Background
Dual specificity phosphatases (DUSPs) are negative regulators of mitogen-activated protein kinases (MAPK) that regulate proliferative signaling pathways, which are often activated in cancer [
1‐
3]. DUSP2 encodes a dual-specificity phosphatase that inactivates ERK1/2 and p38 MAPK [
4,
5]. DUSP2 has also been found to regulate p53- and E2F1-regulated apoptosis [
6,
7]. Previously it has been reported that
DUSP2 expression is markedly reduced or completely absent in many human cancers [
8,
9].
Epigenetic silencing of tumor suppressor genes (TSG) is one of the most relevant molecular alteration that occurs during carcinogenesis [
10]. Promoter hypermethylation of TSG occurs in cancer through methylation at the DNA level at C5 of cytosine (5mC), when found as a dinucleotides with guanine. DNA methylation in CpG islands of TSG leads to epigenetic silencing of the according transcript [
11,
12]. The ten-eleven-translocation methylcytosine dioxygenases (TET1-3) catalyze the oxidation of 5mC and generate cytosine derivatives including 5-hydroxymethylcytosine (5hmC) [
13,
14]. TET proteins are involved in diverse biological processes, as the zygotic epigenetic reprogramming, hematopoiesis and the development of leukemia [
15‐
17]. The frequency of 5hmC suggests that these modified cytosine bases play an important role in epigenetic gene regulation [
18]. Aberrant levels of 5hmC have been reported in human cancer [
19,
20]. Recently it has been shown that TET proteins bind the CCCTC binding factor (CTCF) [
21]. CTCF is associated with altered expression of tumor suppressor genes, such as E-cadherin (CDH1), retinoblastoma 1, RASSF1A, CDKN2A/p16 and TP53 [
22‐
25]. It has also been postulated, that CTCF itself acts as a tumor suppressor [
26,
27].
Here we analyzed the epigenetic inactivation and regulation of the dual specificity phosphate 2 (DUSP2) in human cancers. Our data show that DUSP2 is aberrantly methylated in primary Merkel cell cancer and in different human cancer cell lines. Moreover, we observed that 5-Aza-dC and CTCF induce DUSP2 expression by its promoter demethylation.
Discussion
Previously it has been reported that
DUSP2 expression is downregulated in many human cancers [
8]. However the mechanism of its silencing was not analyzed in details. Deletion of the
DUSP2 locus at 2q11.2 is rather infrequent in cancer. Here we show that the promoter of
DUSP2 is hypermethylated in different human cancer cell lines including lung, breast and skin cancers and in HEK293 cells (Figs.
1 and
2). In primary Merkel cell cancer (MCC) we observed a significant tumor specific methylation of
DUSP2. MCC is one of the most aggressive cancers of the skin and we have reported frequent hypermethylation of the Ras Association Family Members RASSF1A and RASSF10 in this tumor entity [
28,
29]. Hypermethylation of DUSP2 and murine Dusp2 and has been reported in breast cancer cell lines, however methylation in primary human mammary tumors was absent [
50], which was also observed in our study (Additional file
3: Figure S1). By inhibiting DNA methyltransferases with the cytidine analogue 5-aza-dC we found that the
DUSP2 gene is epigenetically reactivated by its demethylation (Fig.
3). The promoter methylation of
DUSP2 in HEK293 consists of 5mC and 5hmC epigenetic marks (Fig.
5). Additionally, we have revealed that CTCF reactivates
DUSP2 and this is associated with demethylation of its CpG island promoter (Figs.
4 and
5b). CTCF is a DNA binding factor well known for its multiple functions in gene regulation. Depending on the participating genetic locus it is involved in transcriptional activation [
51‐
53], transcriptional repression [
54,
55] or enhancer blocking [
56]. Here we show increased binding of CTCF to the
DUSP2 promoter (Fig.
5a) and CTCF-dependent induction of the
DUSP2 promoter activity (Fig.
5c). Thus it will be interesting to analyze the exact mechanism of CTCF induced
DUSP2 expression and promoter dehydroyx-methylation in further details.
DUSP2 encodes a dual-specificity phosphatase that inactivates ERK1/2 and p38 MAPK [
4,
5]. DUSP2 has also been found to regulate p53- and E2F1-regulated apoptosis [
6,
7]. Dual-specificity phosphatases are negative regulators of the MAPK signal transduction, proliferative pathways that are often activated in cancers [
1]. Downregulation of
DUSP2 was detected in human acute leukemia coupled with activation of MEK and hyperexpression of ERK [
9]. Especially in acute myeloid leukemia, translocation and mutations of TET1 and TET2 gene are frequently observed [
57‐
59]. Moreover it has been reported that CTCF binds TET proteins [
21]. Thus it will be interesting to analyze if TET proteins are directly involved in the epigenetic regulation of
DUSP2. Here we observed increased binding of CTCF to its target site in the
DUSP2 promoter after CTCF induction (Fig.
5a). This binding may alter distinct TET- or DNMT-associated chromatin complexes at the
DUSP2 promoter region involved in CTCF-regulated DNA methylation as previously reported [
21,
25,
60,
61] and revealed in Fig.
5b. However CTCF-dependent regulation of
DUSP2 may also involve its function in chromosome configuration, chromatin insulation or transcriptional regulation [
62,
63].
We also observed that the CTCF paralogue CTCFL/BORIS was unable to reactivate
DUSP2 expression (Additional file
4: Figure S2). Since the cancer-testis specific BORIS is aberrantly expressed in cancer, an oncogenic role for BORIS has been proposed [
64,
65]. It was reported that CTCF, unlike BORIS, cannot bind to methylated binding sites [
66]. Therefore, it is interesting to note that the CTCF consensus site in the
DUSP2 promoter sequence lacks CpG sites (Fig.
1a) and ChIP data show an enhanced CTCF binding in CTCF induced TREx293 cells at this site (Fig.
5a). It was also postulated that CTCF itself acts as a tumor suppressor [
26,
27]. CTCF contains a N-terminal PARylation site [
49]. Here we observed that overexpression of CTCF lacking its PARylation site resulted in repression of
DUSP2 expression in H322 and HEK293 cells (Fig.
4d and e). This result suggests that the PARylation site of CTCF is important for its activating function. It has been reported that defective CTCF PARylation and dissociation from the molecular chaperone nucleolin occurs in
CDKN2A- and
CDH1-silenced cells, abrogating its TSG function [
23]. Using CTCF mutants, the requirement of PARylation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation has been demonstrated [
49]. In this model CTCF and Poly(ADP-ribose) polymerase 1 form functional complexes [
49]. Furthermore, CTCF contains two SUMOylation sites [
48]. Overexpresssion of the CTCF construct with mutated SUMOylation sites in the CTCF N-terminus or C-terminus resulted in a lack of
DUSP2 reactivation in the lung cancer H322 and HEK293 cells (Fig.
4d and e). SUMOylation of CTCF has been associated with its tumor suppressive function in
c-myc expression [
48]. There is also a report that CTCF SUMOylation modulates a CTCF domain, which activates transcription and decondenses chromatin [
67].
Competing interests
The authors declare that they have no competing interests. The work was supported by grants (TRR81, LOEWE) from the DFG and Land Hessen to RHD. These organizations had no involvement in the study design, acquisition, analysis, data interpretation, writing of the manuscript and in the decision to submit the manuscript for publication.
Authors’ contributions
RHD has created the study. TH and RHD participated in the design of the study. TH, AMR, APJ and MBS acquired data. TH, MBS, AMR, APJ and RHD controlled analyzed and interpreted data. RHD and TH prepared the manuscript. TH, MBS, AMR, APJ and RHD read, corrected and approved the final manuscript.