In this study, we show that the type IV DUSP, DUSP16, is subject to methylation-dependent transcriptional silencing in BL, resulting in the deregulation of JNK signalling. There are a large number of DUSP
genes in the human genome, but relatively little is known regarding their involvement in cancer. The type I DUSP, DUSP1, has been identified previously in studies using 5′AZA to pharmacologically reverse methylation, yet the CpG island was reported to be unmethylated (Rauhala et al, 2005
). Furthermore, DUSP6 has been reported to be a target for methylation-dependent silencing in pancreatic cancer (Xu et al, 2005
). A single previous study in prostate cancer has reported transcriptional downregulation of DUSP16 expression, but the mechanistic basis of downregulation was not established (Kibel et al, 2004
). The data we present are, therefore, to the best of our knowledge, the first demonstration of epigenetic silencing of a type IV DUSP
gene in human cancer. The ectopic expression of DUSP16 in fibroblasts transformed by BCR-ABL reduces their transforming activity in vivo
accompanied by downregulation of BCR-ABL-induced activation of JNK (Hoornaert et al, 2003
). These studies support the candidacy of DUSP16 as a tumour suppressor and are consistent with our observations of a role for DUSP16 in modulating the intensity and duration of JNK signalling.
Although DUSP16 is commonly methylated in BL cell lines and endemic, sporadic and human immunodeficiency virus-associated BL, methylation was almost never detected in EBV-immortalised LCLs and the gene was abundantly expressed in these cells, implying that methylation is specific to neoplasia. Moreover, we did not detect methylation of DUSP16
in any cancer except BL, including analysis of a panel of NHL cell lines and clinical cases of DLBCL, FL, MCL and marginal zone lymphoma. As such, these results suggest a clear specificity of DUSP16 methylation for BL. Furthermore, analysis of the breast and ovarian cancer cell lines and malignant melanoma cell lines also failed to detect methylation. Why is methylation-dependent silencing of DUSP16 so tightly restricted to BL and not observed in multiple other B lymphoma subtypes? DUSP16 is a direct transcriptional target of B lymphocyte-induced maturation protein, Blimp-1 (Magnusdottir et al, 2007
), suggesting that it may have a role in the normal developmental physiology of B lymphocytes. It remains to be determined, however, whether DUSP16 is downregulated in other cancers by genetic or other mechanisms. In this respect, it is noteworthy that the DUSP16
gene is located on chromosome 12p, an area of frequent chromosomal loss in human tumours. In leukemia, recent data suggests that deletions of 12p are a final event in mature cells (Wiemels et al, 2008
). In non-small cell lung cancer, 12p deletions are an early occurrence (Grepmeier et al, 2005
We previously reported that the Polo-like kinase Snk/Plk2
was subject to methylation-dependent transcriptional silencing at high frequency in BL (Syed et al, 2006
). Methylation of Snk/Plk2
was, however, also detected in multiple other B cell neoplasias, whereas methylation in DUSP16 seems to be tightly restricted to BL. We have sought to address the mechanistic basis favouring the selection of clones lacking DUSP16. Our results reveal that methylation of DUSP16
correlates closely with deregulation of JNK signalling. The activation of JNK signalling has been reported to promote either cell death (Wang et al, 2006
) or cell survival according to different studies, although the reasons for such differences in effect are not known. The data we present show that JNK activation, at least in the context of BL, promotes apoptosis and cytotoxicity in cells challenged with cytotoxic and/or oxidative stress, as ectopic expression of DUSP16 reduced activation of JNK and this correlated with reduced sensitivity to doxorubicin, sorbitol and cisplatin. One implication of the association of JNK activation with cell death is that loss of JNK activation may represent a mechanism of clinical drug resistance. Evidence in acute myeloid leukemia cell lines to support this possibility has recently been published (Lagadinou et al, 2008
). It would be of obvious interest to analyse the expression and epigenetic status of DUSP16 in relapsed BL (at which time there are few active chemotherapeutic options) to determine whether changes in methylation in the DUSP16 CpG island occur with acquisition of clinical drug resistance.
Promotion of apoptosis by activation of JNK has been shown in some systems to result from enhanced degradation of the transcriptional co-repressor CtBP (Wang et al, 2006
). We tested whether degradation of CtBP was increased in BL with DUSP16 methylation, but we observed no evidence to support this hypothesis, CtBP degradation being similar irrespective of DUSP16 status and unaffected by treatment with the proteasome inhibitor MG132.
In conclusion, we have identified DUSP16 as a new epigenetically regulated gene inactivation of which seems specific to BL. We present evidence that expression of DUSP16 expression selectively modulates the activation of JNK and influences cellular sensitivity to chemotherapeutic agents.