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© The Authors Journal compilation © 2010 Biochemical Society
Essays Biochem. (2010) 48, 121–146; doi:10.1042/BSE0480121
9
Epigenetic regulation of cell
life and death decisions and
deregulation in cancer
Nabil Hajji*1 and Bertrand Joseph†
*Department of Experimental Medicine and Toxicology, Division
of Investigative Science, Imperial College, London, U.K., and
†Department of Oncology Pathology, Cancer Centrum Karolinska,
Karolinska Institutet, Stockholm, Sweden
Abstract
For every cell, there is a time to live and a time to die. It is apparent that
cell life and death decisions are taken by individual cells based on their
interpretation of physiological or non-physiological stimuli, or their own
self-assessment of internal damage or changes in their environment. Apoptosis
or programmed cell death is a key regulator of physiological growth control
and regulation of tissue homoeostasis. One of the most important advances
in cancer research in recent years is the recognition that cell death, mostly
by apoptosis, is crucially involved in the regulation of tumour formation and
also critically determines treatment response. The initiation and progression
of cancer, traditionally seen as a genetic disease, is now realized to involve
epigenetic abnormalities along with genetic alterations. The study of epigenetic
mechanisms in cancer, such as DNA methylation, histone modifications and
microRNA expression, has revealed a plethora of events that contribute to the
neoplastic phenotype through stable changes in the expression of genes critical
to cell death pathways. A better understanding of the epigenetic molecular
events that regulate apoptosis, together with the reversible nature of epigenetic
1To
whom correspondence should be addressed (email [email protected]).
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aberrations, should contribute to the emergence of the promising field of
epigenetic therapy.
Introduction
Cell life and death decisions are taken by individual cells based on their
interpretation of physiological or non-physiological stimuli, or their own
self-assessment of internal damage or changes in their environment. Cells that
represent a threat to the organism integrity, such as damaged, infected or aged
cells, are eliminated by a process called PCD (programmed cell death). Thus
PCD is a biological phenomenon which is of fundamental importance to the
integrity of organisms.
PCD
Apoptosis or PCD is a key regulator of physiological growth control and
regulation of tissue homoeostasis. One of the most important advances in
cancer research in recent years is the recognition that cell death, mostly by
apoptosis, is crucially involved in the regulation of tumour formation and
also critically determines treatment response. Killing of tumour cells by
most anticancer strategies currently used in clinical oncology, for example,
chemotherapy, γ-irradiation, suicide gene therapy or immunotherapy, has
been linked to activation of apoptosis signal-transduction pathways in cancer
cells, such as the intrinsic and/or extrinsic pathway. Thus failure to undergo
apoptosis may result in treatment resistance [1]. Understanding the molecular
events that regulate apoptosis in response to anticancer chemotherapy, and
how cancer cells evade apoptotic death, provides novel opportunities for a
more rational approach to develop molecular-targeted therapies for combating
cancer.
The intrinsic pathway
The intrinsic pathway is initiated from within the cell. This is usually in
response to cellular signals resulting from DNA damage, a defective cell
cycle, detachment from the extracellular matrix, hypoxia, loss of cell survival
factors or other types of severe cell stress. The intrinsic pathway, also
known as the mitochondrial pathway, is activated by stimuli that lead to
MOMP (mitochondrial outer membrane permeabilization) and the release of
pro-apoptotic proteins from the mitochondrial IMS (intermembrane space)
[2]. Among these factors, there are several proteins that initiate or regulate
caspase activation, including cytochrome c. In the cytosol, cytochrome c binds
the adaptor Apaf-1 (apoptotic protease-activating factor 1), and in the presence
of dATP it forms a large multiprotein structure known as the apoptosome
[3]. The apoptosome creates a platform that brings together molecules
of the initiator caspase of the intrinsic pathway caspase 9 enabling their
autoactivation, which in turn activates the downstream effector caspases 3, 6
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and/or 7. In addition to cytochrome c, several additional factors released upon
MOMP have been implicated in apoptosis.
The intrinsic apoptotic pathway hinges on the balance of activity between
pro- and anti-apoptotic members of the Bcl-2 (B-cell lymphoma protein-2)
superfamily of proteins, which act to regulate the permeability of the mitochondrial membrane [4,5]. Members of the Bcl-2 family contain signature domains
of homology called BH (Bcl-2 homology) domains (termed BH1, BH2, BH3
and BH4) and can be classified into three main subclasses. Pro-apoptotic Bcl-2
proteins are divided into two subgroups based on the number of BH domains
they contain. There are those with several BH domains (e.g. Bax and Bak),
and then there are those that only have the BH3 domain, such as Bid, Bad,
Bim, Bmf, PUMA and NOXA [6]. BH3-only proteins activate the multi-BH
domain pro-apoptotic proteins Bax and/or Bak. Their activation is believed to
lead to MOMP through a conformational change, inducing their oligomerization and insertion into the outer mitochondrial membrane, where they form
large pores through which proteins from the IMS can access the cytosol [7].
The anti-apoptotic Bcl-2 proteins Bcl2, Bcl-XL, Mcl-1, A1 and Bcl-W, characterized by the presence of all four BH1–4 domains, act to prevent permeabilization of the outer mitochondrial membrane by inhibiting the action of the
pro-apoptotic Bcl-2 proteins Bax and/or Bak. It is worth noting that several
Bcl-2 family members which facilitate mitochondrial permeabilization are transcriptional targets of the p53 tumour suppressor gene, providing a partial explanation for the ability of DNA-damaging agents to induce apoptosis [8].
The extrinsic pathway
The extrinsic pathway begins outside the cell through the activation of
cell-surface-specific cell surface transmembrane receptors (so-called death
receptors). The extrinsic pathway is initiated by pro-apoptotic ligands,
which belong to the TNF (tumour necrosis factor) family. These ligands
include FasL (Fas ligand, also known as CD95L) and TRAIL (TNF-related
apoptosis-inducing ligand/Apo2L) which bind their cognate receptors CD95/
Fas and DR4/DR5 respectively [9]. Engagement of the transmembrane death
receptor by an extracellular death ligand induces receptor clustering and
recruitment of the adaptor protein FADD (Fas-associated death domain) and
the initiator caspases 8 or 10 as pro-caspases, forming a DISC (death-inducing
signalling complex) [10,11]. Formation of the DISC creates a platform that
brings pro-caspase molecules into close proximity to one another, facilitating
their autocatalytic processing and release into the cytoplasm where they
activate effector caspases 3, 6 and/or 7, thereby converging on the intrinsic
pathway. DISC formation is modulated by several inhibitory mechanisms,
including c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like
IL (interleukin)-1β-converting enzyme]-inhibitory protein}, which exerts
its effects on the DISC by interacting with FADD to block initiator caspase
activation; and decoy receptors, which can block ligand binding or directly
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abrogate pro-apoptotic receptor stimulation [12]. It has been shown that
activation of the extrinsic pathway can result in two apoptotic programmes,
termed type I and type II. In type I cells, adequate amounts of active caspase 8
are generated at the DISC to directly activate the effector caspases and execute
cell death [13]. Conversely, in the case of type II cells, which produce minimal
amounts of active caspase 8 at the DISC, an intermediate amplification of the
signal involves the BH3-only Bcl2 family protein Bid. The cleavage of Bid
by caspase 8 results in its translocation to the mitochondria where it activates
pro-apoptotic proteins Bax and Bak and initiates the release of mitochondrial
factors, which in turn augment cell death.
Although apoptosis pathways and signal-transducing molecules have
been shown to play a crucial role in the killing of tumour cells in response to
cytotoxic agents used in the treatment of cancer patients, the significance of
apoptosis as the main mechanism of cancer cell death in response to anticancer chemotherapy has also been challenged. In addition to apoptosis, cancer
cells have also been shown to be effectively eliminated after DNA damage by
necrosis, mitotic catastrophe and autophagic cell death [14].
Epigenetics and PCD
Classical genetics alone cannot explain the diversity of phenotypes within a
population. Nor does classical genetics explain how, despite their identical
DNA sequences, monozygotic twins [15] or cloned animals [16] can have
different phenotypes and different susceptibilities to a disease. The concept of
epigenetics offers a partial explanation of these phenomena. First introduced
by C.H. Waddington in 1939 to name “the causal interactions between genes
and their products, which bring the phenotype into being” [17], epigenetics
was later defined as heritable changes in gene expression that are not due to
any alteration in the DNA sequence [18]. Epigenetic mechanisms are essential
for normal development and maintenance of tissue-specific gene expression
patterns in mammals. Disruption of epigenetic processes can lead to altered
gene function and malignant cellular transformation. Global changes in the
epigenetic landscape are a hallmark of cancer. The initiation and progression
of cancer, traditionally seen as a genetic disease, is now realized to involve
epigenetic abnormalities along with genetic alterations. The study of epigenetic
mechanisms in cancer, such as DNA methylation, histone modifications,
nucleosome positioning and miRNA (microRNA) expression, has revealed a
plethora of events that contribute to the neoplastic phenotype through stable
changes in the expression of genes critical to cell-death pathways.
DNA methylation
Mechanism of action
DNA methylation is a covalent modification formed by the addition of
a methyl group at the 5´ carbon of cytosine in the sequence context CpG
dinucleotides of the DNA molecule. The reaction is catalysed by a family of
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enzymes called DNMTs (DNA methyltransferases) which catalyse the transfer
of a methyl group from the methyl donor SAM (S-adenosyl-L-methionine).
Three active DNMTs, DNMT1, DNMT3A and DNMT3B, have been
identified in mammals. A fourth enzyme previously known as DNMT2
is not a DNMT but is a protein that is closely related to DNMT3A and
DNMT3B structurally and that is critical for DNA methylation, but
appears to be inactive on its own. The modification is generally repressive
to transcription and helps to maintain silencing of transposable elements,
therefore enhancing genome stability. A number of transcription factors
recognize sequences that contain CpG residues [e.g. AP-2, c-Myc/Myn, the
CREB (cAMP-response-element-binding protein), E2F and NF-κB (nuclear
factor κB)], and their binding has been shown to be inhibited by methylation.
DNA methylation attracts specific MBDs (methyl-DNA binding proteins)
(MeCP2, MBD1, MBD2, MBD3, MBD4 and KAISO) which seem to act as a
molecular switch to turn off the expression of genes. Most CpG sequences in
the genome are methylated. However, there are ‘CpG islands’ associated with
gene promoters that escape DNA methylation.
DNA methylation and cancer
A link between DNA methylation and cancer was first demonstrated in 1983,
when it was shown that the genomes of cancer cells are hypomethylated
relative to their normal counterparts [19]. Hypomethylation in tumour cells
is primarily due to the loss of methylation from repetitive regions of the
genome. Global demethylation early in tumorigenesis might predispose cells
to genomic instability and further genetic changes. Aberrant hypermethylation
in cancer usually occurs at CpG islands, and the resulting changes in chromatin
structure effectively silence transcription. Cancer cells usually acquire aberrant
methylation of multiple tumour-related genes that co-operate to confer
survival advantage on neoplastic cells [20]. Therefore DNA-methylationmediated down-regulation of genes involved in cell death could be a significant
mechanism through which tumour cells avoid cell death.
DNA methylation and apoptosis
Evidence gained from studies in different cancers have shown that alteration
of pro- and anti-apoptotic genes by DNA methylation affects all aspect of
apoptosis right from its initiation to its execution (Table 1). DNA methylation
has been shown to contribute to inactivation of both the intrinsic and the
extrinsic apoptotic pathways (Figure 1). DNA hypermethylation is one
mechanism that contributes to the down-regulation of Fas expression which
could afford cells protection from FasL-induced apoptosis and thereby
aid the emergence of a tumorigenic clone during progression from normal
colonic epithelium to adenocarcinoma [21]. DR4 and DR5 are the receptors
for TRAIL and binding of TRAIL to the cognate receptors induces
caspase-dependent apoptosis. In addition, two decoy receptors for TRAIL
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Acute leukaemia
Neuroblastoma and other tumour types
Medulloblastoma tumour
Neuroblastoma and other tumour types
Neuroblastoma and other tumour types
Bladder cancer
Colon carcinoma
B-cell lymphoma cell lines
Paediatric tumours and cell lines
Paediatric tumours and cell lines
Promoter hypermethylation
Promoter hypermethylation
Aberrant H3 and H4 acetylation at
promoter
Promoter hypermethylation
Promoter hypermethylation
Overexpressed miRNA-221
Promoter hypermethylation
Promoter hypermethylation
Promoter hypermethylation
Promoter hypermethylation
Apoptotic peptidase activating factor 1
Death receptor 4
Death receptor 5
Decoy receptors 1 and 2
TNFα-related apoptosis-inducing ligand
Apoptosis-stimulating fragment
Death-associated protein kinase (DAPK)
Cysteine-aspartic proteases 8
Cysteine-aspartic proteases 10
DR5
DcR1 and DcR2
TRAIL
Fas
DAPK
Caspase 8
Caspase 10
XIAP-associated factor 1
B-cell CLL/lymphoma 2
XAF1
Pro-apoptotic genes
Apaf1
DR4
Bcl-2
Classic Hodgkin lymphoma (cHL)
Gastric cancer cells
Glioblastoma (GBM)
Lung cancer
Prostate cancer
Human gastric cancer
Gastric and bladder cancer
Suppressed miR-135a
Suppressed miR-512-5p
Suppressed miR-153
Down-regulated miR-133B
Promoter hypermethylation
Suppressed miRNA miR-34
Promoter hypermethylation
B-cell lymphoma-extra large
Myeloid cell leukaemia-1
Cancer type
Anti-apoptotic genes
Bcl-X(L)
Mcl-1
Epigenetic modification
Full name
Gene symbol
Table 1. Epigenetic modifications of anti and pro-apoptotic genes
[22]
[22]
[118]
[21]
[28]
[119]
[119]
[38]
[22]
[117]
[116]
[110]
[111]
[112]
[113]
[114]
[115]
References
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Bcl-2-interacting mediator of cell death
BIM
Tumour protein 53
Bcl-2 homologous antagonist/killer
p53 up-regulated modulator or apoptosis
BaK
PUMA
p53
Bcl-2-associated X protein
Bax
KAS-6/1 multiple myeloma cell line
Tumorigenesis in Apc− mice
Chronic lymphocytic leukaemia
KAS-6/1 multiple myeloma cell line
KAS-6/1 multiple myeloma cell line
Glioma cells
Chronic myeloid leukaemia
Burkitt’s lymphoma
Human gastric cancer
Acute lymphatic leukaemia patients
Promoter hypermethylation
Possible alteration on H3 and
H4 acetylation
Low expression of miR-34a
Promoter hypermethylation
Promoter hypermethylation
Up-regulated miRNA-221-222
Promoter hypermethylation
Histone modification (H3K27-Me3)
Suppressed miRNA miR-34
Promoter hypermethylation
[122]
[120]
[120]
[123]
[31]
[124]
[125]
[126]
[120]
[121]
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Figure 1. Apoptotic cell death pathways subverted by DNA methylation
Apoptotic genes acting with the extrinsic (death-receptor mediated) and intrinsic
(mitochondria-mediated) cell-death pathways are down-regulated by DNA promoter
hypermethylation. DNA methylation is involved in generating mutations in the coding region
of the p53 gene. Genes silenced by a DNA-methylation-dependent epigentic mechanism
are depicted in red in the Figure. For main details, see the main text. DNA methylation can
contribute in several ways to inactivating the apoptosis pathway.
have been described, DcR1 (decoy receptor 1) and DcR2. DcR1 and DcR2,
which also bind TRAIL, compete with DR4 and DR5 for the ligand, but
do not induce apoptosis. DR4, DR5 and more frequently DcR1 and DcR2
have been shown to be down-regulated by methylation in tumours [22–24].
DR4 promoter methylation is frequent in human astrocytic gliomas and
phaeochromocytoma, and epigenetic silencing of DR4 mediates resistance
to TRAIL/DR4-based therapies [25]. Engagement of these transmembrane
death receptors by their respective ligand promote the formation of the DISC
complex and the activation of caspase 8 within. Inactivation of caspase 8 (and
caspase 10) by DNA methylation has been reported in numerous cancer types
including small cell lung carcinoma, glioblastoma and paediatric tumours
(i.e. rhabdomyosarcoma, medulloblastoma, retinoblastoma and neuroblastoma).
DAPK (death-associated protein kinase) is a Ca2+/calmodulin-regulated,
serine/threonine microfilament-bound kinase shown to be key player in
multiple cell death signalling pathways [26,27]. DAPK also acts as a tumour
suppressor, as its gene silencing via promotor methylation is implicated in
tumorigenesis [28]. DAPK contributes to the extrinsic apoptotic pathway and
hypermethylation of the DAPK promoter attenuates the sensitivity to IFN-γ
(interferon γ)-, TNF-α-, FasL- and TRAIL-induced apoptosis in cancer cells.
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The p53 protein is a key player in the regulation of cell life and death decisions. When a cell suffers DNA damage or another cellular stress, its p53 proteins are activated and send a death signal to the cell. The contribution of DNA
methylation to p53 gene expression is more complex. p53 is the most frequently altered gene in human cancer, with approx. 50% of all types of cancers
carrying a mutation in p53, usually accompanied by LOH (loss of heterozygosity) [29]. Unlike the majority of tumour suppressor genes that are mutated
in human tumours, the wild-type allele of p53 is seldom transcriptionally
silenced through methylation, but is lost from the genome through deletions of
various degrees on chromosome 17 [30]. It is to be noted that even uncommon
p53 gene silencing by DNA hypermethylation has been reported in a group
of acute lymphatic leukaemia patients [31,32]. p53 activates the expression of
HIC1 (hypermethylated in cancer 1), which in turn directly regulates SIRT1
(sirtuin 1) to modulate p53-dependent DNA-damage responses [33,34]. As
its name indicates, HIC1 expression is found in various cancers to be silenced
by DNA hypermethylation. The loss of HIC1 results in up-regulated SIRT1
expression in cancer cells; this deacetylates and inactivates p53, allowing cells
to bypass apoptosis and survive DNA damage.
The BH3-only protein Bim directly binds to Bax, causes its activation
and participates in the intracellular signal transduction pathway of cell death.
Down-regulation of BIM expression by promoter hypermethylation contributes to renal cell carcinoma and chronic myeloid leukaemia apoptosis resistance [31,35].
Downstream of the mitochondria, several other key apoptotic players have
also been seen to be modulated by DNA methylation mechanisms. Inactivation
of the Apaf-1 (apoptotic protease-activating factor 1) gene is implicated in disease progression and chemoresistance of some malignancies. Apaf-1 DNA
methylation was observed at high frequencies in multiple types of leukaemia,
melanoma, gastric, bladder and kidney cancer [36–40]. XAF1 [XIAP (X-linked
inhibitor of apoptosis)-associated factor 1] belongs to a family of proteins that
inhibit IAPs (inhibitors of apoptosis). XAF1 negatively regulates XIAP, which
are potent inhibitors of caspase 3, 7 and 9 and sensitize cells to cell death triggers. Aberrant hypermethylation of the CpG sites in the XAF1 promoter is
strongly associated with lower expression of XAF1 in gastric cancers [41,42].
Histone covalent modification
Histone-modifying enzymes manage post-translational
modifications of histones
In addition to DNA methylation, modifications of histones are important
determinants of the epigenetic state. In eukaryotes, an octamer of histones
H2A, H2B, H3 and H4 is wrapped by 147 bp of DNA to form a nucleosome,
the fundamental unit of chromatin. Histones are the chief protein components
of chromatin, act as spools around which DNA winds and play a role in gene
regulation [43]. Histones are subject to a wide variety of post-translational
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modifications including, but not limited to, lysine acetylation, lysine and
arginine methylation, serine and threonine phosphorylation, and lysine
ubiquitination and SUMOylation, as well as ADP-ribosylation and
carbonylation [44]. These modifications occur primarily within the histone
N-terminal tails protruding from the surface of the nucleosome as well as
on the globular core region [45]. The histone-modifying enzymes affect
histones either locally, through targeted recruitment by sequence-specific
transcription factors [46], or globally throughout the genome in an untargeted
manner affecting virtually all nucleosomes [47]. The best characterized histone
modifications are acetylation, and methylation. In ‘normal’ cells, genomic
regions that include the promoters of tumour-suppressor genes are enriched
in histone-modification marks associated with active transcription, such
as acetylation of H3 and H4 lysine (K) residues (for instance H3K9ac,
H4K5ac, H4K8ac, H4K12 and H4K16ac) and trimethylation of K4 of H3
(H3K4me3) [48]. In the same cells, DNA repeats and other heterochromatic
regions are generally deacetylated, but characterized by trimethylation of
Lys27 and tri- and di-methylation of Lys9 of H3 (H3K27me3, H3K9me2 and
H3K9me3), and trimethylation of Lys20 of H4 (H4K20me3), which function
as repressive marks [49,50]. However, there are no absolute rules and many
active and inactive genes have overlapping patterns of histone modifications.
Nevertheless, it is clear that aberrant regulation of histone modification
can affect gene activity and therefore oncogenic potential exists. Histone
modifications are proposed to affect chromosome function through at least
two distinct mechanisms. The first mechanism suggests that modifications may
alter the electrostatic charge of the histone, resulting in a structural change
in histones or their binding to DNA. The second mechanism proposes that
these modifications are binding sites for protein recognition modules, such as
the bromodomains or chromodomains, which recognize acetylated lysines or
methylated lysine residues respectively.
Histone-modifying enzymes
For each modification, enzymes exist which either lay down the appropriate
mark or remove it. Major players in this regulation are the HATs (histone
acetyltransferases), which acetylate the histone tails and induce chromatin
decondensation, HDACs (histone deacetylases), which remove the acetyl
groups and promote a tighter binding of histones to DNA, HMTs (histone
methyltransferases), which promote or inhibit transcription depending
on the target histone residue, and HDMs (histone demethylases) which
counteract the HMTs. HATs generally contain multiple subunits, and the
functions of the catalytic subunit depend largely on the context of the other
subunits in those complexes. HATs are grouped into different family classes
based on their catalytic domains. Gcn5 is the founding member of the GNAT
(Gcn5 N-acetyltransferase) family, that includes Gcn5, PCAF, Elp3, Hat1a,
Hpa2 and Nut1a. The MYST HATs are named for the founding members
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of this family: Esa1, Mof, Moz, Morf, Ybf2/Sas3, Sas2, Hbo1 and Tip60.
Although these two families of HATs are the predominant ones, other proteins
including p300/CBP (CREB-binding protein), TAFII250 and a number of
nuclear receptor co-activators, have also been shown to possess intrinsic HAT
activity.
HDAC proteins occur in four groups depending on their sequence similarity and cofactor dependency. The first two groups are considered ‘classical’ HDACs whose activities are inhibited by TSA (trichostatin A), whereas
the third group is a family of NAD+-dependent proteins not affected by
TSA. The fourth group is considered an atypical category of its own, based
solely on DNA sequence similarity to the others. Homologues to the first
three groups are found in yeast having the names: Rpd3 (reduced potassium
dependency 3), which corresponds to Class I; hda1 (histone deacetylase 1),
corresponding to Class II; and Sir2 (silent information regulator 2), corresponding to Class III [51]. HDACs are divided into the following classes:
class I (HDAC1–3 and 8), class II (HDAC4–7, 9 and 10), class III (sirtiun1–7)
and class IV (HDAC11).
HMTs are enzymes, HKMTs (histone-lysine N-methyltransferases)
and HRMTs (histone-arginine N-methyltransferases), which catalyse the
transfer of one to three methyl groups from the cofactor SAM to lysine and
arginine residues of histone proteins. HKMT proteins often contain an SET
[Su(var)3-9, Enhancer of Zeste, Trithorax] domain and they make up the
SET-domain protein methyltransferase family. The exception to the rule is
the DOT1 family, members of which methylate Lys79 in the globular region
of histone H3 and which are structurally not related to SET-domain proteins
[52]. Protein arginine methylation, including of histones, is a covalent posttranslational modification carried out by a family of enzymes, the PRMTs
(protein arginine methyltransferases). PRMTs use SAM as a methyl donor
and add one or two methyl groups onto the guanidino nitrogens of the
arginine residue. In humans, the PRMT family consists of nine members [35].
In particular, histones are substrates of PRMT1, CARM1 (also referred to as
PRMT4), and PRMT5, which can be therefore considered a HRMT [53].
Histone methylation is now considered a more dynamic modification
with the discovery of HDMs. Levels of lysine methylation on histone tails
are known to change during processes such as transcriptional regulation.
Therefore it was proposed that specific enzymatic activity might remove the
methyl groups [54]. Indeed, amine oxidation by LSD1 (lysine-specific demethylase 1) and hydroxylation by JmjC-domain-containing proteins (including
the subfamily of proteins: JHDM1, PHF2/PHF8, JARID, JHMD3/JMJD2,
UTX/UTY, JHDM2 and JmjC only) are histone-modifying enzymes that can
remove methyl groups on lysine residues [55]. It was proposed that reversal
of arginine methylation might be catalysed by deiminases. PADI4 is a nuclear
member of the peptidyl arginine deiminase family which deiminate arginine
residues on histones H3 and H4 by converting them into citrulline [39,56,57].
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Aberrant modifications of histones: hallmarks of cancer
Overall, post-translational modification of histones provide an epigenetic
mechanism for the regulation of a variety of normal and disease-related
processes. Thus, considering the fundamental roles of histone modifications,
it is not surprising that aberrations in histone modifications are discovered in
cancer. Aberrant modifications of histones associated with subtelomeric DNA
and other DNA repeats have been reported. These aberrations include lower
levels of histone H4K16ac, H4K20me3 and H3K27me3 [58]. Interestingly,
loss of H4K16 acetylation and H4K20 trimethylation have been suggested to
be common hallmarks of cancer cells [59]. The loss of these repressive marks
leads to a more ‘relaxed’ chromatin conformation in these regions and might
contribute to genome instability. Global enzymatic modifications of single
residues on histone tails can be correlated with the state of stress within the cells
with surprising accuracy [60]. Histone modification patterns based on H3K4me2
and H3K18ac can predict the risk of tumour recurrence for prostate cancer.
Alterations in modifications of histones have also been linked to deregulated expression of many genes with important roles in cancer development
and progression. Promoter CpG-island hypermethylation in cancer cells
is known to be associated with a particular combination of histone marks:
deacetylation of histones H3 and H4, loss of H3K4me3, and gain of H3K9me
and H3K27me3 [61,62]. It is also recognized that certain genes with tumoursuppressor-like properties, such as p21WAF1, are silent at the transcriptional
level in the absence of CpG-island hypermethylation when hypoacetylated
and hypermethylated histones H3 and H4 are present [63].
In addition, growing evidence suggests that the different types of histonemodifying enzymes are found deregulated in human cancers. An extensive
analysis of expression patterns of histone-modifying enzymes was able to
discriminate tumour samples from their normal counterparts and cluster the
tumour samples according to cell type [64]. Genes that encode HAT enzymes
have been found to be translocated, amplified, overexpressed or mutated in
both solid tumours and haematological malignancies [65]. For example, HAT
hMOF (GNAT-MYST family) is frequently down-regulated in primary
breast carcinoma and medulloblastoma [66]. Leukaemias and uterine myomas carry translocations that generate fusion proteins such as CBP–MOZ
and CBP–MORF that disrupt the acetylation of histone H4 [67,68]. Altered
expression and mutations of genes that encode HDACs have been linked
to tumour development since they both induce the aberrant transcription
of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis [69]. Overexpression of HDACs has
also been reported in cancer. For example, there is an increase in HDAC1
expression in gastric, prostate, colon and breast carcinomas. Overexpression
of HDAC2 has been found in cervical, gastric and colorectal carcinoma. Other
studies have reported high levels of HDAC3 and HDAC6 expression in
colon and breast cancer specimens respectively. SIRT1 levels are increased in
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a number of tumour cell types. Mutational inactivation of HDAC2 in human
cancers has also reported to confer resistance to HDAC inhibition induced cell
death.
Several HMTs have been linked to different types of cancer. Of the
approx. 50 arginine and lysine HMTs encoded in the human genome, at least
22 have been associated with cancer or other diseases in humans or in mouse
models, and misregulation of some HMTs has also been associated with cancer
aggressiveness [70]. In parallel, numerous deregulations of HDM functions
have also been recently reported in cancer. Both gain- and loss-of-function for
HDMs are reported [71].
The potential apoptotic histone code
Chromatin condensation paralleled by DNA fragmentation is one of the most
important nuclear events occurring during apoptosis. DNA is associated with
histone proteins; hence, hallmark properties of apoptosis, such as chromatin
condensation, may be regulated by post-translational histone modifications.
Indeed, histone modifications, and in particular phosphorylation and
acetylation, have been suggested to affect chromatin function and structure
during cell death. Histone phosphorylation at specific sites, in particular,
has been shown to be involved in structural changes in chromatin during the
cell cycle. The cdc2/H1 kinase-dependent phosphorylation of H1 histones
during the cell cycle is a dynamic process with a phosphorylation maximum
during mitosis [72]. During induction of apoptosis, but before initiation of
nucleosomal fragmentation, H1 histones are rapidly dephosphorylated, a
process which accompanies condensation of chromatin.
Aurora B kinase and MSK1/2 (mitogen- and stress-activated kinases 1/2)
have been shown to be responsible for phosphorylation of Ser10 and Ser28 on histone H3 (H3-S10ph and H3-S28ph), phosphorylation events that mark the compaction of chromatin during mitosis [73]. It is worth noting that overexpression
of Aurora B kinase promotes tumour cell proliferation and inhibits apoptosis,
whereas inhibition of the kinase induces cell death [74]. MSK1/2 have also been
reported to negatively regulate apoptosis [75]. In parallel, histone H2B phosphorylation at Ser14 (H2B-S14ph) has been proposed as an epigenetic marker of
apoptotic cells associated with DNA degradation [76,77], whereas acetylation
at the adjacent Lys15 (H2B-K15ac) is a property of non-dying cells. The modification appeared to be reciprocal, and deacetylation of H2BK15 is necessary to
allow H2BS14 phosphorylation. Phosphorylation of H2BS14 is catalysed by
Mst1 (mammalian sterile twenty) kinase. Mst1 promotes H2BS14ph in vitro and
in vivo, and the onset of H2BS14 phosphorylation is dependent upon cleavage
and activation of Mst1 by caspase 3. RCC1 (regulator of chromosome condensation 1) reads the histone code created by caspase-activated Mst1 to initiate apoptosis by reducing the level of RanGTP in the nucleus, which leads to inactivation
of the nuclear transport machinery. As a consequence, nuclear-localization-signal-containing proteins, including survival factors such as NF-κB-p65, remain
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bound to importins α and β in the cytoplasm. Apoptosis-induced acetylation on
H2BK12 (H2B-K12ac) has also been reported [78].
Histone H2A.X phosphorylation on Ser139 (H2A-S139ph) in response
to DNA damage is the major signal for the assembly of the so-called
γ-H2AX chromatin domain, a region surrounding an unrepaired DNA
double-strand break that is characterized by the accumulation of a large
number of DNA-damage response proteins [79]. However, γ-H2AX, a
hallmark of the DNA-damage response, also forms early during apoptosis
induced by death receptor agonists (TRAIL and FasL) and staurosporine
targeting the intrinsic apoptotic pathway [80–82]. The recent discovery of
H2AX Tyr142 phosphorylation (H2AX-Y142ph) by the WICH complex and
its dephosphorylation by the EYA1/3 phosphatases may provide substantial
novel insight into this process [83]. WSTF, a subunit of the WICH complex,
bears a novel kinase domain at its N-terminus that constitutively targets
H2AX-Y142ph. This novel histone modification appears to determine the
relative recruitment of either DNA repair or pro-apoptotic factors to sites
of DNA damage and allows it to function as an active determinant of repair/
survival compared with apoptotic responses to DNA damage. Thus the balance of H2AX-Y142ph phosphorylation/dephosphorylation may constitute
a novel switch mechanism to determine cell fate after DNA damage [84].
Collectively these data provide evidence for a potential apoptotic ‘histone
code’. These unique histone modifications occurring during the course of cell
death provide new features to monitor and study apoptosis (Figure 2).
Furthermore, covalent histone modification has also been reported to regulate the expression of the cell-death-related gene. Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb
(EZH2)-based H3K27me3 and PcG (polycomb)-based H3K9me2 [85].
Promoters silenced by epigenetic mechanism include those for the apoptosisrelated gene p16INK4a [86], p57Kip2 [87], GAS2 and PIK3CG and p21Waf [63].
miRNAs
miRNAs: key regulators of gene expression
miRNAs are small non-coding RNAs that regulate the expression of
complementary mRNAs and function as key controllers in a myriad of cellular
processes, including proliferation, differentiation and apoptosis. The miRNA
biogenesis is an organized cellular process involving RNA-mediated gene
silencing (Figure 3).
miRNA alteration in cancer
miRNAs are considered as one of the most abundant classes of regulatory
genes in humans. Curiously, more than half of all miRNA genes are located in
cancer-associated genomic regions or fragile sites, which are frequently altered
during cancer development. Large-scale profiling of miRNAs has revealed that
miRNA deregulation is a marker of cancers. In cancer, miRNA expression is
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Figure 2. Existence of an apoptotic histone code
In ‘normal’ non-dying cells, nucleosomes are enriched in histone-modification marks, such as the
Aurora B/MSK1/2-dependent phosphorylation of Ser10 and Ser28 on histone H3, and the acetylation of Lys15 on histone H2B (H2B-K15ac) carried out by the HAT CBP/p300. In apoptotic cells,
this scenario is disrupted by the loss of the H2B-K15ac marks, which allow the caspase 3-activated Mst1 kinase-dependent phosphorylation of Ser14 on histone H2B. Acetylation of Lys22 on
histone H2B is also reported in apoptotic cells. DNA-PK-dependent-histone H2A phosphorylation on Ser139 (known as γ-H2AX), a hallmark of the DNA-damage response, forms early during
apoptosis at the nuclear periphery, forming the so-called ‘γ-H2AX ring’. H2AX Tyr142 phosphorylation by the WICH complex and its dephosphorylation by the EYA1/3 phosphatases appears
to determine the relative recruitment of either DNA repair or pro-apoptotic factors to sites of
DNA damage and allows it to function as an active determinant of repair/survival compared with
apoptotic responses to DNA damage. Collectively these results provide evidence for a potential
apoptotic ‘histone code’.
highly specific for tissues and developmental stage [88], and that has allowed
for molecular classification of tumors [89], drawing attention to its potential
in cancer diagnosis. For instance, miRNA miR-21 is up-regulated in a wide
variety of cancers, both solid and haematological tumours. This miRNA has
been shown to play a role as a marker of early detection of PDAC (pancreatic
ductal adenocarcinoma). Besides miR-21, other miRs including miR-155,
miR-181b, miR-221 and miR-222 are also frequently up-regulated in cancers
of the blood, brain, thyroid and the GI (gastro-intestinal) systems, and to a
lesser extent in liver cancer, lung cancer and breast cancer.
miRNAs regulating apoptosis as oncogenes and tumour suppressors
One of the most important links between miRNA function and cancer
pathogenesis is supported by the alteration of pro- and anti-apoptotic genes.
Down-regulated miRNAs in cancerous cells are considered as tumour
suppressor genes. Tumour suppressor miRNAs usually prevent tumour
development by negatively inhibiting oncogenes and/or genes that control
cell differentiation or apoptosis. However, miRNA overexpressed in tumours
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Figure 3. miRNA biogenesis and therapeutic strategies in cancer
(A) miRNA biogenesis is an organized process; primary transcripts RNAs (Pri-miRNA) are generated in the nucleus by polymerase II. The transcription product is capped at the 5´ end, polyadenylated to give a (poly)A tail and spliced to form Pri-miRNA. The processing of pri-miRNAs is
mediated by DiGeorge Syndrome Critical Region 8 (DGCR8) protein, the cofactor to drosha.
Together, drosha and DGCR8 form the microprocessor complex and cleave both strands of
the hairpin stem, thus producing pre-miRNAs which are transported to the cytoplasm by the
nuclear membrane protein exportin 5 using Ran-GTP. Once in the cytoplasm, pre-miRNAs
encounter a second RNase II endonuclease, Dicer, which cleaves the double-stranded RNA from
the end, subsequently releasing the mature miRNA. Dicer cleavage of pre-miRNA may generate
an imperfect miRNA, leading to protein level decrease without a decrease in mRNA levels. This
phenomenon is called translational repression. However, in the case of a perfect complementarity of miRNA with mRNA, miRNAs can act as small interfering RNAs (siRNAs), inducing mRNA
degradation, indicating that siRNAs may also be able to function as miRNAs. (B) Several recent
studies identified specific miRNAs that are down-regulated in more than one major tumour cell
line cluster; they also found that the re-expression of those miRNAs in many cancer cell lines
efficiently induce cell death. In miRNA re-expression therapeutic strategy, overexpressed specific pro-apoptotic miRNAs target the anti-apototic proteins and consequently induce apoptosis;
similarly the re-expressed specific anti-apoptotic miRNAs induce pro-apoptotic proteins and as a
result induce apoptosis. ORF, open reading frame.
may be considered as oncogenes and usually promote tumour development by
negatively inhibiting tumour suppressor genes and/or genes that control cell
differentiation or apoptosis. In fact, the let-7 miRNA is generally expressed at
low levels in cancerous lung tissue compared with normal tissue. Furthermore,
the low expression of let-7 correlates inversely with the level of RAS oncogene
proteins, but not RAS mRNA levels, suggesting that let-7 regulation of RAS
is a mechanism for let-7 to function as a tumour suppressor gene in lung
oncogenesis [90]. The miR-17-92 cluster is highly expressed in many tumours,
including lung tumours. This cluster increases the expression of the oncogenic
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c-Myc gene. Consequently c-Myc up-regulation induces down-regulation
of the transcription factor E2F1 protein expression [91]. This level of E2F1
expression results in the inhibition of apoptosis through the ARF-p53
pathway, and causes cancer pathogenesis in several organs, including lung
cancer and lymphomas. In this regulatory mechanism, miR-17-92 functions as
an oncogene.
miRNAs and apoptosis
miRNAs can regulate more than one protein-coding gene in the human
genome. Therefore, in cancer, several miRNAs have been shown to be
implicated in regulating the expression of cell proliferation and apoptosis
proteins. Some miRNAs are implicated only in apoptosis, whereas others may
play roles in both the apoptotic and cell-proliferation pathways [88].
The anti-apoptotic miRNAs
Individual miRNAs such as miR-21 have been found to regulate more than one
phenoytype, including apoptosis and proliferation [92,93]. MiR-21 knockdown
in human glioblastoma cells has been shown to increase apoptosis through
the caspase pathway. This miRNA inhibition highlighted its role as an antiapoptotic factor. Also in glioblastoma cells miR-21 is found to target directly
PTEN (phosphatase and tensin homologue deleted on chromosome 10),
reducing apoptosis [94]. In breast cancer, miR-21 regulates the proto-oncognes
and tumour suppressor protein PDCD4 (programmed cell death 4) [95].
This tumour suppressor whose deregulation leads to tumorigenesis, regulates
important cellular processes, including cell growth, proliferation and apoptosis.
Additionally, miRNA-21 has been shown to acts as anti-apoptotic factor in
regulating IL-6 and STAT3 (signal transducer and activator of transcription
3) [96]. STAT3 plays a central role in IL-6-mediated cell proliferation by
inhibiting apoptosis.
Pro-apoptotic miRNAs
The miR-15-16 cluster induces apoptosis by targeting the anti-apoptotic Bcl-2
at the post-transcriptional level [97]. Bcl-2 repression by miR-15-16-clusterinduced apoptosis seems to be the main regulatory mechanism involved in the
pathogenesis of leukaemic cell line model. In fact, the miRNAs miR-15-16 are
located on human chromosome 13 in a region frequently deleted in B-CLL
(B-cell chronic lymphocyte leukaemia) and its levels are inversely correlated
with Bcl-2 protein levels in CLL patients. Down-regulation of miR-15-16
cluster was also reported in other human malignancies such as pituitary
adenoma and prostate carcinoma. This cluster is considered a natural antisense
of Bcl-2 inhibitor. Furthermore, overexpression of miR-15-16 in a leukaemic
cell line results in decreased Bcl-2 protein expression, activation of the intrinsic
apoptosis pathway and ultimately cell death. Another miRNA down-regulated
is miR-29b, which is associated with lung and prostate cancers. MiR-29b has
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been also reported with target MCL1 (myeloid cell leukaemia sequence 1)
(an anti-apoptotic member of the Bcl-2 family), implying that the function of
miR-29b may be similar to that of miR-15-16. The miR-34 family has been
identified as components of tumour suppressor and pro-apoptotic miRNAs.
Ectopic expression of miR-34 induces cell-cycle arrest in both primary
and tumour-derived cell lines. He et al. [98] were the first to identify that
p53 can induce expression of miR-34. miR-34 inhibition of SIRT1 in colon
cancer cells leads to an increase in acetylated p53 and expression of p21 and
PUMA, transcriptional targets of p53 that regulate the cell cycle and apoptosis
respectively. The perturbation of miR-34 expression as occurs in some human
cancers, may thus contribute to tumorigenesis by attenuating p53-dependent
apoptosis. Therefore a miR-34 re-expression may induce apoptosis in these
cancers [99].
To date, there are a total of 6396 miRs, of which, 678 miRs are found in
humans [100]. However, mRNA targets and regulatory pathways have only
been explored for a few miRNAs. The complexity of understanding miRNA
regulation is due to the high number of target genes that can be regulated by
one single miRNA (200 target genes according to bioinformatics analysis).
Additionally, each cell type may be defined by a unique set of miRNAs that
control gene expression.
epi-drugs induced cancer cell death
Since aberrant epigenetic changes are a hallmark of cancer, the reversible nature
of epigenetic aberrations has led to the emergence of the promising field of
epigenetic therapy, which is already making progress, with the recent USA FDA
(Food and Drug Administration) approval of three epigenetic drugs, known as
epi-drugs, for cancer treatment. Epigenetic therapy of cancer is based mostly on
the use of DNMT inhibitors, HDAC inhibitors and anti-miRNA therapy.
The expression and regulation of DNA methylation and the subsequent
chromatin structure are significantly altered in cancer cells, underlying the
benefits of DNA demethylating agents, DNMT inhibitors, for cancer therapy.
Treatment with DNMT inhibitors, such as the nucleoside analogue pan-dnmt
inhibitor, 5-azaC (5-azacytidine), and its deoxy analogue, 5-azaCdR (5-deoxycytidine), a potent and non-selective DNA methylation inhibitor, lead to the
reactivation of pro-apoptotic genes, and subsequent cell death in numerous
tumour cell types [101].
Since HDAC inhibition prompts tumour cells to enter apoptosis, small
molecule HDAC inhibitors have been developed as a new class of anti-cancer agents, many of which have entered clinical trials. HDAC inhibitors are
a structurally diverse group of molecules, or epi-drugs, which can induce
cancer cell death, whereas normal cells are relatively resistant [102]. HDAC
inhibitors may act through the reactivation of dormant tumour suppressor
genes. They also modulate the expression of several genes related to apoptosis.
Furthermore, as histone acetylation plays a role in the regulation of chromatin
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structure and gene expression, two determinant parameters of the DNAdamage response, HDAC inhibitors have been shown to enhance radio- and
chemo-sensitivity of tumour cells [103,104]. A synergistic effect of the combined use of DNMT and HDAC inhibitors has been observed [105]. HDAC
inhibitors, in combination with DNA-demethylating agents, chemopreventive,
or classical chemotherapeutic drugs, could be promising candidates for cancer
therapy. Actually, in 2006 Vorinostat [SAHA (suberoylanilide hydroxamic
acid)] was the first epi-drug approved by FDA for the treatment of cutaneous T-cell haematological malignancies [106]. Novel polyamine analogues
have been shown to be potent inhibitors of the HDM LSD1, promote histone
methylation and represent a highly promising addition to the armamentarium
of epi-drugs to modulate cancer cell death [107].
Finally, every cellular process, including cell death, is likely to be regulated
by miRNAs, and an aberrant expression signature of these small non-coding
RNAs is a hallmark of several diseases, including cancer [108]. It has been
shown that miRNAs can function as potential oncogenes or tumour suppressor genes, depending on the cellular context and on the target genes they
regulate. Thus the possibility of modulating miRNA expression by developing synthetic pre-miRNA molecules or antisense oligonucleotides have at the
same time provided a powerful tool to develop a deeper comprehension of the
molecular mechanisms regulated by these molecules, and suggested the intriguing and promising perspective of their possible use in therapy [109].
Conclusion
During recent years, epigenetic alterations in the cell-death pathways and their
implication in cancer have received increasing attention. Consequently, the
list of enzymes involved in the epigenome is steadily increasing. In parallel,
the list of genes subject to abnormal epigenetic expression regulation is also
rapidly growing. However, in most cases, our comprehension of the molecular
mechanism that contributes to cancer development or progression is still
quite limited. In addition, even if changes in the epigenetic landscape can be
considered as a hallmark of cancer, epigenetic diversity is also a characteristic
of cancer, and should be considered, if not cell-specific, at least as tumourtype-specific. Further understanding of the different signalling pathways that
control apoptosis in the different tumour types and their epigenetic regulation,
will help with the discovery of novel targeted agents and the design of clinical
trials that are based on the molecular defects specific to the targeted tumour.
Most encouragingly, research in epigenetics has led to improved survival of
patients with certain forms of lymphoma and leukaemias through the use of
drugs that alter DNA methylation and histone acetylation. In addition, there
are numerous other clinical applications of the field being explored in areas
such as cancer screening and early detection, prevention, classification for
epidemiology and prognostic purposes, and predicting outcomes after standard
therapy. In conclusion, despite the complexity and heterogeneity of cancer, the
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field of epigenetic therapy, especially with the development of novel epi-drugs
(HDAC and HDM inhibitors) and miRNA-based therapy which reactivates
the cell death programme, provides, alone or in combination with current
therapeutic modalities, possibilities for treating human cancers.
Summary
•
•
•
•
•
Apoptosis alteration plays a central role in the regulation of tumour
formation and determines treatment response.
Global changes in the epigenetic landscape can be considered as a
hallmark of cancer.
Epigenetic mechanisms in cancer, such as DNA methylation, histone
modifications and miRNA expression, have revealed a plethora of
events that contribute to the neoplastic phenotype through stable
changes in the expression of genes critical to cell-death pathways.
An increased understanding of the mechanisms of epigenetic alterations
in the cell-death pathways and their implication in cancer will help
with the discovery of novel targeted agents and the design of clinical
trials that are based on the molecular defects specific to the targeted
tumour.
Research in epigenetics has led to improved survival of patients with
certain forms of cancers (e.g. lymphoma and leukaemias) through the
use of epi-drugs.
We apologize to authors whose primary references could not be cited owing to
space limitations. We thank Professor Alan R. Boobis for helpful comments. Work in
the authors’ laboratory is supported by the Swedish Research Council, the Swedish
Cancer Society and the Karolinska Institutet Foundations (KI Cancer) (to B.J.), and
the Department of Experimental Medicine and Toxicology, Imperial College London
(to N.H.).
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