MINIREVIEW
Death-associated proteinkinase(DAPK)and signal
transduction: regulationin 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 proteinkinase(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 proteinkinase 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 proteinkinase(DAPK) is a pro-apoptotic serine ⁄ threo-
nine proteinkinase 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 incancer 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-associatedprotein 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 regulationand metastasis are aberrantly
methylated incancer 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 incancer 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 andprotein 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 incancer 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 incancer 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 incancer 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, andin 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). In light of this, it is of fundamental
importance to gain a deeper understanding of the
mechanisms involved in DAPK regulation, as this may
assist rational drug design and suitable therapeutic reg-
imens to enable the outcome of cell fate decisions in
neoplastic cells to be tipped in favour of apoptosis.
Acknowledgements
The authors would like to thank Reginald Clayton for
his critical review of the manuscript, and gratefully
acknowledge financial support from the Medical
Research Council and Tenovus Scotland.
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. 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, . MINIREVIEW Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer Alison M. Michie, Alison M. 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