9, SOX17, ZNF154, HOXD13); (ii) cell signaling (HBP1, SFRP1, VIPR2); and (iii) adhesion (PCDH17, ITGA5, CD34). Three logistic regression models based on the 10 CpGs classified 897 TCGA primary lung tissues with a sensitivity of 95.0~97.8% and a specificity of 97.4~98.7%. However, the classification performance of the models was very poor in bronchial washing samples: the area under the curve (AUC) was equal to 0.72~0.78. The methylation levels of the 10 CpGs in bronchial biopsy were not significantly different from those in surgically resected tumor tissues (P > 0.05, Wilcoxon rank-sum test). However, their methylation levels were significantly different between paired bronchial biopsy and washing (P < 0.05, Wilcoxon signed-rank test). Conclusions: The present study suggests that bronchial biopsy specimen may be used as a surrogate for DNA methylation analysis in patient with inoperable lung cancer.

Overexpression of ß-Catenin and Cyclin D1 is Associated with Poor Overall Survival in Patients with Stage IA-IIA Squamous Cell Lung Cancer Irrespective of Adjuvant Chemotherapy.

INTRODUCTION: This study was aimed at understanding the effect of ß-catenin and cyclin D1 on overall survival in patients with early-stage NSCLC and at evaluating if the prognostic effect can be modified by adjuvant chemotherapy. METHODS: We retrospectively analyzed the expression of ß-catenin and cyclin D1 using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 576 patients with early-stage NSCLC. RESULTS: The median duration of follow-up was 5.1 years. Overexpression of ß-catenin and cyclin D1 was found in 56% and 50% of 576 cases, respectively. Overexpression of ß-catenin and cyclin D1 was significantly associated with poor overall survival (p = 0.003 and p = 0.0009, respectively; log rank test) in squamous cell carcinomas, not in adenocarcinomas. The prognostic significance of each protein in the squamous cell carcinomas was limited to stages IA, IB, and IIA. In addition, simultaneous overexpression of ß-catenin and cyclin D1 in the squamous cell carcinomas synergistically increased hazard ratios (HRs) 15.79 (95% confidence interval [CI] = 1.09-51.23; p =0.04) for stage IA, 10.30 (95% CI = 2.29-46.41; p = 0.002) for stage 1B, and 3.55 (95% CI = 1.22-10.36; p = 0.02) times for stage 2A compared to those without overexpression of the two proteins, after adjusting for confounding factors. In addition, the effect was not dependent on adjuvant chemotherapy. CONCLUSIONS: The present study suggests that simultaneous overexpression of ß-catenin and cyclin D1 may be associated with poor overall survival irrespective of platinum-based adjuvant chemotherapy in stage IA-IIA squamous cell carcinoma of the lung.

Negative effect of cyclin D1 overexpression on recurrence-free survival in stage II-IIIA lung adenocarcinoma and its expression modulation by vorinostat in vitro.

BACKGROUND: This study was aimed at identifying prognostic biomarkers for stage II-IIIA non-small cell lung cancer (NSCLC) according to histology and at investigating the effect of vorinostat on the expression of these biomarkers. METHODS: Expression levels of cyclin D1, cyclin A2, cyclin E, and p16 proteins that are involved in the G1-to-S phase progression of cell cycle were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 372 samples of stage II-IIIA NSCLC. The effect of vorinostat on the expression of these proteins, impacts on cell cycle, and histone modification was explored in lung cancer cells. RESULTS: Abnormal expression of cyclin A2, cyclin D1, cyclin E, and p16 was found in 66, 47, 34, and 51 % of 372 cases, respectively. Amongst the four proteins, only cyclin D1 overexpression was significantly associated with poor recurrence-free survival (adjusted hazard ratio = 1.87; 95 % confidence interval = 1.12 - 2.69, P = 0.02) in adenocarcinoma but not in squamous cell carcinoma (P = 0.44). Vorinostat inhibited cell cycle progression to the S-phase and induced down-regulation of cyclin D1 in vitro. The down-regulation of cyclin D1 by vorinostat was comparable to a siRNA-mediated knockdown of cyclin D1 in A549 cells, but vorinostat in the presence of benzo[a]pyrene showed a differential effect in different lung cancer cell lines. Cyclin D1 down-regulation by vorinostat was associated with the accumulation of dimethyl-H3K9 at the promoter of the gene. CONCLUSIONS: The present study suggests that cyclin D1 may be an independent prognostic factor for recurrence-free survival in stage II-IIIA adenocarcinoma of lung and its expression may be modulated by vorinostat.

RARß2 hypermethylation is associated with poor recurrence-free survival in never-smokers with adenocarcinoma of the lung.

BACKGROUND: This study was aimed at investigating if the effect of RARß2 hypermethylation on recurrence-free survival (RFS) in non-small cell lung cancer (NSCLC) depends on one's smoking status and specific interacting proteins. RESULTS: We retrospectively analyzed the expressions of five proteins using immunohistochemistry in archival formalin-fixed and paraffin-embedded tissues from 578 NSCLC patients who had undergone surgical resection from 1994 through 2004. Promoter methylation of RARß2 was assessed by bisulfite pyrosequencing. Recurrence was found in 268 (46%) of 578 NSCLCs with a median follow-up period of 4.8 years. Overexpression of ß-catenin, c-MET, cyclin D1, and EGFR occurred in 55%, 72%, 51%, and 41% of the patients, respectively. E-cadherin expression was negative in 62% of the patients, and RARß2 hypermethylation was found in 37%. The abnormal expression of c-MET (P = 0.002) and EGFR (P = 0.001) was found to be highly prevalent in never-smokers. RARß2 hypermethylation was significantly associated with poor recurrence-free survival (RFS) in 128 never-smokers with adenocarcinoma (P = 0.01) For parsimonious model building, the five proteins were clustered into three groups (ß-catenin and E-cadherin; c-MET; cyclin D1 and EGFR) by an unsupervised hierarchical clustering and were included in a multivariate analysis. Cox proportional hazard analysis showed that RARß2 hypermethylation was significantly associated with poor RFS in 128 never-smokers with adenocarcinoma (adjusted hazard ratio [HR] = 2.19, 95% confidence interval [CI] = 1.28 to 3.47; P = 0.009), after adjusting for interacting proteins. CONCLUSIONS: The present study suggests that RARß2 hypermethylation may be an independent prognostic factor of RFS in never-smokers with adenocarcinoma of the lung.

HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer.

This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (P<0.0001; Wilcoxon signed-rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P=0.12) and Ki-67 proliferation index (P=0.15). However, patients with HOXA9 hypermethylation had poor recurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC.

HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.

Cohypermethylation of p14 in combination with CADM1 or DCC as a recurrence-related prognostic indicator in stage I esophageal squamous cell carcinoma.

BACKGROUND: The objective of this study was to discover molecular biomarkers associated with the recurrence of esophageal squamous cell carcinoma (ESCC). METHODS: The authors retrospectively analyzed the hypermethylation status of 11 genes using methylation-specific polymerase chain reaction (PCR) and the expression of epidermal growth factor receptor (EGFR), O-6 methylguanine-DNA methyltransferase (MGMT), tumor protein 53 (p53), and transforming growth factor ß (TGFß) using immunohistochemistry in 329 formalin-fixed, paraffin-embedded ESCCs. RESULTS: Recurrence was identified in 151 of 329 ESCCs (46%) at a median follow-up of 4.5 years. The recurrence was associated with hypermethylation of the genes cell adhesion molecule 1 (CADM1) (P = .003), deleted in colon carcinoma (DCC) (P = .04), or cyclin-dependent kinase inhibitor 2A (p14) (P = .02) in patients with stage I ESCC. Thirty-six of 37 Stage I ESCCs (97%) that had cohypermethylation of at least 2 of the 3 genes had hypermethylation of p14 plus either CADM1 or DCC or both CADM1 and DCC. The 5-year recurrence-free survival (RFS) rates were 93% in patients who had stage I disease without hypermethylation of the 3 genes and 56% in those who had cohypermethylation of p14 in combination with CADM1 and/or DCC. Patients who had stage I ESCC with cohypermethylation of p14 in combination with DCC and/or CADM1 had 7.13 times (95% confidence interval, 1.61-31.64 times; P = .009) poorer RFS compared with those who had no hypermethylation of the 3 genes after adjusting confounding factors. Hypermethylation of the other 8 genes and altered expression of 4 proteins were not associated with recurrence across pathologic stages. CONCLUSIONS: The current results suggested that cohypermethylation of p14 in combination with DCC and/or CADM1 may be an independent prognostic factor for recurrence in patients with stage I ESCC.

Association of RASSF1A and p63 with poor recurrence-free survival in node-negative stage I-II non-small cell lung cancer.

PURPOSE: This study was aimed at analyzing the recurrence-related prognostic significance of 12 candidate molecular biomarkers in node-negative stage I-II non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: We retrospectively analyzed promoter methylation of eight genes using methylation-specific PCR in formalin-fixed and paraffin-embedded tissues from 328 node-negative stage I-II NSCLCs. The expression of Bcl-2, E-cadherin, p53, and p63 proteins was also assessed by immunohistochemistry. RESULTS: Recurrence was found in 145 (44%) of 328 node-negative stage I-II NSCLCs with a median follow-up period of 6.2 years. No association was found between recurrence and alteration of individual biomarker in univariate analysis. We defined recurrently divergent groups on the basis of recursive partitioning analyses for 12 biomarkers and found a significant association of co-alteration of RASSF1A and p63 with poor recurrence-free survival (RFS). Cox proportional hazards analysis showed that hypermethylation of RASSF1A and negative expression of p63 was associated with poor RFS [HR, 1.93; 95% confidence interval (CI), 1.13-5.47; P = 0.009] compared with those without co-alteration of RASSF1A and p63, after adjusting for age, adjuvant therapy, histology, and tumor size. Random forest classifier including RASSF1A and p63 showed best performance in the prediction of recurrence in node-negative stage I-II NSCLCs: area under receiver operator characteristic curve for random forest was 0.91 and error rate for the model was 17%. CONCLUSION: The present study suggests that RASSF1A and p63 may be independent prognostic indicators for RFS in node-negative stage I-II NSCLCs.

Aberrant methylation of APC, MGMT, RASSF2A, and Wif-1 genes in plasma as a biomarker for early detection of colorectal cancer.

PURPOSE: To identify epigenetic molecular makers in plasma for the early detection of colorectal cancer. EXPERIMENTAL DESIGN: We retrospectively analyzed the methylation status of 10 genes in fresh-frozen tissues and corresponding plasma samples from 243 patients with stage I and II sporadic colorectal cancer, 276 healthy individuals, and plasma from 64 colorectal adenoma patients using methylation-specific PCR. The methylation score (Mscore) was used to find molecular markers with high sensitivity and specificity. RESULTS: Of the 243 colorectal cancer tissues, methylation was detected in 18% for p14, 34% for p16, 27% for APC, 34% for DAPK, 32% for HLTF, 21% for hMLH1, 39% for MGMT, 24% for RARbeta2, 58% for RASSF2A, and 74% for Wif-1. Receiver operator characteristic curve analysis in plasma from 243 patients with cancer and 276 healthy individuals showed that the M score of any single gene had a sensitivity of <40% after controlling for age, sex, and tumor location. The specificity of the M score was not different between multigene and single gene analyses, but the sensitivity of the M score was significantly increased by multigene analysis. For all patients, the M score in a model including APC, MGMT, RASSF2A, and Wif-1 genes had a sensitivity of 86.5% and a specificity of 92.1% when 1.6 was used as a cutoff. In this model, the M score had a positive predictive value of 90.6% and a negative predictive value of 88.8%. CONCLUSION: The present study suggests that tumor-specific methylation of APC, MGMT, RASSF2A, and Wif-1 genes might be a valuable biomarker in plasma for the early detection of colorectal cancer.

Relationship of phospho-pRb (Ser-807/811) level to exposure to tobacco smoke in primary non-small cell lung cancer.

This study was aimed at understanding the effect of smoking on pRb phosphorylation and the clinicopathological significance of phospho-pRb in non-small cell lung cancers (NSCLCs). Phospho-pRb (Ser-807/811) expression was not detected in 149 (39%) of 382 patients, and the mean phospho-pRb (Ser-807/811) level was 5.7%. Squamous cell carcinoma had higher phospho-pRb (Ser-807/811) levels than adenocarcinoma (7.1%+/-10.4% versus 4.7%+/-7.9%; P=0.003). The association between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke was different according to the statuses of cyclin D1 expression and p16 methylation, suggesting that their statuses might play a role as an effect modifier in the relationship between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke. In stratified multivariate analysis, phospho-pRb (Ser-807/811) levels were not associated with exposure to tobacco smoke in 38 patients with p16 hypermethylation and cyclin D1 expression >5%, after adjusting for confounding factors. However, in the remaining 344 patients, the mean phospho-pRb (Ser-807/811) levels in patients who had smoked >40 pack years increased by 4.65% (P<0.0001) on average than those who had never smoked. No association was found between the phospho-pRb (Ser-807/811) levels and overall survival. In conclusion, the present study suggests that exposure to tobacco smoke is associated with phosphorylation of pRb in NSCLC patients and its relationship depends on the p16 methylation status and cyclin D1 expression levels.

Histone deacetylase inhibitor scriptaid induces cell cycle arrest and epigenetic change in colon cancer cells.

Histone deacetylase inhibitors (HDACIs) are involved in cell growth, apoptosis and differentiation. This study aimed to investigate the effects of HDACI scriptaid on histone modification, demethylation, cell growth, cell cycle and apoptosis in the RKO colorectal cancer cell line and screening for scriptaid-induced genes. RKO cells were treated with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA) or scriptaid at different concentrations. Histone modification and methylation status of a silenced p16 gene were analyzed using chromatin immunoprecipitation and methylation-specific PCR, respectively. Flow cytometry was performed for the analysis of cell cycle and apoptosis. Scriptaid-induced expression was analyzed using Human OneArray chip. Scriptaid resulted in the demethylation and re-expression of a hypermethylated p16 gene along with 5-aza-dC synergistically in the RKO cells, but not alone. Scriptaid induced modifications of core histone tails important in euchromatin structure: increases in acetyl-H3-K9 and dimethyl-H3-K4 and a decrease in dimethyl-H3-K9. Cell growth was inhibited by scriptaid in a dose-dependent manner. Cell cycle analysis showed that scriptaid induced G1 arrest at 0.5 and 1.0 microM concentrations and G1 and G2/M arrest at 2.0 microM. Scriptaid did not have a significant effect on apoptosis in RKO cells. An altered expression of 278 genes was observed in RKO cells in response to scriptaid treatment. In conclusion, the present study suggests that scriptaid may be effective in growth suppression and cell cycle arrest and in the reversal of repressive chromatin marks at the promoter region of a hypermethylated p16 gene in colorectal cancer.

CpG island hypermethylation of E-cadherin (CDH1) and integrin alpha4 is associated with recurrence of early stage esophageal squamous cell carcinoma.

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence-associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif-1, sFRP1, integrin alpha4, CDH1, DAP kinase and RARbeta2 genes in 251 ESCCs. The methylation status was determined by methylation-specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARbeta2, 43% for CDH1, 21% for integrin alpha4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif-1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48-18.67; p = 0.01) and a poor recurrence-free survival after surgery (HR = 3.13, 95% CI = 1.21-8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46-70.41; p = 0.003) times higher in patients with Wif-1 methylation than in those without. For stage II cancers, integrin alpha4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09-8.37; p = 0.03) and a poor recurrence-free survival (HR = 2.12, 95% CI = 1.13-3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin alpha4 genes may be used as recurrence-associated prognostic indicators in stage I and stage II ESCCs, respectively.