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1.  Promoter Methylation of CDKN2A, RARβ, and RASSF1A in Non-Small Cell Lung Carcinoma: Quantitative Evaluation Using Pyrosequencing 
Background
While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, RARβ, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs.
Methods
We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, RARβ, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of p16INK4A immunoexpression.
Results
Hypermethylation of CDKN2A, RARβ, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with p16INK4A immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of p16INK4A immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed p16INK4A immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another.
Conclusion
We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and p16INK4A immunoexpression loss.
doi:10.4046/trd.2012.73.1.11
PMCID: PMC3475475  PMID: 23101020
DNA Methylation; Genes, p16; RASSF1 Protein, Human; Receptors, Retinoic Acid; Sequence Analysis, DNA; Carcinoma, Non-Small Cell Lung
2.  Association between P16INK4a Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis 
PLoS ONE  2013;8(4):e60107.
Background
Aberrant methylation of CpG islands acquired in tumor cells in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates P16INK4a gene promoter hypermethylation is involved in non-small cell lung carcinoma (NSCLC), indicating it may be a potential biomarker for this disease. The aim of this study is to evaluate the frequency of P16INK4a gene promoter methylation between cancer tissue and autologous controls by summarizing published studies.
Methods
By searching Medline, EMBSE and CNKI databases, the open published studies about P16INK4a gene promoter methylation and NSCLC were identified using a systematic search strategy. The pooled odds of P16INK4A promoter methylation in lung cancer tissue versus autologous controls were calculated by meta-analysis method.
Results
Thirty-four studies, including 2 652 NSCLC patients with 5 175 samples were included in this meta-analysis. Generally, the frequency of P16INK4A promoter methylation ranged from 17% to 80% (median 44%) in the lung cancer tissue and 0 to 80% (median 15%) in the autologous controls, which indicated the methylation frequency in cancer tissue was much higher than that in autologous samples. We also find a strong and significant correlation between tumor tissue and autologous controls of P16INK4A promoter methylation frequency across studies (Correlation coefficient 0.71, 95% CI:0.51–0.83, P<0.0001). And the pooled odds ratio of P16INK4A promoter methylation in cancer tissue was 3.45 (95% CI: 2.63–4.54) compared to controls under random-effect model.
Conclusion
Frequency of P16INK4a promoter methylation in cancer tissue was much higher than that in autologous controls, indicating promoter methylation plays an important role in carcinogenesis of the NSCLC. Strong and significant correlation between tumor tissue and autologous samples of P16INK4A promoter methylation demonstrated a promising biomarker for NSCLC.
doi:10.1371/journal.pone.0060107
PMCID: PMC3618325  PMID: 23577085
3.  p16INK4a and p15INK4b gene promoter methylation in cervical cancer patients 
Oncology Letters  2012;3(6):1331-1335.
The aim of this study was to determine the methylation status of the p16INK4a, p14ARF and p15INK4b genes and the subsequent effect of hypermethylation on the expression of these genes in cervical cancer patients. Methylation-specific PCR (MSP) was performed to analyse the methylation status of p16INK4a, p14ARF and p15INK4b genes and was confirmed by sequencing. Reverse transcription PCR (RT-PCR) was carried out to determine changes in the expression of the genes due to hypermethylation. Hypermethylation of the p16INK4a, p14ARF and p15INK4b gene promoters was observed in 36, 8.8 and 11.2%, respectively, of 125 cervical cancer samples from a north Indian population. Methylation of p16INK4a was significantly (P<0.001) associated with the cervical cancer cases. Significant association of p16INK4a hypermethylation with passive smoking and oral contraceptive use was also observed. Methylation of p15INK4b was also found to be significant (P<0.05). Our findings did not reveal any correlation between the promoter methylation of p16INK4a, p14ARF and p15INK4b with factors, including age and human papillomavirus infection. mRNA expression was significantly reduced in patients with a methylated promoter (P<0.001) of p16INK4a compared to patients with an unmethylated promoter. In conclusion, this is the first study demonstrating significant hypermethylation of p15INK4b and p16INK4a genes among cervical cancer patients in a north Indian population, and a significant association of p16INK4a hypermethylation with passive smoking and oral contraceptive use.
doi:10.3892/ol.2012.655
PMCID: PMC3392578  PMID: 22783444
promoter hypermethylation; methylation-specific PCR; reverse transcription PCR; CpG island; human papillomavirus
4.  Clinicopathological significance and potential drug target of T-cadherin in NSCLC 
Background
Previous studies demonstrate that T-cadherin is a candidate tumor suppressor in several types of human tumors, including non-small cell lung cancer (NSCLC). Lack of protein expression of T-cadherin by hypermethylation has been found to play an important role in lung alveolar differentiation regulation and epithelial tumorigenesis. However, the correlation between T-cadherin hypermethylation and clinicopathological characteristics of NSCLC remains unclear. Here we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of T-cadherin hypermethylation on the incidence of NSCLC and clinicopathological characteristics.
Methods
A detailed literature search was carried out for related research publications. Analyses of pooled data were performed. Odds ratio (OR) and hazard ratio (HR) were calculated and summarized, respectively.
Results
Final analysis of 1,172 NSCLC patients from 15 eligible studies was performed. T-cadherin hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, based on the pooled OR from nine studies including 532 NSCLC and 372 normal lung tissue samples (OR=8.19, 95% confidence interval [CI]=5.41–12.39, P<0.00001). T-cadherin hypermethylation may also be associated with pathological types. The pooled OR was obtained from four studies including 111patients with squamous cell carcinoma and 106 with adenocarcinoma (OR=0.35, 95% CI=0.19–0.66, P=0.001), which indicated that T-cadherin hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. We did not find that T-cadherin hypermethylation was correlated with the sex or smoking status, clinical stages, or epidermal growth factor receptor (EGFR) mutation status. However, T-cadherin hypermethylation was found to be significantly higher in poorly differentiated NSCLC than in moderately and highly differentiated NSCLC, and NSCLC patients with T-cadherin hypermethylation had a lower survival rate than those without T-cadherin hypermethylation.
Conclusion
The results of this meta-analysis suggest that T-cadherin hypermethylation is associated with an increased risk and worse survival in NSCLC. T-cadherin hypermethylation, which induces the inactivation of T-cadherin gene, plays an important role in the carcinogenesis, cancer progression, as well as clinical outcome.
doi:10.2147/DDDT.S74259
PMCID: PMC4278732  PMID: 25565774
methylation; lung cancer; meta-analysis; EGFR; odds ratio; hazard ratio
5.  Methylation of the Candidate Biomarker TCF21 Is Very Frequent Across A Spectrum of Early Stage Non-Small Cell Lung Cancers 
Cancer  2010;117(3):606-617.
Background
The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, we sought to characterize the stages and types of non-small cell lung cancer (NSCLC) that are hypermethylated and to define the frequency of hypermethylation and associated “second hits”.
Methods
We determined TCF21 promoter hypermethylation in 105 NSCLC including various stages and histologies in smokers and nonsmokers. Additionally, we examined TCF21 loss-of-heterozygosity and mutational status. We also assayed 22 cancer cell lines from varied tissue origins. We validated and expanded our NSCLC results by examining TCF21 immunohistochemical expression on a tissue microarray containing 300 NSCLC cases.
Results
Overall, 81% of NSCLC samples showed TCF21 promoter hypermethylation and 84% showed decreased TCF21 protein expression. Multivariate analysis showed that TCF21 expression, although below normal in both histologies, was lower in adenocarcinoma than squamous cell carcinoma, and was not independently correlated with gender, smoking and EGFR mutation status, or clinical outcome. Cell lines from other cancer types also showed frequent TCF21 promoter hypermethylation.
Conclusions
Hypermethylation and decreased expression of TCF21 were tumor-specific and very frequent in all NSCLC, even early-stage disease, thus making TCF21 a potential candidate methylation biomarker for early-stage NSCLC screening. TCF21 hypermethylation in a variety of tumor cell lines suggests it may also be a valuable methylation biomarker in other tumor types.
doi:10.1002/cncr.25472
PMCID: PMC3023841  PMID: 20945327
TCF21; methylation; biomarker; lung cancer; screening
6.  Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas 
BMC Cancer  2007;7:47.
Background
High risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25–57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas
Methods
Methylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique.
Results
The extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands). The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors.
Conclusion
Hypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical carcinomas and cannot be an effective marker of cancer cells with up-regulated expression of p16ink4a. Our data confirm other previous studies claiming specific p16INK4a up-regulation in the majority of cervical carcinomas at both the protein and mRNA levels. Cytoplasmic accumulation of p16ink4a is a feature of cervical carcinomas.
doi:10.1186/1471-2407-7-47
PMCID: PMC1831478  PMID: 17359536
7.  The clinicopathological significance and ethnic difference of FHIT hypermethylation in non-small-cell lung carcinoma: a meta-analysis and literature review 
Emerging evidence indicates that FHIT is a candidate tumor suppressor in many types of tumors including non-small-cell lung carcinoma (NSCLC). However, the prognostic value and correlation between FHIT hypermethylation and clinicopathological characteristics of NSCLC remains unclear. In this report, we performed a meta-analysis to evaluate the effects of FHIT hypermethylation on the incidence of NSCLC and clinicopathological characteristics of human NSCLC patients. Final analysis of 1,801 NSCLC patients from 18 eligible studies was performed. FHIT hypermethylation was found to be significantly higher in NSCLC than in normal lung tissue. The pooled odds ratio (OR) from ten studies included 819 NSCLC and 792 normal lung tissues (OR =7.51, 95% confidence interval [CI] =2.98–18.91, P<0.0001). Subgroup analysis based on ethnicity implied that FHIT hypermethylation level was higher in NSCLC tissues than in normal tissues in both Caucasians (P=0.02) and Asians (P<0.0001), indicating that the difference in Asians was much more significant. FHIT hypermethylation was also correlated with sex status, smoking status, as well as pathological types. In addition, patients with FHIT hypermethylation had a lower survival rate than those without (hazard ratio =1.73, 95% CI =1.10–2.71, P=0.02). The results of this meta-analysis suggest that FHIT hypermethylation is associated with an increased risk and poor survival in NSCLC patients. FHIT hypermethylation, which induces the inactivation of FHIT gene, plays an important role in the carcinogenesis and clinical outcome and may serve as a potential diagnostic marker and drug target of NSCLC.
doi:10.2147/DDDT.S85253
PMCID: PMC4760666  PMID: 26929601
FHIT; methylation; tumor suppressor gene; meta-analysis; odds ratio; hazard ratio
8.  Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients 
Journal of Korean Medical Science  2007;22(Suppl):S32-S37.
Promoter hypermethylation of the p16INK4a gene was investigated in 81 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with primary lung cancer, using the modified real-time polymerase chain reaction (PCR)/ SYBR Green detection method. The results showed hypermethylation of p16INK4a in 27.2% of tumor tissues, and in 11.1% of adjacent normal tissue. No significant association was found between the overall aberrant methylation in tumor and corresponding normal specimens (r=0.137, p=0.219). In 22 cases with p16INK4a hypermethylation in tumor tissues, only 4 (18.1%) cases were found to have a hypermethylated normal tissue specimen. The findings of this study show that smoking can influence the methylation level of the promoter region of p16INK4a, and that this occurs in tumor tissues more frequently than in normal tissues. Other clinicopathological characteristics, including age, sex, tumor stage, and histologic type were not found to be correlated with p16INK4a methylation.
doi:10.3346/jkms.2007.22.S.S32
PMCID: PMC2694382  PMID: 17923752
Hypermethylation; p16INK4a; Lung Cancer
9.  Meta-analyses of gene methylation and smoking behavior in non-small cell lung cancer patients 
Scientific Reports  2015;5:8897.
Aberrant DNA methylation can be a potential genetic mechanism in non-small cell lung cancer (NSCLC). However, inconsistent findings existed among the recent association studies between cigarette smoking and gene methylation in lung cancer. The purpose of our meta-analysis was to evaluate the role of gene methylation in the smoking behavior of NSCLC patients. A total of 116 genes were obtained from 97 eligible publications in the current meta-analyses. Our results showed that 7 hypermethylated genes (including CDKN2A, RASSF1, MGMT, RARB, DAPK, WIF1 and FHIT) were significantly associated with the smoking behavior in NSCLC patients. The further population-based subgroup meta-analyses showed that the CDKN2A hypermethylation was significantly associated with cigarette smoking in Japanese, Chinese and Americans. In contrast, a significant association of RARB hypermethylation and smoking behavior was only detected in Chinese but not in Japanese. The genes with altered DNA methylation were likely to be potentially useful biomarkers in the early diagnosis of NSCLC.
doi:10.1038/srep08897
PMCID: PMC4354004  PMID: 25754026
10.  Prognostic significance of CDH13 hypermethylation and mRNA in NSCLC 
OncoTargets and therapy  2014;7:1987-1996.
Aberrant methylation of CpG dinucleotides is a commonly observed epigenetic modification in human cancer. Thus, detection of aberrant gene promoter methylation as a tool for diagnosis of tumors or as a prognostic marker has been widely described for many types of cancers, including nonsmall cell lung cancer (NSCLC). Emerging evidence indicates that CDH13 is a candidate tumor suppressor in several types of human tumors, including NSCLC. However, the correlation between CDH13 hypermethylation and clinicopathological characteristics of NSCLC remains unclear. In the current study, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of CDH13 hypermethylation on the incidence of NSCLC and clinicopathological characteristics. Final analysis of 803 NSCLC patients from eleven eligible studies was performed. CDH13 hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, with the pooled odds ratio (OR) from seven studies including 448 NSCLC and 345 normal lung tissue (OR, 7.85; 95% confidence interval, 5.12–12.03; P<0.00001). CDH13 hypermethylation was also associated with pathological types. The pooled OR was obtained from four studies, including 111 squamous cell carcinoma and 106 adenocarcinoma (OR, 0.35; 95% confidence interval, 0.19–0.66; P=0.001), which indicated that CDH13 hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. NSCLC with CDH13 hypermethylation was found more frequently in poorly differentiated NSCLC patients. NSCLC patients with CDH13 hypermethylation had a lower survival rate than those without CDH13 hypermethylation. In addition, CDH13 mRNA high expression was found to correlate with better overall survival for all NSCLC patients followed for 20 years (hazard ratio, 0.81; P=0.0056). Interestingly, CDH13 mRNA overexpression was found to correlate with better overall survival only in adenocarcinoma patients (hazard ratio, 0.42; P=9.6e–09), not in squamous cell carcinoma patients (hazard ratio, 0.93; P=0.59). The results of this meta-analysis suggest that CDH13 hypermethylation is associated with an increased risk and worse survival in NSCLC. CDH13 hypermethylation and mRNA expression play an important role in carcinogenesis, progression, and development, as well as clinical outcomes.
doi:10.2147/OTT.S67355
PMCID: PMC4222896  PMID: 25382980
prognosis; methylation; lung cancer; tumor suppressor gene; meta-analysis; odds ratio; hazard ratio
11.  Cytological examination of pleural cavity lavage accompanied by the study of gene promoter hypermethylation of p16 and O6-methylguanine-DNA-methyltransferase genes in diagnostics of non-small cell lung cancer metastatic changes into pleura 
Contemporary Oncology  2012;16(4):322-327.
Aim of the study
Metastases of non-small cell lung cancer (NSCLC) into pleura disqualify a patient from surgery and present a bad prognostic index. The aim of the study was to find out whether washing out the pleural cavity in such cases and examining obtained washings for presence of cancer cells will help to detect early NSCLC metastases into pleura, and also whether negative results of the cytology determine whether hypermethylation of these genes will increase the sensitivity of this examination.
Material and methods
The study consisted of the examination of 76 patients, including 59 operated on for NSCLC and 17 operated on for other reasons. Pleural washing fluid collected during the surgery was subjected to cytological examination as well as examined to determine the presence of promoter region hypermethylation of p16 and MGMT genes.
Results
Positive cytological results of pleural lavage were confirmed in 4 persons (7%) with NSCLC. The presence of promoter region hypermethylation of one or both examined genes was found in 3 patients (18%) in the control group and in 47 (80%) in the study group. Sex, occupational exposure, smoking cigarettes, and NSCLC histological type did not have an influence on the presence of cancer cells or hypermethylation in the pleural lavage fluid. Positive cytology results were more frequent at the T4 stage of NSCLC. Hypermethylation was more frequent in the research group (p < 0.01). Cancer cells and hypermethylation did not occur more frequently in pleural lavage fluid of patients with metastases into pleura.
Conclusions
The cytological examination and promoter region hypermethylation assessment of the p16 gene and MGMT gene in pleural lavage cells do not allow one to detect early metastasis of NSCLC into pleura.
doi:10.5114/wo.2012.30061
PMCID: PMC3687432  PMID: 23788902
lung cancer; pleura; cytology; DNA methylation; p16 gene; O(6)-methylguanine-DNA methyltransferase
12.  Clinicopathological significance and potential drug target of RUNX3 in non-small cell lung cancer: a meta-analysis 
Background
Emerging evidence indicates that RUNX3 is a candidate tumor suppressor in several types of human tumors, including non-small cell lung cancer (NSCLC). However, the correlation between RUNX3 hypermethylation and clinicopathological characteristics of NSCLC remains unclear. Here, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of RUNX3 hypermethylation on the incidence of NSCLC and clinicopathological characteristics.
Methods
A detailed literature search was made using Medline, Embase and Web of Science for related research publications written in English. The methodological quality of the studies was evaluated. The data were extracted and assessed independently by two reviewers. Analysis of pooled data was performed. The odds ratio (OR) and hazard ratio were calculated and summarized.
Results
Final analysis of 911 NSCLC patients from 13 eligible studies was performed. We observed that RUNX3 hypermethylation was significantly higher in NSCLC than in normal lung tissue; the pooled OR from seven studies including 361 NSCLC and 345 normal lung tissue (OR 7.08, confidence interval 4.12–12.17, P<0.00001). RUNX3 hypermethylation may also be associated with pathological types. The pooled OR was obtained from eleven studies including 271 squamous cell carcinoma and 389 adenocarcinoma (OR 0.41, confidence interval 0.19–0.89, P=0.02), which indicated that RUNX3 hypermethylation is significantly higher in adenocarcinoma that in squamous cell carcinoma. We did not find that RUNX3 hypermethylation was correlated with clinical stage or differentiated status. However, NSCLC patients with RUNX3 hypermethylation had a lower survival rate than those without RUNX3 hypermethylation.
Conclusion
The results of this meta-analysis suggest that RUNX3 hypermethylation is associated with an increased risk and worse survival in NSCLC. RUNX3 hypermethylation, which induces inactivation of the RUNX3 gene, plays an important role in lung carcinogenesis and clinical outcome.
doi:10.2147/DDDT.S76358
PMCID: PMC4461130  PMID: 26082616
RUNX3; methylation; lung cancer; tumor suppressor gene; meta-analysis; odds ratio; hazard ratio
13.  The clinicopathological significance of FHIT hypermethylation in non-small cell lung cancer, a meta-analysis and literature review 
Scientific Reports  2016;6:19303.
Emerging evidence indicates that FHIT is a candidate tumor suppressor in non-small cell lung cancer (NSCLC). However, the correlation between FHIT hypermethylation and clinicopathological characteristics of NSCLC remains unclear. Thus, we conducted a meta-analysis to quantitatively evaluate the effects of FHIT hypermethylation on the incidence of NSCLC and clinicopathological characteristics. Final analysis of 1717 NSCLC patients from 16 eligible studies was performed. FHIT hypermethylation was found to be significantly higher in NSCLC than in normal lung tissue, the pooled OR from 8 studies including 735 NSCLC and 708 normal lung tissue, OR = 5.45, 95% CI = 2.15–13.79, p = 0.0003. FHIT hypermethylation was also correlated with sex status, smoking status, as well as pathological types. We did not find that FHIT hypermethylation was correlated with the differentiated types or clinical stages in NSCLC patients. However, patients with FHIT hypermethylation had a lower survival rate than those without, HR = 1.73, 95% CI = 1.10–2.71, p  = 0.02. The results of this meta-analysis suggest that FHIT hypermethylation is associated with an increased risk and worsen survival in NSCLC patients. FHIT hypermethylation, which induces the inactivation of FHIT gene, plays an important role in the carcinogenesis and clinical outcome and may serve as a potential drug target of NSCLC.
doi:10.1038/srep19303
PMCID: PMC4726317  PMID: 26796853
14.  Clinicopathological significance and potential drug target of p15INK4B in multiple myeloma 
Multiple myeloma (MM) is a clonal malignancy characterized by the proliferation of malignant plasma cells in the bone marrow and the production of monoclonal immunoglobulin. In addition to genetic changes, gene hypermethylation is an alternative mechanism of tumor suppressor gene inactivation in MM. The cyclin-dependent kinase inhibitor 1 (CDKN2B or p15INK4B) gene lies adjacent to the tumor suppressor gene, cyclin-dependent kinase inhibitor 2 (CDKN2A), and is frequently mutated and deleted in a wide variety of tumors, including MM. However, there is a lack of systematic analysis of p15 epigenetic modification such as methylation in MM from different studies that can provide more powerful estimation of an effect. In this study, we have systematically reviewed the studies of p15INK4B promoter methylation in MM and quantified the association between p15INK4B promoter methylation and MM using meta-analysis methods. We observed that the frequency of p15INK4B methylation is significantly higher in MM patients than in normal healthy controls. The pooled odds ratio (OR) from ten studies including 394 MM and 99 normal individuals is 0.08, while confidence interval (CI) is 0.03–0.21 (P<0.00001). This indicates that p15INK4B inactivation through methylation plays an important role in the pathogenesis of MM. In addition, the frequency of p15INK4B methylation was significantly higher in patients with MM than in those with asymptomatic monoclonal gammopathy of undetermined significance. The pooled OR from four studies is 0.40, 95% CI =0.21–0.78 (P=0.007). These results suggest that silencing of p15INK4B gene expression by epigenetic modification such as promoter hypermethylation plays a role not only in the initiation of MM but also in plasma cell malignant transformation, disease progression, and development.
doi:10.2147/DDDT.S71088
PMCID: PMC4222634  PMID: 25382971
multiple myeloma; asymptomatic monoclonal gammopathy of undetermined significance (MGUS); p15; methylation; meta-analysis
15.  Aberrant p16INK4A methylation: Relation to viral related chronic liver disease and hepatocellular carcinoma 
Background:
Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the third leading cause of cancer related deaths. Several studies have shown that the tumor suppressor gene p16INK4A is frequently downregulated by aberrant methylation of the 5’-cytosine-phosphoguanine island within the promoter region.
Aim:
To find out the frequency of methylated p16INK4A in the peripheral blood of HCC and cirrhotic patients and to evaluate its role in hepatocarcinogenesis.
Patients and Methods:
This study was performed on 58 subjects: 30 HCC patients, 20 cirrhotic patients, and eight healthy volunteers. Methylation of p16INK4A was examined using methylation specific polymerase chain reaction (PCR) (MSP). Comparison of quantitative variables between the study groups was done using Mann-Whitney U test for independent samples when not normally distributed. For comparing categorical data, Chi-square (χ2) test was performed. Exact test was used instead when the expected frequency was less than 5.
Results:
Methylation of p16INK4A was found in 6.7% of HCC patients, 5% of liver cirrhosis (LC) patients, and none of the healthy volunteers; 66.67% of the p16INK4A-methylated cases (2/3) were positive for anti-hepatitis C virus (HCV) antibodies (one of them had HCC). All HCC cases with aberrant p16INK4A methylation show very high serum alpha fetoprotein (AFP) level (9,080; 30,000 μg/mL). There were no significant associations between the status of p16INK4A methylation and tumor size.
Conclusion:
Hypermethylation of p16INK4A was found to be infrequent among Egyptian patients with HCC.
doi:10.4103/2278-330X.126498
PMCID: PMC3961859  PMID: 24665436
Hepatocellular carcinoma; methylation; p16INK4A
16.  Promoter hypermethylation in Indian primary oral squamous cell carcinoma 
We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian-origin and compare with North-American head and neck squamous cell carcinomas (HNSCC). Quantitative-methylation-specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16INK4a and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at-least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16INK4a and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty-two of 72 node positive cases harbored p16INK4a and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16INK4a methylation (47.8%) in this Indian cohort in comparison with a North-American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16INK4a genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers.
doi:10.1002/ijc.25377
PMCID: PMC2946507  PMID: 20473870
hypermethylation; EDNRB; KIF1A; OSCC; p16INK4a; DCC; nodal metastasis
17.  A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype 
eLife  2014;3:e02313.
Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in, and the mechanistic basis of, CIMP is not understood. Among the CIMP genes are the tumor suppressors p14ARF, p15INK4B, and p16INK4A, encoded by the INK4-ARF locus. In this study, we perform an RNA interference screen and identify ZNF304, a zinc-finger DNA-binding protein, as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors, ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1, resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304, which drives DNA binding. Finally, we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells.
DOI: http://dx.doi.org/10.7554/eLife.02313.001
eLife digest
Colorectal cancer, which affects the large intestine, is a leading cause of cancer deaths worldwide, ranking fourth after cancers of the lung, stomach, and liver. Like these other cancers, this disease is caused by mutations to genes that allow cells to multiply in an out of control manner. Mutations that change the gene encoding a protein called KRAS are found in many different types of cancer. Moreover, about 70% of colorectal cancers with a KRAS mutation also have an excess of small chemical marks on other genes, some of which are known to suppress the growth of tumors. These marks ‘switch off’ these genes, and although the identities of the enzymes that typically leave these marks on DNA are known, the link between these enzymes and the KRAS protein is unknown.
Now Serra, Fang et al. have identified a protein, called ZNF304, that is required by KRAS to switch off a large number of genes, including multiple tumor suppressors. In the absence of ZNF304, these tumor suppressor genes remained switched on in cancer cells with the KRAS mutation, so the growth of the tumor was slowed down. ZNF304 is a protein that binds to stretches of DNA, including regions of DNA at the start of several tumor suppressor genes, and it recruits the enzymes that add the chemical marks that switch off these genes.
Serra, Fang et al. found that the levels of ZNF304 protein were elevated in colorectal cancer cells with the mutated KRAS, and showed that this was due to the combined activities of two other proteins that prevented ZNF304 from being broken down in the cell. Mutant KRAS caused an increase in the levels of these two proteins, which in turn caused the elevated ZNF304 levels and the excessive marking of the DNA in the tumor suppressor genes.
Furthermore, some of these same tumor suppressor genes are switched off in the earliest cells in a human embryo—which have the potential to become any of 200 or so cell types in the human body. In these embryonic stem cells, Serra, Fang et al. showed that ZNF304, but not KRAS, was also involved in keeping these genes switched off until the stem cells started changing into specific types of cells.
Since they are a crucial part of the pathway linking a cancer-causing mutation to increased tumor growth, the proteins identified by Serra, Fang et al. could represent promising targets for the development of new anti-cancer drugs.
DOI: http://dx.doi.org/10.7554/eLife.02313.002
doi:10.7554/eLife.02313
PMCID: PMC3949416  PMID: 24623306
CpG island methylator phenotype; INK4-ARF; colorectal cancer; ZNF304; KRAS; DNMT1; human; mouse
18.  Relationships among folate, alcohol consumption, gene variants in one-carbon metabolism and p16 INK4a methylation and expression in healthy breast tissues 
Carcinogenesis  2014;36(1):60-67.
Summary
Alcohol consumption, breast folate concentration and variation in one-carbon metabolism genes may be determinants of p16 INK4a promoter methylation and P16 protein expression in histologically normal breast tissues, and may influence early breast carcinogenic events.
p16 INK4a is a tumor suppressor gene, frequently hypermethylated in breast cancer; this epigenetic silencing of p16 INK4a occurs early in carcinogenesis. The risk factors and functional consequences of p16 INK4a methylation are unknown. Alcohol consumption, a breast cancer risk factor, impedes folate metabolism and may thereby alter gene methylation since folate plays a pivotal role in DNA methylation. In a cross-sectional study of 138 women with no history of breast cancer who underwent reduction mammoplasty, we studied breast cancer risk factors, plasma and breast folate concentrations, variation in one-carbon metabolism genes, p16 INK4a promoter methylation and P16 protein expression. Logistic regression was used to estimate multivariable-adjusted odds ratios (OR) and 95% confidence intervals (CI). p16 INK4a methylation was negatively correlated with P16 expression (r = −0.28; P = 0.002). Alcohol consumption was associated with lower breast folate (P = 0.03), higher p16 INK4a promoter methylation (P = 0.007) and less P16 expression (P = 0.002). Higher breast folate concentrations were associated with lower p16 INK4a promoter methylation (P = 0.06). Genetic variation in MTRR (rs1801394) and MTHFD1 (rs1950902) was associated with higher p16 INK4a promoter methylation (OR = 2.66, 95% CI: 1.11–6.42 and OR = 2.72, 95% CI: 1.12–6.66, respectively), whereas variation in TYMS (rs502396) was associated with less P16 protein expression (OR = 0.22, 95% CI: 0.05–0.99). Given that this is the first study to indicate that alcohol consumption, breast folate and variation in one-carbon metabolism genes are associated with p16 INK4a promoter methylation and P16 protein expression in healthy tissues; these findings require replication.
doi:10.1093/carcin/bgu219
PMCID: PMC4351383  PMID: 25344837
19.  Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis 
BMC Cancer  2002;2:29.
Background
Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes.
Methods
We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors.
Results
While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a , RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%).
Conclusions
Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.
doi:10.1186/1471-2407-2-29
PMCID: PMC139988  PMID: 12433278
20.  Reduced levels of p15INK4b, p16INK4a, p21cip1 and p27kip1 in pancreatic carcinoma 
Molecular Medicine Reports  2012;5(4):1106-1110.
Pancreatic carcinoma is one of the leading causes of cancer mortality worldwide, although the molecular mechanisms of this disease are poorly understood. The aim of this study was to examine the expression of cyclin-dependent kinase inhibitors (CDKIs) and the epigenetic modifications in the promoters of these genes. We also evaluated the correlation between the methylation status of CDKI genes and smoking habit in clinical pancreatic carcinoma specimens. Western blotting and real-time PCR were performed to assess CDKI expression. Methylation-specific PCR was carried out to examine the methylation status of the promoters of CDKI genes. In this study, we revealed that reduced levels of the CDKI proteins, p15INK4b, p16INK4a, p21cip1 and p27kip1, are a prominent feature of pancreatic carcinoma patients. The DNA hypermethylation of the promoter was observed in 40% (2 of 5) of the p15INK4b genes, 60% (3 of 5) of the p16INK4a genes and 60% of the p21cip1 genes, which markedly correlated with their decreased mRNA expression. No hypermethylation was detected in the p27kip1 gene promoter in 5 pancreatic carcinoma patients with markedly decreased expression of p27kip1 mRNA, suggesting an alternative mechanism of p27kip in these patients. In this study, patients with a smoking habit displayed methylation of 2 CDKI genes in their pancreatic carcinoma specimens. We concluded that epigenetic modification via hypermethylation represents a critical mechanism for the inactivation of CDKI genes in pancreatic carcinoma.
doi:10.3892/mmr.2012.771
PMCID: PMC3493078  PMID: 22293850
p15INK4b; p16INK4a; p21cip1; p27kip1; methylation; pancreatic carcinoma
21.  p16INK4a Gene Promoter Hypermethylation in Mucosa as a Prognostic Factor for Patients with Colorectal Cancer 
Molecular Medicine  2008;14(7-8):412-421.
Low gene expression of folylpolyglutamate synthase (FPGS) in colorectal mucosa correlates with low folate levels and poor survival of colorectal cancer (CRC) patients. Because gene-specific hypermethylation is affected by the folate level, the hypermethylation status in mucosa may also be linked to clinical outcome of CRC patients. The tumor suppressor gene p16INK4a (p16) regulates the cell cycle and angiogenic switch. In human neoplastic tissues, the main mechanism of p16 inactivation is promoter methylation. The aim of the study was to determine whether hypermethylation of the p16 promoter could be detected in mucosa of CRC patients (n = 181) and to analyze if hypermethylation was related to survival. The relation between p16 hypermethylation and expression of FPGS and two other folate-associated genes, reduced folate carrier 1 (RFC-1), and thymidylate synthase (TS), was analyzed (n = 63). The results showed that p16 was hypermethylated in 65 (36%) of the mucosa samples and that hypermethylation was age-related (P = 0.029). After adjustment for known risk factors, Cox regression analysis showed that Dukes’ A-C patients with p16 hypermethylation in mucosa had an increased risk of cancer-related death (hazard ratio = 2.9, P = 0.007) and shorter disease-free survival (hazard ratio = 2.5, P = 0.015) compared with patients with no p16 hypermethylation. RFC-1 and FPGS gene expression levels were significantly correlated in patients lacking p16 hypermethylation in mucosa (P = 0.0003), but not at all correlated in patients having hypermethylation in mucosa (P = 1.0). In conclusion, p16 hypermethylation in mucosa of CRC patients was identified as an independent prognostic parameter for cancer-specific survival as well as an independent predictor of DFS. The results suggest that there might be a connection between folate-associated gene expression and p16 methylation status.
doi:10.2119/2007-00096.Wettergren
PMCID: PMC2309648  PMID: 18418463
22.  Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-O-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis 
PLoS ONE  2013;8(11):e79634.
Background
This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 (HS3ST2) hypermethylation in non-small cell lung cancer (NSCLC).
Methodology/ Principal Findings
HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPERTM assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2′-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman’s rank correlation). HS3ST2 hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25–3.58, P = 0.005) compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation.
Conclusions/ Significance
The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.
doi:10.1371/journal.pone.0079634
PMCID: PMC3827134  PMID: 24265783
23.  Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma 
BMC Cancer  2004;4:65.
Background
Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.
Methods
We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.
Results
Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.
Conclusions
Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.
doi:10.1186/1471-2407-4-65
PMCID: PMC520749  PMID: 15367334
24.  Methylation of p15INK4b and Expression of ANRIL on Chromosome 9p21 Are Associated with Coronary Artery Disease 
PLoS ONE  2012;7(10):e47193.
Background
Genome-wide association studies have identified that multiple single nucleiotide polymorphisms on chromosome 9p21 are tightly associated with coronary artery disease (CAD). However, the mechanism linking this risk locus to CAD remains unclear.
Methodology/Principal Findings
The methylation status of six candidate genes (BAX, BCL-2, TIMP3, p14ARF, p15INK4b and p16INK4a) in 205 patients and controls who underwent coronary angiography were analyzed by quantitative MethyLight assay. Rs10757274 was genotyped and expression of INK4/ARF and antisense non-coding RNA in the INK4 locus (ANRIL) was determined by real-time RT-PCR. Compared with controls, DNA methylation levels at p15INK4b significantly increased in CAD patients (p = 0.006). To validate and dissect the methylation percentage of each target CpG site at p15INK4b, pyrosequencing was performed, finding CpG +314 and +332 remarkably hypermethylated in CAD patients. Further investigation determined that p15INK4b hypermethylation prevalently emerged in lymphocytes of CAD patients (p = 0.013). The rs10757274 genotype was significantly associated with CAD (p = 0.003) and GG genotype carriers had a higher level of ANRIL exon 1–5 expression compared among three genotypes (p = 0.009). There was a stepwise increase in p15INK4b and p16INK4a methylation as ANRIL exon 1–5 expression elevated (r = 0.23, p = 0.001 and r = 0.24, p = 0.001, respectively), although neither of two loci methylation was directly linked to rs10757274 genotype.
Conclusions/Significance
p15INK4b methylation is associated with CAD and ANRIL expression. The epigenetic changes in p15INK4b methylation and ANRIL expression may involve in the mechanisms of chromosome 9p21 on CAD development.
doi:10.1371/journal.pone.0047193
PMCID: PMC3473029  PMID: 23091611
25.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187

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