Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma

Background Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs. Methods We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep). Results KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene. Conclusions Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-988) contains supplementary material, which is available to authorized users.


Background
Epigenetic mechanisms play important roles in the regulation of gene expression and phenotypic plasticity. The addition of a methyl group to the cytosine of a CpG dinucleotide (i.e., DNA methylation) in the promoter region of genes commonly mediates gene repression and acts as a silencing mechanism [1]. Post-translational modifications of histone tails are important regulatory markers for generating transcriptionally active and inactive chromatin. For instance, the trimethylation of Lys 4 on histone H3 (H3K4me3) is associated with gene activation, while the methylation of H3K27 (H3K27me3) and H3K9 (H3K9me2 and H3K9me3) is often connected to gene repression [2,3]. These epigenetic modifications dynamically regulate the chromatin architecture of promoter regions leading to the establishment of gene expression patterns. Polycomb repressive complex 2 (PRC2) comprises four core components (Ezh2, Suz12, Eed, and RbAp46/48) and several other proteins [4]. Ezh2 contains histone methyltransfease activity and plays an important role in the methylation of H3K27 mediated by PRC2. Dysregulation of PRC2 has been linked to several human cancers including lymphoma, squamous cell carcinoma, and breast and prostate cancer [5][6][7][8][9].
Oral squamous cell carcinoma (OSCC) is the most common neoplasm of the oral cavity and has poor clinical outcomes associated with recurrence and metastasis [10]. The Keratin 13 (KRT13) gene encodes a type I acidic keratin which is expressed in the differentiated cells of non-cornified stratified squamous epithelia [11][12][13]. Notably, the disappearance of KRT13 is often seen in OSCC lesions, while KRT13 is expressed in normal noncornified oral mucosa [14][15][16][17][18][19]. In addition, KRT13-negative OSCC is associated with a high potential for local recurrence [20]. Although the loss of KRT13 is correlated with the cellular transformation of oral epithelial cells, the epigenetic mechanisms by which KRT13 is repressed in OSCCs remain unclear.
In this study, we examined the epigenetic alterations in OSCC cells by focusing on the silencing mechanisms of the KRT13 gene and showed elevated KRT13 promoter DNA methylation and repressive histone modifications in OSCC cell lines. Furthermore, we found a PRC2 inhibitor effective for restoring KRT13 transcription. Our findings provide molecular insights into the epigenetic silencing of the KRT13 gene in OSCC cells as well as important implications for the development of diagnostic markers and novel therapeutic approaches.

Ethics statement
All experiments in this manuscript have been approved by the Fukuoka Dental College Institutional Biosafety Committee.

Cells and drug treatment
HSC3 and HSC4 cells were cultured as described previously [21]. Immortalized human keratinocyte HaCaT cells and OSCC-derived SAS cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin and maintained at 37°C with 5% CO 2 . The potent PRC2 inhibitor 3-deazaneplanocin A (DZNep) was purchased from Sigma-Aldrich (St. Louis, MO). Cells were seeded the day before the drug treatment, and DZNep (10 μM) was added to the culture medium for 24 h or 72 h.

DNA methylation analysis
Genomic DNA was extracted from cells using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) and subjected to bisulfite modification using the Methyl-Easy Xceed kit (Human Genetic Signatures, Randwick, Australia) according to the manufacturer's instructions. The primers used for amplification of the KRT13 promoter after bisulfite modification were 5′-TGGAGTAGATGAA GTGCTAAGAAGG-3′ and 5′-AACAAAAAGAATGATT CAGAGGGG-3′. Polymerase chain reaction (PCR) was performed with EX-taq DNA polymerase (Takara Bio Inc., Shiga, Japan), followed by TA cloning into a pMD20-T vector (Takara Bio Inc.) and sequencing of individual clones.

Quantitative RT-PCR
Total RNA was extracted from cells using the Nucleo-Spin RNA kit (Macherey-Nagel) and reverse transcription was performed using a PrimeScript RT reagent kit (Takara Bio Inc.) according to the manufacturer's instructions. Quantitative reverse-transcription PCR (RT-PCR) was performed on an ABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA) using SYBR Premix Ex Taq II (Takara Bio Inc.). The primers used for KRT13 were 5′-GACCGCCACCATTGAAAACAA-3′ and 5′-TCCAG GTCATTAGACAGAG-3′. GAPDH was used as a reference gene for normalization. The primers used for GAPDH were 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GG CTGTTGTCATACTTCTCATGG-3′. PCR conditions were as follows: 95°C for 30 sec, followed by 45 cycles of 95°C for 10 sec and 60°C for 1 min. The relative standard curve method was used to quantify relative mRNA levels of KRT13 and GAPDH.

Statistics
All values are presented as the means ± SEM for each group. Statistical analysis was performed using Student's t-test to compare the means of two groups or by a oneway analysis of variance followed by Dunnett's post hoc test for more than three groups. p < 0.05 was considered significant.

KRT13 repression by multiple mechanisms in OSCC cells
First, we examined KRT13 expression levels in the differentiated OSCC cell line (HSC4), in the poorly differentiated OSCC cell lines (HSC3 and SAS), and in the immortalized human keratinocyte HaCaT cell line [22][23][24]. Western blotting showed that KRT13 protein levels were significantly decreased in the HSC4 and HSC3 cells compared with the HaCaT cells, and almost absent in the SAS cells ( Figure 1A). Immunofluorescence microscopy revealed high cytoplasmic expression of KRT13 protein in the HaCaT cells, but not in the OSCC cell lines ( Figure 1B and Additional file 1: Figure S1). To investigate whether the reduction in KRT13 protein levels was due to the reduced expression of KRT13 mRNA, we performed quantitative RT-PCR analysis. As shown in Figure 2, KRT13 mRNA levels were significantly decreased in the HSC3 and SAS cells, but not in the HSC4 cells, compared with HaCaT cells. These results suggest that KRT13 expression is likely to be repressed by translational inhibition or protein degradation in the HSC4 cells and transcriptionally silenced in the HSC3 and SAS cells.

KRT13 promoter methylated in OSCC cells
Since it has been reported that the methylation status of the promoter region is generally linked to gene repression [1], we examined the DNA methylation status of the KRT13 promoter in the OSCC cells. The CpG sites within the proximal promoter region (−470 to −190) were analyzed by bisulfite sequencing ( Figure 3A). As shown in Figure 3B, the KRT13 promoter was partially methylated (<50%) in the HaCaT cells and highly methylated (≥90%) in the three OSCC cell lines (HSC4, HSC3, and SAS). These data show that the KRT13 promoter was aberrantly hypermethylated in all of the examined OSCC cell lines.
Alterations of histone H3 methylation patterns in the KRT13 promoter in OSCC cells DNA methylation of the KRT13 promoter did not correlate with KRT13 gene repression. Thus, it is likely that alterations of histone modifications are responsible for KRT13 gene repression in OSCC cells. It has been reported that H3K4me3 is associated with active transcription, while H3K27me3 is generally linked to gene repression [2,3]. We therefore examined histone H3 methylation patterns at the KRT13 promoter in the HaCaT cells and the three OSCC cell lines. ChIP analysis demonstrated that high levels of H3K4me3 but low levels  of K27me3 were detected in the HaCaT and HSC4 cells, in which KRT13 mRNA was highly expressed. On the other hand, HSC3 and SAS cells showed a histone code switch to high H3K27me3 but low K4me3 ( Figure 4A). Additionally, we observed high levels of H3K9me2 and K9me3, and low occupancy of RNA polymerase II (RNAP II) at the KRT13 promoter in the SAS cells compared with the HaCaT cells (Additional file 2: Figure S2A and B). Since PRC2 is known to play an important role in gene repression through the demethylation of H3K4 and the trimethylation of H3K27 and is linked to human cancer [5][6][7][8][9], we examined the expression levels of PRC2 component proteins (Ezh2 and Suz12) by western blotting. As shown in Figure 4B, we found no significant differences in the levels of Ezh2 and Suz12 proteins among the four cell lines. These data suggest that the histone H3 methylation patterns in the KRT13 promoter of the poorly differentiated OSCC cell lines (HSC3 and SAS) are distinct from those of the HaCaT and HSC4 cells and that the aberrant targeting of PRC2 to the KRT13 promoter may cause this sitespecific alteration.

Reactivation of KRT13 transcription by the PRC2 inhibitor DZNep in OSCC cells
To determine whether PRC2-mediated transcriptional repression is responsible for KRT13 gene silencing, we examined the effects of the potent PCR2 inhibitor DZNep on KRT13 mRNA expression in the SAS cells. As shown by western blotting (Figure 5A), treatment of the SAS cells with 10 μM DZNep for 24 h decreased the Ezh2 and Suz12 protein levels and H3K27 trimethylation levels, and KRT13 mRNA expression was markedly increased by almost 30-fold after DZNep treatment ( Figure 5B). In addition, the H3K27me3 levels in the KRT13 promoter were reduced in the SAS cells treated with DZNep ( Figure 5C). We then investigated the reversibility of DZNep-induced KRT13 transcription in SAS cells, which had been treated with DZNep for 24 h. KRT13 mRNA levels were increased after additional exposure to DZNep for 48 h, whereas the mRNA levels were reversed for 48 h after withdrawing DZNep ( Figure 6A). As shown in Figure 6B, the Ezh2 and Suz12 protein levels were decreased by DZNep treatment, and increased more than normal levels after drug removal. Taken together, our data suggest that DZNep induces the down-regulation of Ezh2 proteins and reactivates the transcription of KRT13 gene in SAS cells and that the effects of DZNep treatment on SAS cells are not heritable changes.

Discussion
This study provides molecular insights into the epigenetic silencing of the KRT13 gene in OSCC cells. Hypermethylation of CpG island promoters is known to play a critical role in transcriptional silencing [1], and it has recently been reported that DNA methylation can directly silence genes with non-CpG island promoters [25]. We therefore investigated the DNA methylation status of the KRT13 promoter. According to the defining criteria of CpG islands [26], the KRT13 promoter is classified as a non-CpG promoter. Bisulfite sequencing revealed that the KRT13 promoter was hypermethylated in all of the transformed OSCC cells tested compared with immortalized keratinocytes (Figure 3). Interestingly, Marsit et al. [27] reported that the KRT13 gene was identified as one of the methylated genes associated with aggressive bladder cancer. It is possible that the aberrant DNA methylation patterns of the KRT13 promoter are a hallmark for certain types of cancer.
We evaluated KRT13 expression levels by western blotting, immunocytochemistry, and quantitative RT-PCR. KRT13 protein levels were significantly reduced in all three OSCC cell lines (Figure 1), while KRT13 transcription levels were decreased in the poorly differentiated OSCC cell lines (HSC3 and SAS), but not in the differentiated OSCC cell line HSC4 (Figure 2). This finding is consistent with a previous immunohistochemistry study using paraffin sections of oral carcinoma in situ [16]. Thus, it is likely that OSCC cells repress KRT13 through multiple mechanisms. Our bisulfite sequencing data suggest that DNA methylation in the KRT13 promoter does not directly mediate transcriptional silencing, and that additional events, such as histone modifications, are required for KRT13 gene silencing in the poorly differentiated OSCC cells.
Epigenetic changes of histone modifications (low H3K4me3 and high H3K27me3) in the KRT13 promoter were identified in the HSC3 and SAS cells (Figure 4). We focused on PRC2-mediated H3K27me3, since it has been reported that H3K27me3 and the PRC2 component Ezh2 are involved in squamous cell differentiation [28], and that ) and H3K27me3 were examined by western blotting. Fold change in PRC2 proteins and H3K27me3 were normalized to β-actin and total histone H3, respectively, and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student's t-test. **p < 0.01. (B) KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student's t-test. **p < 0.01. (C) H3K27me3 levels in the KRT13 promoter were analyzed by ChIP assay. Fold enrichment of histone H3K27me3 was normalized to that of total histone H3 and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3-5) are shown. Statistical analysis was performed using Student's t-test. *p < 0.05. deregulation of PRC2 proteins is closely related to several human tumors [5][6][7][8][9]. To examine the recruitment of PRC2 proteins to the KRT13 promoter in SAS cells, we pharmacologically depleted PRC2 proteins. DZNep was previously reported to deplete the cellular levels of PRC2 proteins and inhibit H3K27 trimethylation [29]. DZNep treatment reduced PRC2 proteins and H3K27me3 levels and reactivated the KRT13 transcription in the SAS cells (Figures 5 and 6). KRT13 is also expressed in the differentiated epithelial cells of the suprabasal layer [11][12][13], while Ezh2 is expressed in the basal layer of stratified squamous epithelia [28]. In addition to the loss of KRT13, the overexpression of Ezh2 has been reported to correlate with malignant potential and poor prognosis in human OSCCs [6]. Our data suggest that KRT13 is one of the PRC2-repressed target genes in OSCC cells and that the reciprocal expression of KRT13 and PRC2 serves as a useful biomarker for phenotypic characterization of OSCC cells.

Conclusions
In summary, our study highlights the epigenetic mechanisms of KRT13 silencing in OSCC cells. We identified epigenetic alterations in the KRT13 promoter in OSCC cells and showed that the pharmacological inhibition of PRC2 restored KRT13 expression. To our knowledge, this is the first report on PRC2-mediated epigenetic silencing of KRT13. Our findings may be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

Additional files
Additional file 1: Figure S1. Cytoplasmic expression of KRT13 protein in the HaCaT cells. HaCaT cells were fixed and probed with anti-keratin 13 (EPR3671; Abcam; dilution, 1:100) or normal rabbit IgG (#2729; Cell Signaling Technology; dilution, 1:100), followed by anti-rabbit IgG antibody conjugated with Alexa Fluor 488. Expression of KRT13 protein (green) was analyzed using a confocal microscope (LSM710; Carl Zeiss MicroImaging GmbH, Jena, Germany), and nuclei were visualized with DAPI staining (blue). KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed by a one-way analysis of variance followed by Dunnett's post hoc test. **p < 0.01, N.S. (not significant). (B) Ezh2 and Suz12 protein levels were examined by western blotting. Representative images are shown. Fold change in Ezh2 and Suz12 proteins was normalized to β-actin and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 4) are shown. Statistical analysis was performed by a one-way analysis of variance followed by Dunnett's post hoc test. **p < 0.01.