Biochemical and Biophysical Research Communications
Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells
Highlights
► Elafin expression is epigenetically silenced in human melanoma cells. ► Foxa2 expression in melanoma cells is silenced by promoter hypermethylation. ► Foxa2 directs activation of the elafin promoter in vivo. ► Foxa2 expression induces apoptosis of melanoma cells via elafin re-expression.
Introduction
Elafin, also known as skin-derived antileukoproteinase (SKALP) or peptidase inhibitor 3 (PI3), is an inhibitor targeting serine proteases, including human neutrophil elastase, with documented anti-microbial and anti-inflammatory functions [1], [2]. Initial studies have associated elafin with inflammation, and subsequent investigations have shown differential expression of elafin in many human malignancies [3] and association of its expression with overall survival in some cancer types [3], [4], [5]. We have recently shown that elafin induces apoptosis in human melanoma cells through the p53-dependent intrinsic pathway, and expression of the protein is repressed in human melanoma tissue specimens and cell lines, but not normal melanocytes, suggesting that silencing of elafin expression in melanoma contributes to progression of the disease [6]. However, the underlying mechanism of elafin suppression in melanoma remains to be established.
Chromosomal aberrations in the region encompassing the elafin gene, as exemplified by amplification in ovarian and breast cancers [7], [8] and deletion in oral squamous cell carcinoma [9], may confer cancer- or tissue-specific expression patterns. High-degree polymorphism of the elafin promoter sequence [10] may promote the differential binding of transcription factors in various tissues. At the molecular level, a number of transcription factors play roles in elafin expression, implying complex transcriptional regulation of the elafin promoter. Alterations in the activating/inactivating ratio of the CCAAT/enhancer binding protein β (C/EBPβ) isoforms, leading to their differential availability for binding to the elafin promoter, confer variations in elafin expression patterns between normal and breast cancer cells [11]. Elafin expression is activated by inflammatory cytokines through binding of nuclear factor-κB (NF-κB) to its promoter region in ovarian cancer cells [5] and pulmonary epithelial cells [12]. Induction of Ap1 in breast cancer cells leads to transactivation of the elafin gene through the Ap1 site in the promoter [13]. A quantitative chromatin immunoprecipitation screening identified the elafin promoter as a high-affinity target for c-Myc [14].
In the majority of human cancers, aberrant epigenetic regulation is associated with altered transcription of genes critical for tumor initiation and progression, and altered DNA methylation is a key component of the cancer epigenome [15]. As a member of the forkhead box containing transcription factor family, Foxa2 has been shown to play crucial roles in embryonic development and epithelial differentiation [16]. DNA methylation of the Foxa2 promoter has been reported in several human cancer types including pancreatic, thyroid, lung and breast cancers [17], [18], [19], [20].
We show here that elafin expression is indirectly silenced in melanoma cells by methylation-mediated repression of Foxa2, and that Foxa2 induces cellular apoptosis by directing elafin expression.
Section snippets
Cell lines and materials
Human melanoma cell lines and normal human epidermal melanocytes (NHEM) were described previously [6]. To construct the Foxa2-expressing plasmid pcDNA-Foxa2, the coding region of Foxa2 gene was amplified by PCR using primers 5′-ACTACGAATTCATGCTGGGAGCGGTGAAGATGG-3′ and 5′-ACTACGGATCCTCAATGGTGATGGTGATGA-3′ and cloned into the EcoRI and XbaI sites of pcDNA3.1(+)-Myc/His (Invitrogen, Carlsbad, CA). The Foxa2-expressing lentiviral vector pLenti-Foxa2 was constructed by subcloning the Foxa2 gene
Elafin expression is epigenetically silenced in human melanoma cells
Previously, we showed that elafin is transcriptionally repressed in human melanoma tissues and cell lines [6]. To understand the underlying mechanism of the repression, we explored the possibility of epigenetic regulation of elafin expression, which was prompted by the earlier finding that elafin is upregulated in bladder cancer cells after 5-Aza-CdR treatment [21]. Quantitative real-time RT-PCR and Western analyses revealed that treatment of melanoma cell lines (A375, SKMEL5 and WM2664) with
Discussion
We provide here lines of evidence indicating that silencing of elafin expression in melanoma cells occurs as a result of methylation-mediated suppression of the transcription factor Foxa2. Our findings support a role of Foxa2 as a tumor suppressor in melanoma, consistent with previous reports on Foxa2 functions in other cancer types. In thyroid cancer cells, Foxa2 expression is silenced due to promoter methylation, and forced expression of Foxa2 leads to suppression of cell growth [18].
Conflict of interest
All the authors declare no conflict of interest.
Acknowledgments
This work was supported by the Research Program for New Drug Target Discovery Grant (20090083331) from the Ministry of Education, Science and Technology, Republic of Korea. We thank Hye Jin Min and Eun Hye Park for helpful discussion and technical assistance.
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