Identification of novel DNA methylation markers in cervical cancer

Abstract

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...

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

“…18 For analyses of host gene combinations with HPV testing that have included genes PAX1, SOX1, LMX1, NKX6-1 and WTI or CADM1, MAL and miR-124-2, the sensitivity was shown to rise for CIN31. 12,19 In the current study we observed a gain in performance with a linear predictor for the cancer vs. CIN2/3 and CIN2/3 vs. NILM/CIN1 contrasts. By simulating with our data the contrasts that would be of interest in a triage situation to help decide if HPV161 women must be referred for colposcopy implied setting the methylation level of the markers at a threshold that permitted high sensitivity to detect CIN2/3 or cancer.…”

“…13 We report herein our analysis of methylation of two HPV16 L1 sites and host CpG sites in genes EPB41L3 and LMX1 that have in recent studies shown promising ability to discriminate among pre-invasive cervical lesion stages. 6,16,[18][19][20][21] We investigated the predictive biomarker ability of these methylation sites across the spectrum of HPV16 positive cervical lesions, from negative for intraepithelial lesions or malignancy (NILM) to cervical cancer.…”

Viral epigenomes in human tumorigenesis

“…In fact, DNA methylation in host cell genes has been investigated as a potential biomarker for early detection of cervical cancer by extensive screening of premalignant and invasive cervical carcinoma samples (Lai et al, 2008;Whiteside et al, 2008;Fernandez et al, 2009;Ferrari et al, 2009;Wentzensen et al, 2009).…”

The homeobox NKX and Neoplasms in Digestive system: a systematic review.

“…This poor prognosis is mainly attributed to it often being diagnosed at the late clinical stage when surgical treatment and other medical therapies are limited [70] . Serval previous studies show that NKX6.1, a member of the NKX homeodomain-containing family, plays an important roles not only in neuronal tumors [71] , cervical tumors [72][73][74] , lymphoma [75] or islet tumor [76][77] , but also in HCC and may be a predictive risk factor for unfavorable prognosis in patients with HCC [78][79] .…”