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1.  Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma 
Cell Cycle  2013;12(7):1058-1070.
Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood.
To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.
doi:10.4161/cc.24050
PMCID: PMC3646862  PMID: 23435205
bladder cancer; cigarette smoke extract; epigenetics; in vitro transformation; smoking; urothelial cell carcinoma
2.  Expression Microarray Analysis Reveals Alternative Splicing of LAMA3 and DST Genes in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e91263.
Purpose
Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.
Experimental Design
We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.
Results
After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).
Conclusion
Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.
doi:10.1371/journal.pone.0091263
PMCID: PMC3967989  PMID: 24675808
5.  Association of Promoter Methylation of VGF and PGP9.5 with Ovarian Cancer Progression 
PLoS ONE  2013;8(9):e70878.
Purpose
To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC).
Experimental Design
PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated.
Results
PH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI)  = 0.42–0.84, p = 0.004), and 0.73 (95%CI = 0.55–0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39–0.82, p = 0.003) and 0.72 (95%CI 0.54–0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43–0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41–0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth.
Conclusions
Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.
doi:10.1371/journal.pone.0070878
PMCID: PMC3785492  PMID: 24086249
6.  AIM1 PROMOTER HYPERMETHYLATION AS A PREDICTOR OF DECREASED RISK OF RECURRENCE FOLLOWING RADICAL PROSTATECTOMY 
The Prostate  2011;72(10):1133-1139.
Purpose
To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3 and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy.
Patients and Methods
Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3+4=7 and a minimum follow up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed using the Log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used.
Results
At a median follow-up time of 10 years, 48 patients (50.5%) had evidence of recurrence: biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: older age, HR=0.95 (95% CI: 0.91, 1.0) (P=0.03), positive lymph nodes HR=2.11 (95%CI: 1.05, 4.26) (P=0.04) and decreased hypermethylation of AIM1 HR=0.45 (95%CI: 0.2, 1.0) (P=0.05).
Conclusions
Methylation status of AIM1 in the prostate cancer specimen may predict for time to recurrence in Gleason 3+4=7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.
doi:10.1002/pros.22461
PMCID: PMC3360823  PMID: 22127895
7.  Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer 
Epigenetics  2012;7(7):710-719.
Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.
doi:10.4161/epi.20524
PMCID: PMC3414391  PMID: 22694820
BRAF; RARβ2; RASSF1A; TIMP3; biomarkers; hypermethylation; thyroid cancer; thyroid tissue
8.  SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling 
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
doi:10.1007/s00109-012-0955-3
PMCID: PMC3691869  PMID: 22968441
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
9.  Downregulation of insulin-like growth factor-binding protein 7 in cisplatin-resistant non-small cell lung cancer 
Cancer Biology & Therapy  2012;13(3):148-155.
Cisplatin is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLCs), but development of resistance is the primary impediment in cancer treatment. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted tumor suppressor that is inactivated in human lung cancer. IGFBP7 is known to alter sensitivity to interferon-based anticancer therapy, and here, we examined loss of IGFBP7 as a potential contributor to chemo-resistance to cisplatin. The transcriptional level of IGFBP7 was decreased in cisplatin-resistant human cancer cell lines and NSCLC xenografts. IGFBP7 knock-down increased cellular resistance to cisplatin and increased the level of mitogen-activated protein kinase phosphatases (MKP) 3 levels. The expression of MKP3 increased in a cisplatin-resistant NSCLC cell line and lung xenografts. MKP3 knock-down increased IGFBP7 level, indicating that MKP3 regulates IGFBP7. These findings suggest a novel molecular mechanism responsible for the tumor suppressive function of IGFBP7 in cisplatin-resistant human lung cancer and could lead to the development of IGFBP7 as a cisplatin-sensitizing agent.
doi:10.4161/cbt.13.3.18695
PMCID: PMC3336071  PMID: 22277373
cisplatin resistance; IGFBP7; MKP3; NSCLC; human xenografts; Stat3; Erk
10.  Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers 
PLoS ONE  2012;7(9):e44951.
The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.
doi:10.1371/journal.pone.0044951
PMCID: PMC3459978  PMID: 23028699
11.  Detection of Promoter Hypermethylation in Salivary Rinses as a Biomarker for Head and Neck Squamous Cell Carcinoma Surveillance 
Purpose
Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP).
Experimental Design
Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1.
Results
We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8–80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2–6.5; P = 0.016).
Conclusions
We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.
doi:10.1158/1078-0432.CCR-11-0324
PMCID: PMC3215270  PMID: 21628494
12.  An epigenetic marker panel for detection of lung cancer using cell-free serum DNA 
PURPOSE
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
EXPERIMENTAL DESIGN
To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.
RESULTS
Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).
CONCLUSION
This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.
doi:10.1158/1078-0432.CCR-10-3436
PMCID: PMC3131425  PMID: 21610147
DNA methylation/epigenetics; serum; lung cancer
13.  Cancer epigenetics: above and beyond 
Toxicology Mechanisms and Methods  2011;21(4):275-288.
Epigenetics refers to the study of mechanisms that alter gene expression without altering the primary DNA sequence. Epigenetic mechanisms are heritable and reversible. Over the last few decades, epigenetics has obtained a large importance in cancer research. Epigenetic alterations are widely described as essential players in cancer progression. They comprise DNA methylation, histone modifications, nucleosome positioning, and small, noncoding RNAs (miRNA, siRNA). They are involved in transcriptional changes and decisive events that will determine cell fate and phenotype. Epigenetics not only offers light into cancer biological processes, but also represents an attractive opportunity of reverting cancer-specific alterations, which may lead, in the future, to a possibility of stopping this disease. Epigenetic changes have been identified as putative cancer biomarkers for early detection, disease monitoring, prognosis, and risk assessment. Other epigenetic alterations are promising therapeutic targets and even therapeutic agents. Emerging discoveries in this area are already contributing to cancer management and monitoring, and a lot more progresses are expected in the future.
doi:10.3109/15376516.2011.562671
PMCID: PMC3309610  PMID: 21495866
Cancer; epigenetics; DNA methylation; histones modifications; microRNA
14.  A Survey of Methylated Candidate Tumor Suppressor Genes in Nasopharyngeal Carcinoma 
Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In the present study we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumor's biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation-specific PCR in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fisher's exact test and quantitative values were optimized using cut off values derived from receiver-operator characteristic curves. The methylation status of AIM1, APC, CALCA, DCC, DLEC, DLC1, ESR, FHIT, KIF1A, and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A, and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.
doi:10.1002/ijc.25443
PMCID: PMC2955155  PMID: 20473931
15.  ΔNp63α confers tumor cell resistance to cisplatin through the AKT1 transcriptional regulation 
Cancer research  2011;71(3):1167-1176.
Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g. ovarian, head/neck and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are up regulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1 and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.
doi:10.1158/0008-5472.CAN-10-1481
PMCID: PMC3076926  PMID: 21266360
ΔNp63α; AKT1; CDDP; chemoresistance
16.  Genetic and epigenetic analysis of erbB signaling pathway genes in lung cancer 
Prognosis for patients with non-small cell lung cancer (NSCLC) is poor. The potential value of modulating EGFR for treatment is reflected by the recent approval of specific drugs that inhibit its activity. Mutations in EGFR were reported in lung cancer and generated interest, once they enable the identification of lung cancers likely to respond to various targeted small molecules.
We tested 3 key genetic and epigenetic alterations (EGFR, RASSF1A, and BRAF) of this pathway on a series of primary NSCLC [Total 111; adenocarcinoma 49, squamous cell carcinoma (SCC) 48 and others 14]. The mutational status of KRAS (and p53) was known for these samples. The purpose of this study was to define the pattern of erbB pathway alterations in NSCLC and to test for associations with clinical parameters.
Five EGFR mutations were identified: 3 in adenocarcinoma (6 %), 1 in SCC (2%) and 1 in adenocarcinoma with bronchoalveolar component tumor (7%). EGFR mutations included 3 in-frame deletions in exon 19 and 2 point mutations in exon 21. Promoter methylation of RASSF1A was detected in 25 of 45 adenocarcinomas and 18 of 46 SCC. Mutations of EGFR, BRAF and KRAS in adenocarcinoma were mutually exclusive and inversely correlated with RASSF1A methylation (p = −0.394; p=0.007). Overall, genetic and/or epigenetic alterations of erbB pathway genes were detected in 80% (39/49) of adenocarcinomas.
Nearly half of primary adenocarcinoma harbor molecular alterations of the erbB pathway. Careful characterization of these alterations and response to anti-EGFR therapies is warranted to determine better and accurate determinants of clinical response.
doi:10.1097/JTO.0b013e3181f77a53
PMCID: PMC3057519  PMID: 21102258
EGFR mutation; DNA methylation/epigenetics; RASSF1A
17.  Association between Lifestyle Factors and CpG Island Methylation in a Cancer-Free Population 
Background
Many risk factors have been associated with cancer, such as age, family history, race, smoking, high-fat diet, and poor nutrition. It is important to reveal the molecular changes related to risk factors that could facilitate early detection, prevention, and overall control of cancer.
Methods
We selected six cancer-specific methylated genes that have previously been reported in primary tumors and have also been detected in different bodily fluids of cancer patients. Here, we used quantitative fluorogenic real-time methylation-specific PCR in plasma DNA samples for the detection of methylation changes from an asymptomatic population who do not have any known cancer.
Results
The promoter methylation frequencies of the studied genes were as follows: APC (7%), CCND2 (22%), GSTP1 (2%), MGMT (9%), RARβ2 (29%), and P16 (3%). Promoter methylation of at least one of the genes analyzed was observed in ~46% (72 of 157) of the samples by binary dichotomization. Promoter hypermethylation of at least two genes was detected in 17% (26 of 157) of the samples. RARβ2 methylation was observed in 45% of subjects who had a high-fat diet in contrast with those who had a low-fat diet (23%; P = 0.007).
Discussion
Our findings may help to elucidate early methylation changes that may lead to cancer development. These methylation changes could be due to exposure to risk factors and may be useful for cancer prevention measures such as changes in lifestyle. Longitudinal follow-up of a high-risk population is needed to understand the association of methylation of candidate genes in cancer development.
doi:10.1158/1055-9965.EPI-08-1245
PMCID: PMC2878974  PMID: 19861513
18.  KIF1A and EDNRB are differentially methylated in primary HNSCC and salivary rinses 
Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.
doi:10.1002/ijc.25248
PMCID: PMC2946472  PMID: 20162572
methylation; saliva; head and neck cancer; KIF1A; EDNRB
19.  Aberrant Promoter Methylation of Multiple Genes during Pathogenesis of Bladder Cancer 
Purpose
The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features.
Experimental Design
We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and χ2 or Fisher’s exact tests as appropriate. All statistical tests were two-sided.
Results
Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion.
Conclusion
Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.
doi:10.1158/1055-9965.EPI-08-0192
PMCID: PMC2778751  PMID: 18843024
20.  TIMP3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer 
The Journal of urology  2007;179(2):743-747.
Purpose
Tissue inhibitor of metalloproteinases-3 (TIMP-3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from bladder cancer patients with well characterized clinicopathological parameters including patient outcome.
Materials and methods
We examined urine sediment DNA for aberrant methylation of 9 genes including TIMP-3 by quantitative fluorogenic real-time PCR.
Results
Using an optimal cutoff value by Taqman quantitation, we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (hazard ratio [HR] =1.99, 95% confidence interval [CI] =1.12 to 3.27; p= 0.01 and HR=1.66, 95% CI=1.00 to 2.76; p=0.05 respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between risk of death and stage 3 tumor (HR=2.73, 95% CI=1.58 to 4.72; p=0.003 and the presence of metastasis (HR=3.32, 95% CI=1.98 to 5.57; p=0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p=0.001 and 0.02 respectively).
Conclusion
These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.
doi:10.1016/j.juro.2007.09.019
PMCID: PMC2674621  PMID: 18082200

Results 1-20 (20)