AP-2a Induces Epigenetic Silencing of Tumor Suppressive Genes and Microsatellite Instability in Head and Neck Squamous Cell Carcinoma Kristi L. Bennett1, Todd Romigh 1 , Charis Eng1,2* 1 Genomic Medicine Institute, Lerner Research Institute and Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, United States of America, 2 Department of Genetics and Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America Abstract Background: Activator protein 2 alpha (AP-2a) is involved in a variety of physiological processes. Increased AP-2a expression correlates with progression in various squamous cell carcinomas, and a recent publication found AP-2a to be overexpressed in ,70% of Head and Neck Squamous Cell Carcinoma (HNSCC) patient samples. It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPa), and its binding site correlated with upstream methylation of the C/EBPa promoter. Therefore, we investigated the potential for AP-2a to target methylation to additional genes that would be relevant to HNSCC pathogenesis. Principal Findings: Stable downregulation of AP-2a stable by shRNA in HNSCC cell lines correlated with decreased methylation of its target genes’ regulatory regions. Furthermore, methylation of MLH1 in HNSCC with and without AP-2a downregulation revealed a correlation with microsatellite instability (MSI). ChIP analysis was used to confirm binding of AP- 2a and HDAC1/2 to the targets. The effects of HDAC inhibition was assessed using Trichostatin A in a HNSCC cell line, which revealed that AP-2a targets methylation through HDAC recruitment. Conclusions: These findings are significant because they suggest AP-2a plays a role not only in epigenetic silencing, but also in genomic instability. This intensifies the potential level of regulation AP-2a has through transcriptional regulation. Furthermore, these findings have the potential to revolutionize the field of HNSCC therapy, and more generally the field of epigenetic therapy, by targeting a single gene that is involved in the malignant transformation via disrupting DNA repair and cell cycle control. Citation: Bennett KL, Romigh T, Eng C (2009) AP-2a Induces Epigenetic Silencing of Tumor Suppressive Genes and Microsatellite Instability in Head and Neck Squamous Cell Carcinoma. PLoS ONE 4(9): e6931. doi:10.1371/journal.pone.0006931 Editor: Michael J. Pazin, National Institute on Aging (NIA), National Institutes of Health (NIH), United States of America Received April 14, 2009; Accepted August 6, 2009; Published September 9, 2009 Copyright: 2009 Bennett 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 in part by the Doris Duke Distinguished Clinical Scientist Award and Cleveland Clinic Strategic Investment Funds to the Genomic Medicine Institute (both to CE)[Grant number 20020346]. CE is the Sondra J. and Stephen R. Hardis Chair of the Cancer Genomic Medicine at the Cleveland Clinic. 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: engc@ccf.org Introduction The Activator Protein-2 family is composed of five members (a, b, c, e, d). These genes are transcription factors that play roles in apoptosis, cell cycle regulation, tissue differentiation, and develop- ment [1]. Activator protein 2 alpha (AP-2a) is involved in a variety of processes, including adipogenesis [2], neuronal development [1], and is suspect in cancer progression [3]. AP-2a binds as a homo or heterodimer to targets that contain the motif ‘‘GCCNNNGGC’’ in their regulatory regions [4]. After binding, AP-2a can either function as a transcriptional activator or repressor [5,6]. It is normally expressed at very low levels in keratinocytes, and it is elevated in proliferating, malignant keratinocytes of squamous cell carcinoma of the skin [7]. Furthermore, UVA irradiation increases AP-2a expression and results in malignant transformation [3,8]. Because AP-2a transcriptionally regulates such genes as p21 and CDH1, it is feasible to propose its relevance in tumorigenesis [9]. In fact, AP-2a has been shown to act as both a tumor suppressor and an oncogene in different cancer types [3,5,8]. It appears this is determined by the type and quantity of AP-2 isoforms and AP-2 modulating factors present in the cell [9]. Histone and promoter DNA methylation have been revealed in the past decade to be a frequent cause of gene silencing of tumor suppressors in a variety of malignancies [10,11]. DNA methylation sterically inhibits transcription factor binding, thereby inhibiting gene activation [12]. Conversely, decreased gene transcription has been shown in some cases to precede and propogate DNA methylation, resulting in more stable gene downregulation. For example, a transcriptional suppressor protein that competes with a transcriptional activator for the same binding site could result in decreased activation through increased methylation [13]. AP-2a is one such example, providing transcriptional repression to some genes which are also epigenetically modified[14,15]. It competes with the activator SP1 for binding to these target promoters, thereby repressing transcription [2,16]. AP-2a has previously been shown to recruit HDACs to specific gene promoters following retinoic acid induction [15], and a recent study observed that regions of frequent methylation in Acute Lymphoblastic Leukemia often contained PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e6931