http://orcid.org/0000-0002-0125-8930
Figures
Abstract
Background
Methylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses transcription of that gene. Interferon regulatory factor 6 (IRF6) is associated with the expression of interferon, which is used as an effective adjuvant therapy for melanoma, and is regarded as a tumor suppressor. However, little is known about the methylation status of the IRF6 gene in melanoma.
Objective
The purpose was to determine the methylation status of the CGI located in the 5' region of IRF6 (5' IRF6 CGI) in melanoma.
Methods
Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to examine IRF6 gene methylation status. Quantitative real-time reverse transcription-PCR (RT-PCR) was performed to examine IRF6 expression.
Results
The methylation level of the 5' IRF6 CGI was completely inversely correlated with cell sensitivity to interferon-β in eight examined melanoma cell lines. These methylation levels were high in the melanoma cell lines with suppression of IRF6 expression and were low in the cell lines with IRF6 expression. The methylation levels of the 5' IRF6 CGI ranged widely from 0.0% to 65.4% in 21 clinical melanoma samples but showed a narrow range of low levels between 0.0% to 7.2% in 24 clinical melanocytic nevus samples. These methylation levels were not associated with clinical parameters except for melanoma subtypes.
Citation: Nobeyama Y, Nakagawa H (2017) Silencing of interferon regulatory factor gene 6 in melanoma. PLoS ONE 12(9): e0184444. https://doi.org/10.1371/journal.pone.0184444
Editor: Javier S. Castresana, University of Navarra, SPAIN
Received: July 14, 2017; Accepted: August 23, 2017; Published: September 6, 2017
Copyright: © 2017 Nobeyama, Nakagawa. 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.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
DNA methylation is a DNA modification resulting from the covalent binding of a methyl group to a DNA nucleotide such as the cytosine of a CpG dinucleotide, where a 5' cytosine is adjacent to a 3' guanine [1,2]. The methylation status of individual CpG sites is faithfully copied into daughter cells [3]. CpG islands (CGIs) are dense clusters of CpGs that are often located in the 5' regions of genes. Methylation of a CGI located in the 5' region of a gene suppresses transcription of that gene [4]. In normal cells, the CGIs located in the 5' regions of most genes are unmethylated, resulting in the expression of those genes [4]. However, in malignant cells, a number of CGIs located in the 5' regions of genes, including tumor-suppressor genes, may be methylated, resulting in the suppression of the transcription of those genes [4,5].
Melanoma is a malignant tumor that develops through transformation of melanocytes [6]. Advanced melanomas often show dismal outcomes [7,8], although immune checkpoint modulators such as anti-programmed death receptor-1 antibodies and anti-cytotoxic T-lymphocyte-associated protein 4 antibodies as well as molecular-targeted agents such as BRAF inhibitors and MEK inhibitors have been reported to improve the prognosis of patients with advanced melanoma to some degree [9]. Based on these dismal outcomes, adjuvant therapies to enhance the inhibition of recurrence and metastases are still needed.
Interferon (IFN) is in widespread use as an adjuvant therapy, mainly for stage II and resectable stage III melanoma (according to the staging system of the American Joint Committee on Cancer) [10] due to its effective inhibition of melanoma progression. Eggermont et al. reported that high-dose IFN-α-2b and pegylated IFN can improve relapse-free survival in prospective, randomized, multicenter treatment trials [10]. In addition, Yamamoto et al. suggested that local administration of natural-type IFN-β potentially improves prognosis including the 5-year survival rate [11]. Suppression or low expression of specific genes in the interferon pathway is associated with poor prognosis of melanoma [12].
Interferon regulatory factors (IRFs), which include nine members, are associated with tumor suppression and cell differentiation [13,14,15,16,17]. IRFs exert their functions through various molecular mechanisms including: (i) regulation of type 1 IFN expression via toll-like receptor signaling [18]; (ii) interaction with the NF-κB signaling pathway [19]; and (iii) interaction with the mammary serine protease inhibitor (maspin), which is regarded as a tumor suppressor [20]. Among the nine IRF members, the tumor-suppressive functions of IRF6 have been supported by many studies, in addition to its important function in the development process of palatal fusion [21]. Thus, Botti et al. reported that IRF6 exhibits a tumor-suppressive function in squamous cell carcinomas [22]; Restivo et al. reported that IRF6 exerts tumor-suppressive functions in keratinocytes through mediation of Notch pro-differentiation [23]; Bailey et al. reported that IRF6 expression is decreased in invasive breast cancer cell lines and breast tumors [20] and that IRF6 facilitates mammary epithelial cell entry into the G0 phase of the cell cycle in collaboration with maspin [24].
Despite these diligent studies, little is known regarding the regulation of IRF6 by DNA methylation in melanomas or regarding the association between IRF6 methylation status and sensitivity to IFN. The present study was conducted to clarify the expression and methylation status of the CGI in the 5' region of IRF6 (5' IRF6 CGI) in melanomas.
Materials and methods
Ethics statement
The ethics committee of The Jikei University School of Medicine granted approval for this study, and written informed consent for the use of tissue samples was obtained from reachable donors or their legal guardians. The ethics committee of The Jikei University School of Medicine waived the requirement for consent from unreachable donors.
Cell lines, clinical samples, and extraction of nucleic acid
The human melanoma cell lines A375, Hs294T, SH-4, SK-MEL-2 SK-MEL-5, SK-MEL-28 and RPMI-7951, and two normal human cultured epidermal melanocytes (MC1 and MC2) were purchased from the American Type Culture Collection (Manassas, VA). The melanoma cell line G-361 was purchased from the Riken BioResources Center (Tsukuba, Japan). Twenty-one paraffin-embedded melanoma samples were obtained from patients with melanoma (Table 1). Twenty-four melanocytic nevus samples were obtained from patients without any history of melanoma (Table 2).
Melanoma and melanocytic nevus were diagnosed histopathologically by at least two experienced board-certified dermatologists and pathologists. The TNM classification of cutaneous melanoma was evaluated according to the American Joint Committee on Cancer melanoma staging system (7th edition). Melanoma samples were classified into four subtypes consisting of melanoma arising from non-chronically sun-damaged skin (NSCD), melanoma arising from chronically sun-damaged skin (CSD), mucosal melanoma and acral melanoma [25].
To extract DNA from paraffin-embedded samples, the samples were sliced into 4 to 10 μm-thick sections, deparaffinized, and then dissected with a fine needle. Genomic DNA was extracted by using a standard phenol/chloroform extraction and ethanol precipitation procedure or the QIAamp DNA mini kit (Qiagen, Valencia, CA). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan).
Treatment with 5-aza-2'-deoxycytidine
For 5-aza-2'-deoxycytidine (5-aza-dC) treatment, melanoma cells were seeded at a density of 1.0×105 to 2.5×105 cells per 10-cm dish on day 0 and were exposed to medium containing 1.0 μM 5-aza-dC (Sigma-Aldrich, St Louis, MO) on days 1 and 3. The cells were harvested on day 5. All cell lines showed mild growth suppression on day 5, when compared with untreated cells.
Treatment with natural type IFN-β
To obtain cells treated with natural type IFN-β, melanoma cells were seeded at a density of 2.0×105 to 5.0×105 cells per 10-cm dish on day 0 and were exposed to medium containing 1.0×103 international units/ml of natural type IFN-β (Toray Medical, Tokyo, Japan) or phosphate buffered saline (PBS) on day 1. On day 3, the cell numbers were harvested.
To evaluate growth of cells treated with natural type IFN-β, melanoma cells were seeded at a density of 2.0×105 to 5.0×105 cells per 10-cm dish on day 0 and were exposed to medium containing 1.0×103 international units/ml of natural type IFN-β or PBS on days 1 and 3. On day 5, the cell numbers were counted. Growth rate was defined as the value of the number of IFN-β-treated cells divided by the number of PBS-treated cells.
Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing
One μg of the BamHI-digested genomic DNA was modified by sodium bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA) according to the instruction manual, and was dissolved in 40 μl of buffer.
For RT-MSP, 1.0 μl of the sodium bisulfite-treated DNA was amplified with the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) using a mixture of primer sets that were specific to the methylated or unmethylated DNA sequence (M or U set, respectively) and the SYBR Green PCR Master Mix I (Toyobo, Osaka, Japan). The sequences of the M set primers were: 5'-GTAGGGTGGGACGTTGGACGGAC-3' for the forward primer and 5'-TAACCACGCCCCCCGACGTTCG-3' for the reverse primer, which were designed to amplify from ‒127 bp to ‒22 bp of the IRF6 gene based on the major IRF6 start site (based on the IRF6 sequence in NC_000001.11). The sequences of the U set primers were: 5'-GGGTTGGGTAGTTTAGAAATGT-3' for the forward primer and 5'-TATAACCACACCCCCCAACA-3' for the reverse primer, which were designed to amplify from ‒98 bp to ‒20 bp of the IRF6 gene based on the major IRF6 start site. The number of molecules of a specific sequence in a sample was measured by comparing its amplification with that of standard samples, which contained 101 to 108 copies of the molecule. The methylation level was defined as the number of methylated DNA molecules divided by the total number of methylated and unmethylated DNA molecules. DNA methylated with SssI methylase (New England Biolabs, Beverly, MA) and DNA amplified with the GenomiPhi DNA amplification kit (GE Healthcare Bioscience, Little Chalfont, UK) were used as methylated and unmethylated DNA controls, respectively, under specifically amplified conditions for M and U sets.
For bisulfite sequencing, 1.0 μl of the sodium bisulfite-treated DNA was used for PCR with primers common to methylated and unmethylated DNA sequences. The sequences of these primers were: 5'-GGAGTTAGAAGYGGAGGAGTAG-3' for the forward primer, in which Y indicates a C or a T, and 5'-ACGCCTCCCAAATATAACCAC-3' for the reverse primer, which were designed to amplify from ‒145 bp to ‒8 bp of the IRF6 gene based on the major IRF6 start site. The PCR products were cloned into a cloning vector, and 12 clones were cycle-sequenced for each sample.
Quantitative real-time reverse transcription-PCR (RT-PCR)
Total RNA was treated with DNase I (Ambion, Austin, TX) and cDNA was synthesized from 1.0 μg of total RNA using a Superscript III kit (Life Technologies, Rockville, MD). RT-PCR was performed using the SYBR Green PCR Master Mix I (Toyobo, Osaka, Japan) and the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The sequences of the primers were: 5'-CCGTTTGAGATCTACTTATGCT-3' for the forward primer and 5'-GATCATCCGAGCCACTACTG-3' for the reverse primer. The number of molecules of a specific gene in the sample was measured by comparing its amplification with that of standard samples, which contained 101 to 108 copies of the gene. The mRNA quantity of each gene was normalized with that of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH).
Statistical analysis
Statistical analysis was performed using the commercially available software, SPSS version 18 (SPSS Japan, Tokyo, Japan). Significant differences in laboratory data were analyzed by linear regression analysis, the Kruskal-Wallis test, the Mann-Whitney U test and the Log-rank test. P < 0.05 was considered statistically significant.
Results
The methylation level of the 5' IRF6 CGI is completely inversely correlated with melanoma cell sensitivity to IFN-β
The methylation status of the 5' IRF6 CGI in melanoma cell lines was analyzed using methylation-specific PCR, and the sensitivity of the same melanoma cell lines to IFN-β was analyzed using cell growth assay (Fig 1). Statistical analysis revealed a significant correlation between 5' IRF6 CGI methylation levels and sensitivity to IFN-β (linear regression analysis, P = 0.033). Thus, the methylation level of the 5' IRF6 CGI was completely inversely correlated with IFN-β sensitivity in melanoma cell lines.
The left bar graph indicates the methylation levels (%) of the 5' IRF6 CGI in the indicated melanoma cell lines, normal cultured melanocytes (MC1 and MC2) and controls including methylated DNA (M) and unmethylated DNA (U). The second bar graph from the left indicates the expression levels of IRF6 divided by the expression levels of GAPDH (×10−4) in each of the melanoma cell lines and normal cultured melanocytes (MC1 and MC2) that were not treated with IFN-β. The expression level of MC1 was 1.1×10−3, which is higher than the 3.0×10−4 upper limit of the graph. The third bar graph from the left indicates the expression levels of IRF6 divided by the expression levels of GAPDH (×10−4) in each of the melanoma cell lines treated with IFN-β. The fourth bar graph from the left indicates the cell growth rate which was defined as the value of the number of IFN-β-treated cells divided by the number of PBS-treated cells. All four bar graphs correspond to each other with respect to the melanoma cell lines and normal cultured melanocytes.
The methylation status of the 5' IRF6 CGI regulates the expression of IRF6
The association between the methylation status of the 5' IRF6 CGI (S1 Fig) and transcription of IRF6 as analyzed by RT-MSP and RT-PCR, respectively (Fig 1), for each of the melanoma cell lines (RPMI-7951, A375, Hs294T, SK-MEL-28, SK-MEL-2, SK-MEL-5, SH-4 and G-361) and normal cultured melanocytes (MC1 and MC2) was as follows. Some of the cell lines with expression of IRF6, including the RPMI-7951, Hs294T, and SK-MEL-2 cell lines and normal cultured melanocytes (MC1 and MC2), exhibited a considerable amount of unmethylated DNA molecules (methylation levels ranging from 0.0% to 53.3%). Other cell lines with high methylations levels (ranging from 87.0% to 97.2%), including A375, SK-MEL-5, SH-4 and G-361 cells, exhibited suppression of IRF6 expression (Fig 1). Treatment with the demethylating agent 5-aza-dC resulted in transcriptional induction of IRF6 in SH-4, G-361 and SK-MEL-5 cells, which had exhibited high methylation levels and suppression of IRF6 before this treatment (Fig 2).
The left bar graph indicates the methylation levels (%) of the 5' IRF6 CGI and the right bar graph indicates the expression levels of IRF6 divided by those of GAPDH (×10−4), in SH-4, G-361 and SK-MEL-5 cells that were treated with the demethylating agent (+) or with PBS (-). Both bar graphs correspond to each other with respect to the cell lines.
IFN-β enhances IRF6 expression in unmethylated melanoma cells
We then determined the association between IFN-β treatment and IRF6 expression in the individual melanoma cell lines according to their 5' IRF6 CGI methylation statuses as shown in Fig 1. IFN-β enhanced IRF6 expression in the melanoma cell lines in which there was a considerable amount of unmethylated 5' IRF6 CGI DNA molecules but had a lower effect on IRF6 expression in melanoma cell lines that had only a small amount of unmethylated DNA molecules.
The 5' IRF6 CGI is methylated in vivo in melanoma but not in melanocytic nevus
The in vivo methylation status of IRF6 in melanomas and melanocytic nevi was analyzed by performing RT-MSP of 21 clinical melanoma samples and 24 clinical melanocytic nevus samples. The methylation levels ranged widely from 0.0% to 65.4% in clinical melanoma samples including sample #17 with a methylation level of 65.4% and sample #10 with a level of 64.2%, while there was a narrow range of low methylation levels, ranging from 0.0% to 7.2%, in clinical melanocytic nevus samples (Tables 1 and 2 and Fig 3). Consequently, six out of 21 melanoma samples (28.6%) showed higher methylation level than the highest methylation level in the melanocytic nevus samples.
Each methylation level was determined using RT-MSP. The vertical and horizontal axes indicate the methylation level (%) and sample ID, respectively. M and U indicate controls of methylated and unmethylated DNA, respectively.
Data from bisulfite sequencing are compatible with those from RT-MSP
The data obtained using RT-MSP were confirmed by bisulfite sequencing using samples with representative RT-MSP results. For this analysis, clinical melanoma samples #10 and #17 were examined as representatives of samples with high methylation levels. Normal cultured melanocytes MC2 and clinical melanocytic nevus samples #23 and #34 were examined as representatives of samples with low methylation levels (Fig 4). The data showed that CpG sites within the 5' IRF6 CGI were densely methylated in samples #10 and #17, while they were sparsely methylated in MC2 cells and in sample #23 and #34. Thus, the bisulfite sequencing data were compatible with the results of RT-MSP.
The results of bisulfite sequencing of the 5' IRF6 CGI for representative samples are shown. The clinical melanoma samples #10 and #17 were examined as representatives of samples with high methylation levels. Normal cultured melanocytes MC2 and clinical melanocytic nevus samples #23 and #34 were examined as representatives of samples with low methylation levels. Closed and open circles indicate methylated and unmethylated CpG sites, respectively. Closed and open triangles indicate the location of the RT-MSP primer sets specific to the methylated and unmethylated DNA sequences, respectively. The vertical and horizontal numbered rows indicate each clone and CpG site, respectively.
The methylation level of the 5' IRF6 CGI is associated with melanoma subtype
The correlations between the methylation status of the 5' IRF6 CGI and clinical parameters such as age, sex, subtype and outcome were analyzed by statistical analyses (Table 3). The Kruskal-Wallis test indicated a significantly different distribution of the methylation levels of the 5' IRF6 CGI among the subtypes of melanoma (P = 0.035); the methylation levels of the acral type tended to be higher than those of the other types. On the other hand, Kaplan-Meier survival analysis and the log-rank test indicated no significant difference in outcome between the patients who donated melanoma samples whether the samples were with or without methylated DNA molecules. In this analysis, the cut-off value for methylation was set at 7.2%, which was the highest methylation level of the 24 melanocytic nevus samples. Similarly, the clinical parameters tested, including age and sex, were not associated with methylation levels of the 5' IRF6 CGI.
Discussion
The present study indicated for the first time that (i) the 5' IRF6 CGI is sometimes methylated, resulting in silencing of IRF6, in melanoma but not in melanocytic nevus, and (ii) the methylation status of the 5' IRF6 CGI is potentially associated with the sensitivity of melanoma to IFN-β.
The in vitro examinations showed that (i) some melanoma cell lines with high methylation levels of the 5' IRF6 CGI exhibited suppression of the gene, (ii) other melanoma cell lines with low methylation levels of this CGI exhibited expression of the gene, and (iii) treatment with a demethylating agent restored IRF6 gene expression in the melanoma cell lines that had high methylation levels and suppression of IRF6 before the treatment. These data indicated that (i) the methylation status of the analyzed region within the CGI located in the 5' region of IRF6 regulates the expression of the gene, and (ii) IRF6 can be silenced through methylation of the 5' IRF6 CGI in melanoma. These in vitro data encouraged us to investigate the in vivo methylation status of IRF6 in melanoma.
The in vivo examinations indicated that the clinical melanocytic nevus samples exhibited a narrow and range of low methylation levels, which ranged from 0.0% to 7.2%, and that the clinical melanoma samples exhibited a wide range of methylation levels, which ranged from 0.0% to 65.4% and included a number of samples with high methylation (e.g. 65.4% methylation in sample #17 and 64.2% in #10). Six of the 21 clinical melanoma samples exhibited a methylation level that was higher than the highest methylation level of the clinical melanocytic nevus samples. These results suggested that a certain percentage of melanomas may develop in association with the silencing of IRF6 unlike nevus melanocytic.
The present study also demonstrated that (i) the methylation level of the 5' IRF6 CGI was completely inversely correlated with the sensitivity to IFN-β of the eight melanoma cell lines examined, and (ii) IFN-β enhanced IRF6 expression in the melanoma cell lines that had a considerable amount of unmethylated 5' IRF6 CGI DNA molecules. These observations strongly suggested that cell sensitivity to IFN-β is dependent on the methylation status of the 5' IRF6 CGI, which regulates the IRF6 gene expression level. This consideration led to the hypotheses that (i) IFN-β treatment enhanced IRF6 expression in the melanoma cells with an unmethylated 5' IRF6 CGI, with the result that IRF6-associated tumor suppressors, including maspin and IRF6-induced endogenous IFNs, inhibit the growth of melanoma cells with an unmethylated 5' IRF6 CGI, and (ii) IFN-β treatment cannot induce IRF6 expression in the melanoma cells with methylated 5' IRF6 CGI, with the result that IRF6-associated tumor suppressors do not inhibit the growth of melanoma cells with a methylated 5' IRF6 CGI. Therefore, methylation analysis of the 5' IRF6 CGI may be useful for the selection of IFN-β treatment-sensitive melanomas.
The in vitro data indicated that the melanoma cell lines with suppression of IRF6 exhibited a methylation level >68.5%, while the in vivo data indicated that the highest value of such methylation levels among melanocytic nevus samples was 7.2%, and that the methylation levels of 21 clinical melanoma samples ranged from 0.0% to 65.4%. The clinical melanoma samples included a considerable amount of tumor-surrounding tissues with normally low methylation levels. Therefore, clinical samples with a methylation level >7.2% should be regarded as having a highly methylated 5' IRF6 CGI, even though their methylation levels are ≤68.5%.
Statistical analyses indicated a significantly different distribution of the methylation levels of the 5' IRF6 CGI among the subtypes of melanoma (Kruskal-Wallis test, P = 0.035); the acral type tended to be higher than those of the other types. These results suggested that (i) Curtin's classification of melanoma may be associated with epigenetic abnormalities as well as genetic abnormalities [25], and (ii) IFN-β-resistant melanomas are more frequent in acral melanomas than in the other types of melanomas.
Bisulfite sequencing was performed to confirm the results of RT-MSP. The data were generally consistent with those of RT-MSP. This examination indicated that the CpG sites within the 5' IRF6 CGI were densely methylated in the clinical melanoma samples in which high methylation levels were detected by RT-MSP (e.g., sample #10 with a 64.2% methylation level and sample #17 with a 65.4% level). Conversely, this examination indicated that these CpG sites were sparsely methylated in the samples in which low methylation levels were detected by RT-MSP (e.g., MC2 cells with a 0.1% methylation level and samples #23 and #34 with a 0.0% level). These data suggested that RT-MSP is a reliable and reasonable procedure to use for determination of the methylation status of the 5' IRF6 CGI.
One limitation of this study is that only 21 advanced stage melanoma samples were analyzed. Our data derived from the statistical analyses should be confirmed in a larger number of melanoma samples. Another limitation of this study is that data regarding the IRF6 protein level were not provided. The association between the methylation status of the 5' IRF6 CGI and IRF6 protein expression should be concurrently evaluated.
In conclusion, IRF6 is aberrantly silenced due to DNA methylation of the CGI located in the 5' region of the IRF6 gene in melanomas, but not in melanocytic nevi. The methylation levels of 5' IRF6 CGI are potentially associated with the sensitivity of melanoma cells to IFN.
Supporting information
S1 Fig. Gene structure of the 5' region of IRF6.
The gray square indicates exon 1. The long vertical line indicates the transcriptional start site. Vertical short lines show individual CpG sites (top) and GpC sites (bottom). The regions analyzed using the methylated DNA-specific primer set and the unmethylated-DNA sequence-specific primer set in RT-MSP are shown as a black and a gray horizontal line, respectively. The region analyzed by bisulfite sequencing is shown as a double horizontal line.
https://doi.org/10.1371/journal.pone.0184444.s001
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