MINIREVIEW Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer Alison M. Michie, Alison M. McCaig, Rinako Nakagawa and Milica Vukovic Section of Experimental Haematology, Division of Cancer Sciences, Faculty of Medicine, University of Glasgow, UK Introduction Death-associated protein kinase (DAPK) is a cal- cium ⁄ calmodulin-regulated serine ⁄ threonine protein kinase, located on chromosome 9q21.33, which is com- posed of several functional domains, including a kinase domain, an ankyrin repeat domain and a death domain [1,2]. DAPK was first identified as a mediator of interferon-c-mediated apoptosis [3,4]. Subsequently, DAPK has been found to participate in a number of additional apoptosis-inducing pathways downstream of CD95 (Fas), tumour necrosis factor-a and transform- ing growth factor-b [5,6]. The death domain regulates the pro-apoptotic function of DAPK in part by interacting with netrin-1 receptor UNC5H2 [7], the mitogen-activated protein kinase extracellular signal- regulated kinase (ERK) [8] and members of the tumour necrosis superfamily TNFR1 and FADD [5,9]. Activation of p53, initiated by DNA-damaging agents or oncogene expression, leads to an elevation in DAPK expression [10]. Additionally, DAPK over- expression upregulates p53 expression, suggesting that an autoregulatory feedback loop exists between DAPK and p53 that controls apoptosis [4,10,11]. In support of this, DAPK inactivation reduces the induction of p19 ARF ⁄ p53, thus inactivating the p53-dependent path- way for apoptosis [11]. These findings suggest that attenuation of p53 by loss of DAPK may be an impor- tant factor in transformation in vivo. As well as regulating apoptosis, DAPK has been shown to be involved in the control of autophagy [12]. Autophagy is a mechanism adopted by cells during Keywords cancer; CpG methylation; DAPK; ERK–mitogen-activated protein kinase; post-translational regulation Correspondence A. M. Michie, Section of Experimental Haematology, Division of Cancer Sciences, Faculty of Medicine, University of Glasgow, Paul O’Gorman Leukaemia Research Centre, Gartnavel General Hospital, 21 Shelley Road, Glasgow G12 0XB, UK Fax/Tel: +44 141 301 7898 E-mail: A.Michie@udcf.gla.ac.uk (Received 11 March 2009, revised 28 May 2009, accepted 17 June 2009) doi:10.1111/j.1742-4658.2009.07414.x Death-associated protein kinase (DAPK) is a pro-apoptotic serine ⁄ threo- nine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regula- tion or function of DAPK. Abbreviations CLL, chronic lymphocytic leukaemia; DAPK, death-associated protein kinase; Dnmt, DNA methyltransferase; ERK, extracellular signal-regulated kinase; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma. 74 FEBS Journal 277 (2010) 74–80 ª 2009 The Authors Journal compilation ª 2009 FEBS conditions of stress to maintain cellular homeostasis, by catabolizing cellular components to provide emergency nutrients [13]. Under these conditions, inhibition of autophagy can lead to apoptosis, suggesting that a potential outcome of autophagy is survival [14]. Inter- estingly, studies in Caenorhabditis elegans demonstrated that starvation-induced autophagy is regulated in part through a DAPK signalling pathway and that DAPK levels are critical for modulating cell fate decisions that lead to survival or death, highlighting the capacity of DAPK to integrate signals from apoptotic and auto- phagic pathways ([15–18]). These findings have impor- tant implications during cellular transformation when DAPK expression levels are reduced, as this could con- vert a death-inducing signal (when DAPK expression is high in nonmalignant cells) into a pro-survival signal (Fig. 1). Therefore, it is critical to delineate the mecha- nisms utilized by cancer cells to subvert DAPK func- tion during the initiation of neoplasmic transformation. Regulation of DAPK function The attenuation of DAPK function in a number of cancers has mainly been attributed to hypermethylation of the DAPK promoter region [1]. However, the corre- lation between the levels of methylation observed and the extent of DAPK repression either at the transcript or protein level is not always consistent, suggesting that additional regulatory mechanisms may be responsible for reducing DAPK function within specific cancers. Epigenetic regulation of DAPK Deregulation of epigenetic control of the gene expres- sion is heavily implicated in the development and pro- gression of cancer. The aberrant mechanisms include increasing methylation of DNA, deacetylation of core histone proteins and RNA interference. Methylation of CpG islands in the promoter regions of genes, by addi- tion of a methyl group to the cytosine ring to form methylcytosine, is a mechanism utilized by cells to selectively silence genes. A number of distinct genes involved in the regulation of apoptosis, DNA repair, cell cycle regulation and metastasis are aberrantly methylated in cancer cells, resulting in their transcrip- tional repression [19]. Indeed, a plethora of publica- tions have reported that DAPK promoter regions are hypermethylated in a wide range of cancer cells com- pared with normal tissues, including lung, bladder, head and neck, kidney, breast and B cell malignancies [1,20–24]. In most cases this results in attenuated expression and, therefore, reduced function of DAPK. In the majority of cell lines assessed, DAPK promoter hypermethylation could be reversed upon treatment with DNA methyltransferase (Dnmt) inhibitors, result- ing in re-expression of DAPK [25,26]. Moreover, DAPK repression was also alleviated upon treatment of the cell lines with histone deacetylase inhibitors, although in fewer cases, suggesting that dysregulation of histone acetylation also has a role in DAPK repres- sion [25,26]. Defining the methylation levels of selected genes may permit a prediction of the clinical outcome for specific cancers. Indeed, restoration of DAPK expression in lung carcinoma cells was shown to suppress their meta- static potential in vivo [4]. Moreover, there appears to be an association between DAPK promoter hyper- methylation and the metastatic potential of particular cancers, such as head and neck tumours, non-small cell lung cancer (NSCLC) and pancreatic adencarcinoma Nonmalignant cell Malignant cell Unmethylated DAPK promoter Hypermethylated DAPK promoter ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG Transcription of DAPK mRNA Reduced transcription of DAPK mRNA Apoptosis Survival metastasis Transcription Translation P DAPK DAPK inhibition DAPK activation DAPK gene deletion DAPK promoter point mutations Autophagy Src kinase activation RSK activation p53 inactivation CD95, TGF IFN , TNF p53 or ERK activation Fig. 1. Mechanisms utilized by cancer cells to evade DAPK-medi- ated cell death. DAPK function can be reduced at multiple levels. Reduced expression of DAPK at the mRNA level can occur because of: (a) aberrant hypermethylation of the DAPK promoter (dark stars – methylated CpG islands) in a malignant cell can result in a reduction in gene transcription compared with reduced ⁄ absent methylation (white stars) in nonmalignant cells; (b) deletions of the DAPK gene; (c) point mutations in the promoter region of DAPK. Additional mechanisms exist to modulate DAPK activity at the pro- tein level via site-specific phosphorylation of DAPK by upstream kinases, which may play a central role in cellular transformation: abrogation of DAPK activity resulting in increased cell survival and metastasis. A. M. Michie et al. Regulation of DAPK in cancer FEBS Journal 277 (2010) 74–80 ª 2009 The Authors Journal compilation ª 2009 FEBS 75 [27–29]. Collectively, these studies suggest that a corre- lation exists between the invasive and metastatic poten- tial of tumours and the methylation status of DAPK, and that methylation of DAPK may represent a bio- marker of prognostic significance for selected cancers. An assessment of hypermethylation in primary cancer cells and cell lines has revealed that DAPK is not glob- ally methylated in all cancers. Indeed, reports indicate that the promoter of DAPK is unmethylated in T cell malignancies such as T acute lymphoblastic leukaemia compared with significantly higher levels of methylation observed in B cell malignancies [23,24]. Interestingly, a study assessing the level of DAPK methylation in nor- mal peripheral blood lymphocytes revealed that periph- eral IgM ) B lymphocytes exhibited a higher level of methylation at the DAPK promoter than IgM + B lym- phocytes or T cells, monocytes or neutrophils [30]. These findings demonstrate that DAPK is differentially methylated in distinct healthy cell types, and suggest that certain cells may be more predisposed to exhibit hypermethylation of DAPK as a feature of malignancy. Although the mechanism responsible for hyperme- thylation of the DAPK promoter in cancer cells has not been elucidated, a recent study demonstrated that Daxx, a modulator of apoptosis, represses DAPK in an NF-jB-dependent manner. This repression was mediated through the interaction of Daxx with Dnmt family members, and subsequent recruitment of Dnmt to RelB (an NF-jB family member) target promoters. This in turn increased the methylation of RelB target genes, such as DAPK. DAPK repression was alleviated by treating the cells with the Dnmt inhibitor 5-azacity- dine [31]. This study may have wider implications in the field of cancer biology, particularly in light of the fact that a number of cancers are reported to exhibit constitutively active NF-jB [32,33]. Thus, this may provide a mechanism for epigenetic changes in tumour suppressor gene promoters. Germline mutations/polymorphisms in DAPK Although hypermethylation of the DAPK promoter elicits gene silencing in the majority of cases, there are instances when DAPK expression is evident in the pres- ence of hypermethylation, or DAPK expression is reduced in the absence of methylation. These findings indicate that additional mechanisms inactivate DAPK function. A limited number of reports describe homozy- gous deletions, allelic deletions and point mutations resulting in the attenuation of DAPK expression. Indeed, a homozygous deletion was uncovered in the CpG region of the DAPK promoter in a small number of pituitary adenomas, due to a lack of correlation between methylation levels and protein expression [34]. Additionally, allelic loss in the region of the DAPK gene in 50–55% of NSCLC cell lines has been reported [35]. Chronic lymphocytic leukaemia (CLL) is a clonal expansion of B lineage cells that behaves heteroge- neously in patient cohorts, as some patients survive over a decade with stable disease requiring little or no treatment, whereas in others the leukaemia behaves aggressively, with survival measured in months despite treatment. However, CLL cell clones display a restricted usage of immunoglobulin heavy chain vari- able regions, and share common gene expression pat- terns consistent with antigen-experienced B cells [36], suggesting that CLL may arise as the result of a single genetic event. In support of this, Raval et al. [24] recently reported that downregulation of DAPK gene expression correlated with both familial and sporadic CLL in the majority of cases, suggesting that DAPK may behave as a tumour suppressor gene in CLL. In sporadic CLL cases, downmodulation of DAPK expression was ascribed to promoter methylation in the majority of cases assessed. However, the authors also analysed the DAPK gene in an extended family in which members had been diagnosed with CLL. Inter- estingly, a disease haplotype was defined in the pro- moter of DAPK, which was present only in family members affected with CLL and resulted in signifi- cantly reduced DAPK expression. Furthermore, hyper- methylation of the DAPK promoter was found in the CLL cells of affected family members, further reducing the expression of DAPK. The ‘CLL haplotype’ resulted in an increased binding of the transcription factor HOXB7 to the DAPK promoter, which in turn repressed DAPK expression [24]. Of note, additional cohorts of familial CLL cases did not possess this par- ticular CLL haplotype, suggesting that additional hapl- otypes may be uncovered that modulate DAPK function in CLL. Interestingly, a relatively high pene- trant germline polymorphism in the death domain of DAPK (N1347S) has been identified that can attenuate ERK-dependent apoptosis [37], perhaps by shifting the equilibrium of DAPK signalling to allow the mutant DAPK to drive autophagic ⁄ survival signalling. This polymorphism has not, as yet, been associated with a specific disease model. Collectively, these findings sug- gest that although mutations in the DAPK gene are quite rare, further analysis is justified. Post-translational regulation Recent studies indicate that DAPK protein expression can be detected in lung and renal cell carcinoma Regulation of DAPK in cancer A. M. Michie et al. 76 FEBS Journal 277 (2010) 74–80 ª 2009 The Authors Journal compilation ª 2009 FEBS (RCC), suggesting that additional, post-translational mechanisms exist to hinder DAPK function [26,38,39]. Indeed, studies in primary NSCLC tissue and cell lines established that although expression of DAPK was reduced compared with normal lung tissue, DAPK expression was observed in the presence of significant hypermethylation. Moreover, Toyooka et al. [26] defined a subset of cell lines in which DAPK expres- sion was reduced in the absence of hypermethylation, possibly due to the existence of a germline deletion in DAPK, as noted above [34,35]. These findings suggest that expression levels of DAPK can only be partially related to the level of hypermethylation and that addi- tional, as yet undefined, mechanisms exist. In support of this study, Wethkamp et al. [38] noted that DAPK was expressed at the protein level in the majority of RCCs in vivo. However, further analysis of RCC cell lines revealed that DAPK protein was inactive, sug- gesting that the kinase activity of DAPK was inacti- vated in cancer cells [38]. Our own studies assessing protein expression of DAPK in freshly isolated CLL cells demonstrated that, although reduced in a cohort of sporadic CLL patient samples compared with nor- mal mature B lymphocytes, DAPK is clearly detectable (A. M. Michie and T.R. Hupp, unpublished observa- tions). These data are distinct from published data indicating that DAPK expression is low or absent in CLL cells [24]. An interesting mechanism for the inactivation of DAPK activity has been recently described. Wang et al. [40] demonstrated that DAPK is a substrate for leukocyte common antigen-related tyrosine phospha- tase and that dephosphorylation of Y491 ⁄ Y492, located in the ankyrin repeat domain, resulted in acti- vation of the pro-apoptotic activities of DAPK. Recip- rocally, phosphorylation of Y491 ⁄ Y492 by Src kinase inhibited DAPK activity. Indeed, in response to epidermal growth factor stimulation, Src kinase was activated and leukocyte common antigen-related was decreased, resulting in DAPK inactivation [40]. A number of cancer types are known to possess either an overexpression of or constitutively active Src kinase activity, including colon, NSCLC, pancreatic and breast cancers [41,42]. Moreover, a positive correlation was found between DAPK hyperphosphorylation and raised Src kinase activity in colon cancer cell lines and primary tissue [40], suggesting that this may represent a novel mechanism for the inactivation of DAPK activity in additional cancer models. As mentioned previously, ERK activation has been shown to lead to an increase in DAPK activity, due to phosphorylation at S735 [8]. Interestingly, DAPK activity has also been shown to be negatively regulated in both healthy and Ras⁄ Raf- transformed cells, through p90 ribosomal S6 kinase-mediated phosphory- lation at S289, upon activation of the Ras-ERK signal- ling pathway [43]. However, this modification has not been shown to contribute to tumorigenesis. These studies illustrate that differential regulation of the ERK-mediated signalling pathway leads to divergent effects on DAPK activity, and subsequently cell fate decisions. Potential therapeutic avenues The reversibility of the deregulated epigenetic modifi- cations in cancer cells represents an interesting thera- peutic avenue for promoting re-expression of silenced genes [24–26]. Although such a therapeutic strategy inhibits methylation and deacetylation globally and cannot be gene specific, there has been some success with these inhibitors, particularly in haematological malignancies [44,45]. Clinical trials assessing the treat- ment of leukaemia and myelodysplastic syndrome patients with decitabine (5-aza-2¢-deoxycytidine) revealed an antineoplastic activity that correlated with changes in gene expression [46]. Conversely, studies indicate that Dnmt inhibitors in monotherapy have little clinical benefit in the treatment of solid tumours and are now being tested in combination therapies [47]. Tyrosine kinase inhibitors are widely and success- fully used in the treatment of cancer, and in the context of this review, may inhibit tumour growth⁄ metastasis or target tumour cells for apoptosis by activating DAPK activity [40]. Indeed, bosutinib, an investigational Src kinase inhibitor, has been shown to inhibit the migration and invasion of breast cancer cells [48]. In addition, dasatinib, a dual Src ⁄ Abl kinase inhibitor that has been shown to have a potent antitu- mour activity in chronic myeloid leukaemia, can inhi- bit the invasion of head and neck, melanoma and lung cancer cell lines [49,50]. In light of these reports, DAPK may represent an important downstream target of Src kinase inhibitors, by reactivating the DAPK- dependent pro-apoptotic signalling pathways. Conclusion Recent publications indicate that DAPK functions as a molecular rheostat, responsible for regulating cell fate decisions. Indeed, the critical nature of DAPK in regu- lating apoptosis under normal conditions is highlighted by the findings that malignant cells employ multiple mechanisms to abolish DAPK function, thus creating pro-survival conditions and promoting the initiation of A. M. Michie et al. Regulation of DAPK in cancer FEBS Journal 277 (2010) 74–80 ª 2009 The Authors Journal compilation ª 2009 FEBS 77 cancer (Fig. 1). 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McCaig, Rinako Nakagawa and Milica Vukovic Section. 2009) doi:10.1111/j.1742-4658.2009.07414.x Death-associated protein kinase (DAPK) is a pro-apoptotic serine ⁄ threo- nine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which