Silencing of interferon regulatory factor gene 6 in melanoma

RESEARCH ARTICLE Silencing of interferon regulatory factor gene 6 in melanoma Yoshimasa Nobeyama*, Hidemi Nakagawa Department of Dermatology, The Jikei University School of Medicine, Minato-ku, Tokyo, Japan * Abstract Background a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 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 OPEN ACCESS 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 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. Received: July 14, 2017 Results Accepted: August 23, 2017 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. 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 Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Conclusion IRF6 is aberrantly silenced by DNA methylation of the 5’ IRF6 CGI in melanoma. The methylation status of IRF6 is potentially associated with the sensitivity of melanoma to interferon. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0184444 September 6, 2017 1 / 14 Silencing of IRF6 in melanoma 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-lymphocyteassociated 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 PLOS ONE | https://doi.org/10.1371/journal.pone.0184444 September 6, 2017 2 / 14 Silencing of IRF6 in melanoma donors or their legal guardians. The ethics committee of The Jikei University School of Medicine waived the requirement for consen (...truncated)