Testing for DNA methylation has potential in cancer screening. Most previous studies of DNA methylation in cervical cancer used a candidate gene approach. The aim our study was to identify novel genes that are methylated in cervical cancers and to test their potential in clinical applications. We did a differential methylation hybridization using a CpG island (CGI) microarray containing 8640 CGI tags to uncover methylated genes in squamous cell carcinomas (SCC) of the uterine cervix. Pooled DNA from cancer tissues and normal cervical swabs were used for comparison. Methylation-specific polymerase chain reaction, bisulfite sequencing and reverse transcription polymerase chain reaction were used to confirm the methylation status in cell lines, normal cervices (n 5 45), low-grade lesions (n 5 45), high-grade lesions (HSIL; n 5 58) and invasive squamous cell carcinomas (SCC; n 5 22 from swabs and n 5 109 from tissues). Human papillomavirus (HPV) was detected using reverse line blots. We reported 6 genes (SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1) more frequently methylated in SCC tissues (81.5, 94.4, 89.9, 80.4, 77.8 and 20.4%, respectively) than in their normal controls (2.2, 0, 6.7, 11.9, 11.1 and 0%, respectively; p < 0.0001). Parallel testing of HPV and PAX1 methylation in cervical swabs confers an improved sensitivity than HPV testing alone (80% vs. 66%) without compromising specificity (63% vs. 64%) for HSIL/SCC. Testing PAX1 methylation marker alone, the specificity for HSIL/SCC is 99%. The analysis of these novel DNA methylations may be a promising approach for the screening of cervical cancers. ' 2008 Wiley-Liss, Inc.Key words: cervical cancer; epigenetics; hpv; methylation; microarray In addition to genetic changes, epigenetic alterations such as DNA methylation and histone modifications can result in heritable gene silencing without changes to genetic sequences and are recognized as important causes of cancer.1-3 DNA methylation mostly occurs at the 5 0 cytosine in the palindromic sequence, 5 0 -CpG-3 0 . CpG islands are CpG-rich areas of 1 kb that are usually located in the vicinity of genes, often near the promoters of widely expressed genes.4,5 Methylation of CpG sites in the human genome is catalyzed by a family of DNA methyltransferases (DNMTs). DNMT1 is a maintenance methyltransferase with a preference for hemimethylated DNA whereas DNMT3a and DNMT3b are de novo methyltransferases with approximately equal preferences for methylated and unmethylated DNA.2,6 The addition of methyl groups by DNMTs recruits complexes with transcription repressors that modify histones and thus silence genes. Global DNA hypomethylation and site-specific hypermethylation result in genomic instability and transcriptional gene inactivation, respectively, both of which are associated with cancer. 7,8 As epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly observed in human cancers, DNA methylation could serve as a marker for early diagnosis of cancer and as a means of assessing the prognos...

The limitations of current anti-angiogenic therapies necessitate other targets with complimentary mechanisms. Here, we show for the first time that soluble E-cadherin (sE-cad) (an 80-kDa soluble form), which is highly expressed in the malignant ascites of ovarian cancer patients, is a potent inducer of angiogenesis. In addition to ectodomain shedding, we provide further evidence that sE-cad is abundantly released in the form of exosomes. Mechanistically, sE-cad-positive exosomes heterodimerize with VE-cadherin on endothelial cells and transduce a novel sequential activation of β-catenin and NFκB signaling. In vivo and clinical data prove the relevance of sE-cad-positive exosomes for malignant ascites formation and widespread peritoneal dissemination. These data advance our understanding of the molecular regulation of angiogenesis in ovarian cancer and support the therapeutic potential of targeting sE-cad. The exosomal release of sE-cad, which represents a common route for externalization in ovarian cancer, could potentially be biomarkers for diagnosis and prognosis.

A recent hypothesis for cancer chemoresistance posits that cytotoxic survival of a subpopulation of tumor progenitors drives the propagation of recurrent disease, underscoring the need for new therapeutics that target such primitive cells. To discover such novel compounds active against drug-resistant ovarian cancer, we identified a subset of chemoresistant ovarian tumor cells fulfilling current definitions of cancer-initiating cells from cell lines and patient tumors using multiple stemness phenotypes, including the expression of stem cell markers, membrane dye efflux, sphere formation, potent tumorigenicity, and serial tumor propagation. We then subjected such stem-like ovarian tumor-initiating cells (OTIC) to high-throughput drug screening using more than 1,200 clinically approved drugs. Of 61 potential compounds preliminarily identified, more stringent assessments showed that the antihelmintic niclosamide selectively targets OTICs in vitro and in vivo. Gene expression arrays following OTIC treatment revealed niclosamide to disrupt multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation. These studies support niclosamide as a promising therapy for ovarian cancer and warrant further preclinical and clinical evaluation of this safe, clinically proven drug for the management of this devastating gynecologic malignancy.

BACKGROUND: DNA methylation may be used a potential biomarker for detecting cervical cancer. The authors of this report used quantitative methylation analysis of 4 genes in a full spectrum of cervical lesions to test its potential clinical application. METHODS: This hospital-based, retrospective, case-control study was conducted in 185 patients and included patients who had a normal uterine cervix (n ¼ 53), cervical intraepithelial neoplasm type 1 (CIN1) (n ¼ 37), CIN2 (n ¼ 22), CIN3 (n ¼ 24), carcinoma in situ (CIS) (n ¼ 22), squamous cell carcinoma (SCC, n ¼ 20), and adenocarcinoma (AC) (n ¼ 7). Methylation levels of the genes sex-determining region Y, box 1 (SOX1); paired box gene 1 (PAX1); LIM homeobox transcription factor 1a (LMX1A), and NK6 transcription factor-related locus 1 (NKX6-1) were determined by using real-time methylation-specific polymerase chain reaction (PCR) amplification. Cutoff values of the percentage of methylation reference (PMR) for different diagnoses were determined to test the sensitivity and specificity and to generate receiver operating characteristic (ROC) curves. Two-sided Mann-Whitney U tests were used to test differences in PMR between groups. RESULTS: The PMRs of the 4 genes were significantly higher in CIN3 and worse (CIN3þ) lesions than the PMRs in specimens of normal cervix and CIN1 or CIN2 (P < .001). ROC curve analysis demonstrated that the sensitivity, specificity, and accuracy for detecting CIN3þ lesions were 0.88, 0.82, and 0.95, respectively, for SOX1; 0.78, 0.91, and 0.89, respectively, for PAX1; 0.77, 0.88, and 0.90, respectively, for LMX1A; and 0.93, 0.97, and 0.97, respectively, for NKX6-1. CONCLUSIONS: The current results indicated that quantitative PCR-based testing for DNA methylation of 4 genes holds great promise for cervical cancer screening and warrants further population-based studies using standardized DNA methylation testing. Cancer 2010;116:4266-74.

BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.

Cancer cells exhibit phenotypic plasticity during epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat further reveal cell state-dependent plasticity upon GRHL2 overexpression. Overall, we demonstrate that epithelial genes are subject to epigenetic control during intermediate phases of EMT/MET involving GRHL2.

BackgroundDespite of the trend that the application of DNA methylation as a biomarker for cancer detection is promising, clinically applicable genes are few. Therefore, we looked for novel hypermethylated genes for cervical cancer screening.Methods and FindingsAt the discovery phase, we analyzed the methylation profiles of human cervical carcinomas and normal cervixes by methylated DNA immunoprecipitation coupled to promoter tiling arrays (MeDIP-on-chip). Methylation-specific PCR (MSP), quantitative MSP and bisulfite sequencing were used to verify the methylation status in cancer tissues and cervical scrapings from patients with different severities. Immunohistochemical staining of a cervical tissue microarray was used to confirm protein expression. We narrowed to three candidate genes: DBC1, PDE8B, and ZNF582; their methylation frequencies in tumors were 93%, 29%, and 100%, respectively. At the pre-validation phase, the methylation frequency of DBC1 and ZNF582 in cervical scraping correlated significantly with disease severity in an independent cohort (n = 330, both P<0.001). For the detection of cervical intraepithelial neoplasia 3 (CIN3) and worse, the area under the receiver operating characteristic curve (AUC) of ZNF582 was 0.82 (95% confidence interval  = 0.76–0.87).ConclusionsOur study shows ZNF582 is frequently methylated in CIN3 and worse lesions, and it is demonstrated as a potential biomarker for the molecular screening of cervical cancer.

This study demonstrates the potential use of methylated BHLHE22/CDO1/CELF4 panel for endometrial cancer screening of cervical scrapings. Clin Cancer Res; 23(1); 263-72. ©2016 AACR.