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Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma
© Naganuma et al.; licensee BioMed Central. 2014
Received: 8 August 2014
Accepted: 16 December 2014
Published: 20 December 2014
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.
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).
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.
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.
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 . 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 . 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 [5–9].
Oral squamous cell carcinoma (OSCC) is the most common neoplasm of the oral cavity and has poor clinical outcomes associated with recurrence and metastasis . The Keratin 13 (KRT13) gene encodes a type I acidic keratin which is expressed in the differentiated cells of non-cornified stratified squamous epithelia [11–13]. Notably, the disappearance of KRT13 is often seen in OSCC lesions, while KRT13 is expressed in normal non-cornified oral mucosa [14–19]. In addition, KRT13-negative OSCC is associated with a high potential for local recurrence . 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.
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 . 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% CO2. 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 MethylEasy 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′-TGGAGTAGATGAAGTGCTAAGAAGG-3′ and 5′-AACAAAAAGAATGATTCAGAGGGG-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.
Total RNA was extracted from cells using the NucleoSpin 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′-TCCAGGTCATTAGACAGAG-3′. GAPDH was used as a reference gene for normalization. The primers used for GAPDH were 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-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.
Cells were scraped and resuspended in RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Noidet P-40, 5 mM EDTA, 1% sodium deoxycholate). Total protein extracts (5–10 μg) were resolved by electrophoresis on 4–20% polyacrylamide-SDS gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 4% (w/v) ECL Prime blocking agent (GE Healthcare Life Sciences, Backinghamshire, UK) in 0.1% Tween-TBS and probed with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. The specific antigen-antibody interactions were detected on a LAS-2000 imaging system (Fuji Film, Tokyo, Japan) using an ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences). Quantification of band intensity was performed using ImageJ 1.47v (National Institute of Health, Bethesda, MD). Relative expression levels were normalized to β-actin or histone H3. The following primary antibodies were used in this study: anti-keratin 13 (EPR3671; Abcam, Cambridge, UK; dilution, 1:1000), anti-Ezh2 (#5246; Cell Signaling Technology, Danvers, MA; dilution, 1:1000), anti-Suz12 (#3737; Cell Signaling Technology; dilution, 1:1000), anti-histone H3 (#4499; Cell Signaling Technology; dilution, 1:1000), anti-trimethyl histone H3 (Lys27) (MAB323B; MAB Institute Inc., Sapporo, Japan; dilution, 1:1000), and anti-β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX; dilution, 1:2000).
Cells were plated on Nunc Lab-Tek chamber slides (177429; Thermo Scientific, Waltham, MA), incubated for 24 h, fixed with 4% paraformaldehyde in PBS for 20 min at 21–25°C, permeabilized with 0.1% Triton X-100 for 30 min, and washed three times with PBS. Cells were blocked with 1% bovine serum albumin and 0.1% Tween 20 in PBS and probed with anti-keratin 13 (EPR3671; Abcam, dilution; 1:100), followed by anti-rabbit immunoglobulin-G (IgG) antibody conjugated with Alexa Fluor 488 (A-11034; Life Technologies, Carlsbad, CA; dilution, 1:800). Nuclei were counterstained with DAPI (P36935; Life Technologies). Fluorescence imaging was performed and images were captured using a Biorevo microscope (BZ-9000; Keyence, Osaka, Japan).
Cells were fixed in culture medium containing 1% formaldehyde for 10 min at 21–25°C and incubated in NP-40 buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% NP-40) for 5 min at 21–25°C. Cell were harvested and resuspended in SDS lysis buffer (50 mM Tris–HCl pH 8.0, 1% SDS, 10 mM EDTA), followed by 5-fold dilution in chromatin immunoprecipitation (ChIP) dilution buffer (50 mM Tris–HCl pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate). Chromatin was sonicated using a Bioruptor (Cosmo Bio Co., Tokyo, Japan) at medium power ten times for 20 sec. Ten micrograms of soluble sheared chromatin was incubated overnight at 4°C with protein G magnetic beads (#9006; Cell Signaling Technology) bound to 2 μg anti-histone H3 (#4620; Cell Signaling Technology), anti-trimethyl histone H3 (Lys4) (#9751; Cell Signaling Technology), anti-trimethyl histone H3 (Lys27) (#9733; Cell Signaling Technology), or control IgG (Cell Signaling Technology #2729), followed by sequential washing with low salt RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), high salt RIPA buffer (50 mM Tris–HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), wash buffer (50 mM Hepes-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate), and TE buffer. Immune complexes were then eluted by incubation for 20 min at 65°C with ChIP direct elution buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1% SDS), and the cross-linking was reversed by incubating overnight at 65°C. DNA was purified using the GenElute PCR Clean-up kit (Sigma-Aldrich) and subjected to quantitative PCR on an ABI 7500 real-time PCR system (Applied Biosystems). The primers used for the KRT13 promoter were 5′-TTGTGGGAAACAGAAGTGTAGTTGGC-3′ and 5′-GGTGAGAGCAGGATTGAGAGCAGGT -3′.
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 one-way 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
KRT13 promoter methylated in OSCC cells
Alterations of histone H3 methylation patterns in the KRT13 promoter in OSCC cells
Reactivation of KRT13 transcription by the PRC2 inhibitor DZNep in OSCC cells
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 , and it has recently been reported that DNA methylation can directly silence genes with non-CpG island promoters . We therefore investigated the DNA methylation status of the KRT13 promoter. According to the defining criteria of CpG islands , 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.  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. 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 , and that deregulation of PRC2 proteins is closely related to several human tumors [5–9]. 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 . 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 [11–13], while Ezh2 is expressed in the basal layer of stratified squamous epithelia . 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 . 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.
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.
We thank Drs. Tsuyako Ohkubo, Kazuhiko Okamura, and Kenichi Kato for engaging in helpful discussions. This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (Grant Number 25462906).
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