Autophagy is a mechanism adopted by cells during Keywords cancer; CpG methylation; DAPK; ERK–mitogen-activated protein kinase; post-translational regulation Correspondence A.. However, r
Trang 1Death-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 over-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 p19ARF⁄ 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 funcregula-tion 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.
Trang 2conditions 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 initiafunc-tion of neoplasmic transformafunc-tion
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 methycorre-lation 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
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.
Trang 3[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
Trang 4(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
Trang 5cancer (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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