AP-2a Targets Methylation Figure 5. Decreased AP-2a expression reduces proliferation; and AP-2a is required for target remethylation following demethylation. a) AP-2a western blot analysis of SCC22B cells with AP-2a overexpression. b) Cell cycle analysis via flow cytometry comparing WT, AP-2a-overexpressing, and AP-2a-downregulated SCC22B cells. * P-value = 0.0003. c) Methylation sensitive HhaI digests were performed on DNAs from SCC22B cells with and without AP-2a downregulation, revealing no distinguishable difference in global DNA methylation. ‘‘M’’ = methylated; ‘‘U’’ = unmethylated. d) 1 mM 5-aza-29deoxycytidine was applied to the cells for 96 hours, changing the media daily. After 96 hours, the 5-aza- 29deoxycytidine was removed and the cells were continued in culture for 10 days, collecting cells at 0, 7, and 10 days post-treatment. Combined bisulfite restriction analysis on bisulfite-treated DNA obtained from these cells at 0, 7, and 10 days. Increased methylation at 10 days is seen in the AP-2a-expressing cells (i.e. the digested band indicated by the arrow). doi:10.1371/journal.pone.0006931.g005 Amyloid Precursor Protein (APP), and these two genes have both been found to be increased in squamous cell carcinomas [25]. Furthermore, AP-2a interacts with SMAD2/3 to affect TGFB signaling, which is frequently disrupted in HNSCC [26]. These findings reveal the potential importance of AP-2a’s transcriptional regulation of target genes in the pathogenesis of HNSCC. It has been suggested that AP-2a’s ability to repress target genes results in cellular transformation and tumorigenesis [27], and decreased transcriptional suppression of AP-2a targets via abolished target binding has been shown to favor cellular differentiation [2,28]. In agreement, the results of this study in HNSCC reveal that downregulating AP-2a increases target gene expression by decreasing promoter DNA methylation and results in decreased cellular proliferation. In addition to its effects on proliferation, AP- 2a also has been shown to potentially impact microsatellite instability through its interaction with p53 and subsequent RAD51 suppression [29]. In this study, we demonstrate that AP-2a expression correlates with increased MSI in HNSCC cell lines, which correlates with MLH1 methylation and decreased expression. It has been demonstrated that AP-2a may help to recruit HDACs to the promoters to which it is bound [15]. Therefore, we investigated the association between AP-2a and HDAC binding to target promoters. This revealed a correlation between AP-2a and HDAC binding, and HDAC inhibition via TSA yielded similar effects on target demethylation as AP-2a downregulation. In fact, in some cases such as SCC11B and SCC17as, deacetylation via HDAC recruit- ment may be the primary means of target silencing by AP-2a (since AP-2a downregulation yielded no accountable methylation changes). Therefore, this study reveals that AP-2a acts as a suppressor for certain ‘‘tumor suppressive’’ genes in HNSCC by targeting promoter methylation and/or deacetylation via HDAC recruitment. Perhaps most interesting is the novel discovery that AP-2a is not only involved in the epigenetic silencing of tumor suppressor and DNA repair genes, but also appears to contribute to microsatellite instability in HNSCC. In this manner, AP-2a allows for both increased tumor proliferation and increased genomic instability. Furthermore, this phenomenon in HNSCC is distinct from the colon cancer cell lines used in this study, in which MSI appears to be unaffected by AP-2a expression changes. In light of a recent publication demonstrating AP-2a overexpression in 69% of HNSCC patients [14], these findings emphasize the impact AP- 2a has in HNSCC pathogenesis. Therefore, clinical treatment that targets AP-2a expression in the context of the MSI-positive HNSCC tumor would potentially deter tumor proliferation (ie. via PTEN and RASSF1A reexpression) and resolve genomic instability (ie. via MLH1 reexpression). Methods Cell lines, antibodies, and plasmids The established human HNSCC cell lines used in the study (SCC22B, SCC11B, and SCC17as) were kindly provided by Dr. Thomas Carey, University of Michigan. Cell lines were main- tained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin (S/P) antibiotics. MLH1 (abcam; ab9144), CDH1 (Cell Signaling; 4065), RASGRF1/2 (Santa Cruz; sc-28580), RASSF1A (abca- m;ab23950), a-TUBULIN (Cell Signaling Technologies; Danvers, MA), PTEN (monoclonal antibody clone 6H2.1; Cascade Biosciences, Portland, OR), AP-2a (abcam; ab18112), and acetyl Histone H4 Lys 5 (Upstate Cell Signaling Solutions; 07–327). The AP-2a shRNA construct was previously described [14]. The PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e6931