Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis Samuel Guenin1*., Mustapha Mouallif1,2., Rachel Deplus3, Xavier Lampe3, Nathalie Krusy1, Emilie Calonne3, Katty Delbecque4, Frederic Kridelka5, Franc¸ ois Fuks3, My Mustapha Ennaji2, Philippe Delvenne1 1 Laboratory of Experimental Pathology, Groupe Interdisciplinaire de Ge´noprote´omique Applique´e (GIGA)-Cancer, University of Lie`ge, Lie`ge, Belgium, 2 Laboratory of Virology and Hygiene and Microbiology, University Hassan II-Mohammedia, Mohammedia, Morocco, 3 Laboratory of Cancer Epigenetics, Free University of Brussels, Brussels, Belgium, 4 Department of Pathology, CHU Sart Tilman, Lie`ge, Belgium, 5 Department of Obstetrics and Gynecology, CHU Sart Tilman, Lie`ge, Belgium Abstract Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-29-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. Citation: Guenin S, Mouallif M, Deplus R, Lampe X, Krusy N, et al. (2012) Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis. PLoS ONE 7(8): e42704. doi:10.1371/journal.pone.0042704 Editor: Javier S. Castresana, University of Navarra, Spain Received February 23, 2012; Accepted July 10, 2012; Published August 3, 2012 Copyright: 2012 Guenin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Program of the Walloon Region (Neoangio Nu 616476) and the Belgian Fund for Medical Scientific Research (FNRS). Samuel Guenin and Xavier Lampe are supported by Neoangio. Mustapha Mouallif is supported by a grant from the Averroes Program of the European Commission and by a Te´le´vie grant from the F.R.S-FNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: samuel.guenin@gmail.com . These authors contributed equally to this work. Introduction Cervical cancer is the third cause of cancer-related death in women worldwide after breast and colon cancer [1]. Persistent infection with an oncogenic type of human papillomavirus (HPV) is a necessary factor for the development of invasive squamous cell carcinoma (SCC) of the cervix [2]. SCC are preceded by squamous intraepithelial lesions (SIL). Although the majority of low grade SIL (LSIL, corresponding to CIN1 and condyloma) spontaneously regress, high grade SIL (HSIL, corresponding to CIN2 and CIN3), if left untreated, can progress to SCC [3]. One of the hallmarks of carcinogenesis is the specific hypermethylation of CpG islands within the promoter of tumor suppressor genes, which usually results in the silencing of these genes leading to anarchical cell growth, proliferation and ultimately to the formation of invasive tumor and metastasis [4,5]. It has also been established that embryonic development and cancer pathogenesis share several molecular features. Accordingly, regulation of embryonic stem cell (SC) markers became of particular interest since it has been proposed that they are expressed by a small subpopulation of cells within tumor mass. The so-called tumor initiating cells or cancer stem cells (CSC) drive all the invasive properties of the tumor [6,7]. The presence of CSC has been recently suggested in cervical cancers [8,9]. However, the expression pattern and epigenetic regulation of SC markers in human epithelial tumors, as well as their clinical significance, are still unclear. Recent investigations about OCT4 gene have shown that, despite its promoter hypermethylation in cervical cancer cell lines, this gene is expressed whereas another study indicated the absence of Oct4A, the specific isoform of SC, in epithelial cancer cell lines [10,11]. Moreover, confusing data on OCT4 and NANOG could be related to the existence of multiple isoforms and pseudogenes (at least 6 and 10 pseudogenes for OCT4 and NANOG, respectively) [12,13]. In prostate tumors, CD133 expression, another CSC marker, was not correlated to its promoter methylation status, whereas in colon cancer, the CD133 negative cells were shown to form tumors much more aggressive than their CD133 positive counterparts [14,15]. In PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42704