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1.  An epigenetic marker panel for recurrence risk prediction of low grade papillary urothelial cell carcinoma (LGPUCC) and its potential use for surveillance after transurethral resection using urine 
Oncotarget  2014;5(14):5218-5233.
By a candidate gene approach, we analyzed the promoter methylation (PM) of 8 genes (ARF, TIMP3, RAR-β2, NID2, CCNA1, AIM1, CALCA and CCND2) by quantitative methylation specific PCR (QMSP) in the DNA of 17 non-recurrent and 19 recurrent noninvasive low grade papillary urothelial cell carcinoma (LGPUCC) archival tissues. Among the genes tested, by establishing an empiric cutoff value, CCND2, CCNA1, NID2, and CALCA showed higher frequency of methylation in recurrent than in non-recurrent LGPUCC: CCND2 10/19 (53%) vs. 2/17 (12%) (p=0.014); CCNA1 11/19 (58%) vs. 4/17 (23.5%) (p=0.048); NID2 13/19 (68%) vs. 3/17 (18%) (p=0.003) and CALCA 10/19 (53%) vs. 4/17 (23.5%) (p=0.097), respectively. We further analyzed PM of CCND2, CCNA1, and CALCA in urine DNA from UCC patients including LGPUCC and controls. The frequency of CCND2, CCNA1, and CALCA was significantly higher (p<0.0001) in urine of UCC cases [38/148 (26%), 50/73 (68%) and 94/148 (63.5%) respectively] than controls [0/56 (0%), 10/60 (17%) and 16/56 (28.5%), respectively)]. Most importantly we found at least one of the 3 markers were methylated positive in 25 out of 30 (83%) cytology negative LGPUCC cases. We also explored the biological function of CCNA1 in UCC. Prospective confirmatory studies are needed to develop a reliable tool for prediction of recurrence using primary LGPUCC tissues and/or urine.
PMCID: PMC4170626  PMID: 24980822
LGPUCC; Recurrence; Epigenetics; Biomarkers; DNA methylation
2.  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
3.  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
4.  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
5.  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
6.  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
7.  OGDHL Is a Modifier of AKT-Dependent Signaling and NF-κB Function 
PLoS ONE  2012;7(11):e48770.
Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multi-enzyme OGDH complex (OGDHC) whose malfunction is associated with neuro-degeneration. The essential role of this complex is in the degradation of glucose and glutamate and the OGDHL gene (one component of OGDHC) is down-regulated by promoter hypermethylation in many different cancer types. These properties suggest a potential growth modulating role of OGDHL in cancer; however, the molecular mechanism through which OGDHL exerts its growth modulating function has not been elucidated.
Here, we report that restoration of OGDHL expression in cervical cancer cells lacking endogenous OGDHL expression suppressed cell proliferation, invasion and soft agar colony formation in vitro. Knockdown of OGDHL expression in cervical cancer cells expressing endogenous OGDHL had the opposite effect. Forced expression of OGDHL increased the production of reactive oxygen species (ROS) leading to apoptosis through caspase 3 mediated down-regulation of the AKT signaling cascade and decreased NF-κB phosphorylation. Conversely, silencing OGDHL stimulated the signaling pathway via increased AKT phosphorylation. Moreover, the addition of caspase 3 or ROS inhibitors in the presence of OGDHL increased AKT signaling and cervical cancer cell proliferation.
Taken together, these data suggest that inactivation of OGDHL can contribute to cervical tumorigenesis via activation of the AKT signaling pathway and thus support it as an important anti-proliferative gene in cervical cancer.
doi:10.1371/journal.pone.0048770
PMCID: PMC3495966  PMID: 23152800
8.  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
9.  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
10.  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
11.  Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome 
Cancer research  2008;68(8):2661-2670.
DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells “the cancer methylome” would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.
doi:10.1158/0008-5472.CAN-07-5913
PMCID: PMC3102297  PMID: 18413733
12.  Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer 
Purpose
Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2′-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer.
Experimental Design
Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes.
Results
Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P < 0.0001, Fisher’s exact test).
Conclusions
This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.
doi:10.1158/1078-0432.CCR-08-1304
PMCID: PMC3082476  PMID: 19228724
13.  Forced Cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells 
Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.
doi:10.1002/ijc.24701
PMCID: PMC2988469  PMID: 19569044
Mitochondria; Cytochrome B mutation; apoptosis; mtDNA content

Results 1-13 (13)