Abbreviations aSMAase, acid sphingomyelinase; DISC, death-inducing signaling complex; DNMTs, DNA methyltransferases; FADD, FAS-associated death domain; FASL, FAS ligand; gld, generalized
Trang 1DNA methylation-mediated nucleosome dynamics and
oncogenic Ras signaling
Insights from FAS, FAS ligand and RASSF1A
Samir K Patra1,* and Moshe Szyf2
1 Cancer Epigenetics Research, Kalyani, India
2 Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada
DNA methylation and chromatin modification and
remodeling are currently center stage in studies of the
epigenetic regulation of genome function in normal
physiology, disease states and development [1–25]
Sev-eral isoforms of enzymes catalyzing both DNA and
histone modifications have been characterized
Con-comitant with differentiation, cell-type-specific patterns
of DNA methylation and histone modification are
gen-erated and are believed to program cell-type-specific
physiological functions, including memory formation
in neurons [2,18,20] These elaborate epigenetic programs may be difficult to reverse and rebuild during animal cloning procedures, because the signals and mechanisms for gene-specific hypermethylation and global demethylation patterns are not completely understood [2] In eukaryotes, the chromatin is orga-nized as euchromatin and heterochromatin Euchroma-tin encompasses the majority of single-copy genes, it replicates during early S phase and contains acetylated histones Heterochromatin is composed of long
Keywords
apoptosis; cancer; DNA methylation;
epigenetics; FAS; FAS ligand; H-Ras; K-Ras;
nucleosome dynamics; RASSF1A
Correspondence
S K Patra, Cancer Epigenetics Research,
Kalyani (B-7 ⁄ 183), Nadia, West Bengal, India
Fax: +91 332 582 8460
Tel: +91 943 206 0602
E-mail: skpatra_99@yahoo.com
*Present address
Division of Biochemistry, Department of
Experimental Medicine, University of Parma,
Italy
(Received 5 June 2008, revised 8 August
2008, accepted 22 August 2008)
doi:10.1111/j.1742-4658.2008.06658.x
Cytosine methylation at the 5-carbon position is the only known stable base modification found in the mammalian genome The organization and modification of chromatin is a key factor in programming gene expression patterns Recent findings suggest that DNA methylation at the junction of transcription initiation and elongation plays a critical role in suppression
of transcription This effect is mechanistically mediated by the state of chromatin modification DNA methylation attracts binding of methyl-CpG-binding domain proteins that trigger repression of transcription, whereas DNA demethylation facilitates transcription activation Under-standing the rules that guide differential gene expression, as well as tran-scription dynamics and transcript abundance, has proven to be a taxing problem for molecular biologists and oncologists alike The use of novel molecular modeling methods is providing exciting insights into the chal-lenging problem of how methylation mediates chromatin dynamics New data implicate lipid rafts as the coordinators of signals emanating from the cell membrane and are converging on the mechanisms linking DNA meth-ylation and chromatin dynamics This review focuses on some of these recent advances and uses lipid-raft-facilitated Ras signaling as a paradigm for understanding DNA methylation, chromatin dynamics and apoptosis
Abbreviations
aSMAase, acid sphingomyelinase; DISC, death-inducing signaling complex; DNMTs, DNA methyltransferases; FADD, FAS-associated death domain; FASL, FAS ligand; gld, generalized lymphoproliferative disorder; lpr, lymphoproliferative disorder; MAPK, mitogen activated protein kinase; MBD, methyl-CpG-binding domain proteins; MGMT, O6-methylguanine methyltransferase; RESE, Ras epigenetic silencing effectors; TNF, tumor necrosis factor.
Trang 2stretches of DNA repeats, replicates in late S phase
and contains lower levels of acetylated histones and
higher levels of DNA methylation [1–3,5–7,9,13–17,21–
24] Cytosine methylation is implicated in controlling
transcription, maintaining genome stability, parental
imprinting and X chromosome inactivation [1,2,21,
24,25]
The DNA methylation-mediated repression of
sev-eral genes, including those encoding proteins involved
in cell-cycle regulation and apoptosis, is a major cause
of tumor development and cancer progression [1–9] In
addition to the gene-specific hypermethylation of
sev-eral genes in many cancers, genomes of tumor cells are
globally hypomethylated and several genes critical for
tumor metastasis and progression are activated by
demethylation [2,3,6,11–16] The enzymes and
cofac-tors responsible for demethylation in cancer cells are
currently unknown It is known, however, that during
early development asymmetric DNA demethylation of
the paternal genome is observed just after fertilization
[2,17–19] Similar to the global hypomethylation
observed in cancer, the enzymes responsible for this
demethylation are unknown
Although the DNA methylation pattern is
pro-grammed during development, it remains highly
sensi-tive to both the chemical and social environment [10]
Studies have suggested that bioactive components of
food, both essential and nonessential nutrients, can
modify DNA methylation patterns in complex ways
For example, consumption of a methyl-deficient diet
led to hypomethylation of specific CpG sites within
several oncogenes (such as c-myc, c-fos and c-H-Ras),
resulting in high expression of these genes [26] Recent
studies have shown that tea catechins are effective
inhibitors of human DNA methyltransferase
(DNMT)-mediated DNA methylation in vitro, and re-expression
of a few genes in cultured cancer cells is observed in
response to tea catechins [27,28] Thus, DNA
methyla-tion may be viewed as an interface between the
envi-ronment and the human genome It stands to reason
that it might play critical role in several human
pathol-ogies, in particular age-related disease
DNA methylation enzymes
In mammalian cells, DNA methylation is catalyzed
by two classes of DNMT DNA-methyltransferase-1
(DNMT1; EC 2.1.1.37) is essential for maintaining
DNA methylation patterns in proliferating cells and is
also involved in establishing new DNA methylation
patterns; de novo methylation Members of the second
class of methyltransferases, DNMT3a and DNMT3b
are required for de novo methylation during embryonic
development [2,25], whereas DNMT3L cooperates with the DNMT3 family to establish maternal imprints in mice [29] DNMT1 and DNMT3B interact among themselves [30] and DNMT3A interacts with histone methyltransferases SETDB1 in the promoters of silenced gene during cancer development [16] Catalytic mechanisms of DNMTs involve the formation of a covalent bond between a cysteine residue in the active site of the enzyme and carbon 6 (C6) of cytosine in DNA The mechanisms involved have been described recently [2,25,31–39] Very recent data suggest that DNMTs may also be involved in the deamination of methylated cytosines to thymines [31,32] The mis-matched thymidine is then removed by base⁄ nucleotide excision repair resulting in repair to an unmethylated cytosine [2] This has been proposed to serve as a mechanism for dynamic DNA methylation [31,32]
A different type of DNA methyltransferase is O6-methylguanine DNA methyltransferase (MGMT;
EC 2.1.1.63) This enzyme does not methylate DNA but is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guan-ine O6-Methylguanine often mispairs with thymine during replication Following DNA replication this would result in conversion of a guanine–cytosine (GC) pair to an adenine–thymine (AT) pair Thus, repairing O6-methylguanine adducts is essential for the integrity
of the genome Interestingly this DNA methyltransfer-ase is regulated by DNA methylation Hypermethyla-tion of the MGMT promoter is associated with loss of MGMT expression⁄ function in many tumor types [1,4,40] MGMT hypermethylation is an example of the emerging field of pharmacoepigenomics The impact of chemotherapy would be dependent on the epigenetic state of cardinal genes such as MGMT [1,41] Knowing the state of methylation of critical repair genes is critical for the proper planning of a chemotherapeutic protocol
Removal of the methyl group (MeC-DNA demethy-lation) from critical positions in promoters is essential for the transcription of many genes A long line of evidence suggests that active enzymatic MeC-DNA demethylation occurs in nonreplicating cells to induce the transcription of specific genes at distinct time points [2] The mechanisms of demethylation are unknown and the enzymes involved are not firmly established One possible mechanism is through forma-tion of a cytosine–Michael adduct⁄ complex with MBD2 protein but this would require a cofactor [2,42– 48] which is not known The current notion of Michael adduct chemistry is that in such types of complex the SN2 mechanism would not occur In principle, water added across the 4C–5C double bond with the
Trang 3hydroxyl group attacking carbon 4, followed by
elimi-nation of ammonia will yield thymidine [2,25,34,46]
Epigenetic consequences of DNA
modifications – nucleosome dynamics
There is a bilateral relationship between DNA
methyl-ation and chromatin structure [2,16,49–58] Promoters
of genes and important regulatory sequences are
asso-ciated with hyperacetylated histones, whereas silent
genes are associated with hypoacetylated histones
Acetylated histones are associated with unmethylated
DNA and are rarely present in methylated DNA
regions [59] In addition to histone acetylation, which
plays a critical role in gene regulation, other histone
modifications such as methylation, phosphorylation
and ubiquitination play a similar role in regulating
genome functions [49–56] A combinatorial
arrange-ment of these modifications is believed to constitute a
‘histone code’ Methylation of DNA and deacetylation
of histones H3 and H4, combined with methylation of
K27 residue on the H3-histone tail in upstream
regula-tory regions leads to inactivation⁄ repression of gene
expression, whereas selective acetylation of histones
H1, H3, H4, methylation of H3K4 and DNA
deme-thylation are associated with activation of
transcrip-tion [2,5,6,12,16,22,23] (Fig 1)
How does DNA methylation signal for repression of
transcription? Repression of transcription may occur
through different mechanisms One simple mechanism
is that DNA methylation interferes with the binding of
transcriptional activators [24,25,53,55,59] A second
mechanism involves recruitment of
methyl-CpG-bind-ing domain proteins (MBDs), such as MBD1, MBD2,
MBD3, MBD4 and MeCP2 MBDs recruit
co-repres-sor complexes to methylated genes, which include
histone-modifying enzymes such as histone
deacetylas-es and histone methyltransferasdeacetylas-es precipitating an
inac-tive chromatin structure [16,21,39,49,51,56] This
mechanism provides an explanation for the correlation
between DNA methylation and inactive chromatin
configuration
DNA methylation and histone modification act in
concert to program gene expression Figure 1 presents
a model of the inhibition of gene expression by DNA
methylation Cytosine methylation at the DNA
sequence d(GGCGCC)2 triggers an extended eccentric
double-helix structure called E-DNA Like B-DNA,
E-DNA has a long helical rise and the base is
perpen-dicular to the helix axis The 3¢-endo sugar
conforma-tion provides the characteristic deep major groove and
shallow minor groove of A-DNA [60] Analysis of the
hydration pattern around methylated CpG sites in
crystal structures of A-DNA decamers at three high resolutions (1.7, 2.15 and 2.2 A˚) reveals that the methyl groups of cytosine residues are well hydrated with a higher amount of Mg2+ in their vicinity [61], which facilitates the interaction of MBD proteins and chromatin remodeling machines with the MeCpG sites The MBD–MeCpG complex then brings about deacety-lation of histones H3 and H4 [2,22] by recruiting class I histone deacetylases, which may be co-recruited with DNA-topoisomerase II [62] (Fig 1) Indeed, it has been shown experimentally that methylation of DNA brings about general deacetylation of histones H3 and H4, prevents methylation at H3K4 and induces methylation of H3 K9 [2,52–56] Histone H3K4 trimethylation is associated with transcription-ally active genes [59,63–73] MeCP2 has also been shown to recruit the histone methyltransfaerase SUV39 which targets H3 K9 [74] Okitsu & Hsieh observed a tight correlation between depletion of H3K4Me2 and regions of DNA methylation, and pro-posed that DNA methylation dictates a closed chroma-tin structure devoid of H3K4Me2 [59] The recent discovery of histone demethylases has challenged the originally held belief that histone methylation is static Histone demethylases specific for mono-, di- and trimethylated histone H3K4 are now known and their structures have been described [54,70–73,75–78] It is possible that the recently discovered histone demethy-lase LSD1 also participates in maintaining methylated regions of DNA devoid of H3K4 methylation [56,78] DNA methylation immediately downstream of the transcription start site has a dramatic impact on tran-scription, affecting transcription elongation rather than initiation Recent findings suggest that DNA methyla-tion at the juncmethyla-tion of transcripmethyla-tion initiamethyla-tion and elongation is most critical in transcription suppression and this effect is mechanistically mediated through chromatin structure [53,56,59,78] Although some important ideas have been suggested in other studies [64,68–70], it is still difficult to predict the effect of methylated DNA segments on transcription because differences in the size and position of the methylated DNA regions may differentially affect transcription For example, although a methylated coding region positioned 1 kb downstream of the promoter has little impact on transcription initiation, as observed by Lorincz et al [65], the same methylated sequence might have a much larger impact on transcription initiation if positioned immediately downstream of the promoter [54,64,68–70,78] The context seems to be critical [56] Recent data suggest that not only can DNA methyl-ation direct the formmethyl-ation of inactive chromatin struc-ture, but also that histone signals can direct DNA
Trang 4Fig 1 Cytosine base in DNA – the amazing switch for the regulation of gene expression and chromatin remodeling Cytosine, extended from the sugar-phosphate backbone (black circles–pink lines) and expanded manifold beyond the scale, is the only base in mammalian chro-mosomes which is stably modified by methylation at the carbon-5 position (formation of - Me CpG-) after replication DNA methylation inhibits gene expression affecting chromatin structure [2,6,22,59–71], because the presence of methyl groups on DNA affect the structure of DNA and the interaction of other proteins and enzymes with local nucleosomes [2,60] Methylation of DNA (MeCpG-) brings about a general hydra-tion of DNA [61], which facilitates the methyl-CpG-sequence binding (MBD) proteins to recognize the Me CpG- sites in nucleosomes for remodeling into a repressive complex DNMT- Me CpG- influence deacetylation of histones H3 and H4 by recruiting class I histone deacety-lases (HDACs); prevents methylation at H3K4, and induce methylation of H3 K9 in eukaryotes [56–69] HDACs may be co-recruited with DNA-topoisomerase II [62] Histone H3K4 methylation is associated with transcriptionally active nucleosomes of chromatin in which K4 of H3 are trimethylated, whereas H3 K27 methylation is associated with inactive chromatin [56,59,63–68,79] Methylation of histones is revers-ible and histone demethylases specific for di- and trimethylated histone H3K4 are discovered; for example, LSD1 represses transcription through demethylation of H3K4 Me3 [72,75,77] Okitsu & Hsieh [59] observed a tight correlation between the depletion of H3K4Me2 in the regions of DNA methylation Conversely, the level of H3K4Me2 remains high in the unmethylated DNA regions regardless of the presence
of RNA Pol II It can be proposed that Me CpG- dictates a closed chromatin structure that is devoid of H3K4Me2 and inhibits transcription, and that the presence of H3K4me2 marks an open chromatin structure that would permit transcription if all other conditions for active tran-scription are fulfilled [56,58] In early development, genomic methylation is erased and the somatic methylation pattern is re-established at the time of implantation The initiation of DNA demethylation-dependent nuclear processes is highly dependent on unfolding of chromatin structure In this context, acetylation of lysine ⁄ arginine of histone tails of H3 and ⁄ or H4 at the respective Me
CpG-rich nucleosome depends
on histone acetyl transferases (HATs) [48–50] In addition to methylation, H3 K9, H3 K14, H3 K23 and H3 K27 are also prone to acetylation, whereas as H3 K18 is only acetylated [22,49–59,63–69] This implies that nucleosome position is biased by the DNA sequence to facilitate access to initiation factors and activators by hundreds of histone modification (deacetylation, demethylation and also phosphorylations at ser-ine ⁄ threonine residues) Also, activation of specialized domains by removal of loosely associated mobile proteins, including HMG, HP and H1, partly regulates the expression of independent genes modulating the access of the above factors [2,22] Note: all the modifications men-tioned here are not require for activation of a particular type of gene Histone decoding, DNA modifications and the accessory factors are predominantly dependent on the types of signals a cell receives for activation ⁄ repression of a specific gene or for a particular class of gene.
Trang 5methylation For example, methylation of tumor
sup-pressor genes in cancer usually occurs in regions of
DNA associated with H3-histone K27 methylation, a
suppressive histone mark [2,12,22,54] The histone
methyltransferase EZH2 recruits the DNA
methyl-transferase to targets of EZH2 in the genome [1]
Thus, there is a bilateral relationship between DNA
methylation and inactive chromatin configuration
DNA demethylation is also tightly associated with
chromatin structure; histone acetylation of H3 and H4
histone tails is a hallmark of active chromatin
configu-ration and transcriptionally active regions of the
gen-ome [22,48,52,53,55,57–59] Hypgen-omethylation of DNA
[2,11,14] is found in regions associated with
hyperacet-ylated histones, and pharmacological histone
acetyla-tion could induce DNA demethylaacetyla-tion [48,79–81]
Thus, DNA demethylation, like DNA
hypermethyla-tion, has a bilateral relationship with chromatin
modi-fication
Ras oncogenes and oncoproteins
Ras, Rho, Rab, Arf and Ran are the five major classes
of monomeric GTPases whose biological functions are
regulated by the Ras family GTPases The cellular Ras
oncogene encodes a 21-kDa guanine
nucleotide-bind-ing protein, which plays a role in the regulation of
growth and differentiation in eukaryotic cells [1,82]
Despite profound improvements in our understanding
of the molecular and cellular mechanisms of action of
the Ras proteins, the expanding list of downstream
effectors and the complexity of the signaling cascades
that they regulate suggest that much remains to be
learnt [83] The study of Ras proteins and their
func-tions in cell physiology has led to many insights not
only into tumorigenesis but also into many
develop-mental disorders [82–84]
Although Ras binds both GDP and GTP with very
high affinity, the GTP-bound form is active and the
GDP-bound form is inactive The rate of intrinsic
nucleotide exchange and GTPase activity is very slow
Ras–GDP predominates in resting cells, but when
Ras is activated, specific guanine nucleotide exchange
factors enhance nucleotide exchange, increasing the
Ras–GTP complex Ras–GTP then activates
down-stream effectors such as Raf-1 GTPase-activating
pro-tein, however, causes the precipitation and
accumulation of inactive Ras–GDP within a cell and
may deregulate cellular physiology when overexpressed
[1,82–90] The retroviral oncogene, V-Ras, encodes a
protein that differs from the C-Ras product by a point
mutation that maintains this Ras protein constitutively
active [83] The Ras-related GTPase, Rho is required
for transmission of a proliferative signal by Ras If Rho is inhibited, the constitutively active Ras induces the cyclin-dependent kinase inhibitor p21(Waf1)⁄ Cip1, which blocks entry into the DNA synthesis phase of the cell cycle Rho activity suppresses induction of p21(Waf1)⁄ Cip1 by Ras, thus overcoming the block on entry into the S phase of the cell cycle Cells lacking p21(Waf1)⁄ Cip1 activity do not require Rho for the induction of DNA synthesis by activated Ras [1,83]
Lipid rafts and Ras signaling
Palmitoylation of N-Ras, K-Ras-4A and H-Ras, but not K-Ras-4B, in their C-terminal hypervariable regions is commonly required for their membrane relo-cation After relocation to the membrane, H-Ras and K-Ras-4A are translocated to lipid rafts; however, K-Ras-4B remains in the non-raft portion of the mem-brane [83] Hypervariable regions are responsible for targeting the isoforms to different microdomains in membrane Ras proteins localize to different plasma membrane microdomains, lipid rafts, formed by segre-gation of lipids based on their dissimilar biophysical properties [83,91] A comprehensive model of how Ras proteins are clustered for amplification, internalized and transmit their signals has recently been proposed [1] Eisenberg et al [92], employing fluorescence recov-ery after photobleaching, demonstrated coupling between membrane domains (rafts) in the external and internal leaflets of the plasma membrane and showed that this coupling modulated transbilayer signal trans-duction
Ras circulates between the Golgi, the endoplasmic reticulum and the plasma membranes H-Ras local-ized in the membranes of the endoplasmic reticulum and Golgi apparatus is activated by epidermal growth factor [1,91–93] After post-translational modification
in the endoplasmic reticulum, K-Ras is transported to the plasma membrane by a desorption–absortion mechanism [92,94] Ras detachment from lipid rafts requires GTP hydrolysis [92,93] H-Ras and N-Ras are transported to different sub-compartments by vesicular traffic, or by a nonvesicular pathway involv-ing a constitutive deacylation–reacylation cycle [1,83,92,95–97]
Inter-relationship of genetics and epigenetics in Ras oncogenic signaling
There is a bilateral relationship between genetic and epigenetic mechanisms in the activation of oncogenic Ras signaling Activation of K-Ras and H-Ras in human cancers results in DNA hypermethylation
Trang 6of target genes [1,6,31,83,98] Epigenetic
deregula-tion of critical repair genes can, however, increase the
rate of mutation of Ras genes (Fig 2) For example,
silencing of the repair methyltransferase MGMT would
result in an increased rate of mutation of Ras and
other oncogenes Figure 2 represents a scheme of how
the loss of MGMT expression would result in G to
A transitions in the K-Ras oncogene and in p53
[1,41,99] Indeed, MGMT promoter hypermethylation
is significantly pronounced in tumours that also bear a
G to A mutation in p53 suggesting a link between
epigenetic and genetic events and apoptosis related
diseases [99]
Genetic activation of Ras would also cause a change
in the state of methylation of several genes
Constitu-tive activation of Ras induces DNMT1 expression at
the transcriptional level through activation of cJUN
[100–102] The excess of unscheduled DNMT levels
would target certain genes for hypermethylation It is
believed that promoters marked by H3-K27
methyla-tion are targets of hypermethylamethyla-tion perhaps through
recruitment of DNMTs by EZH2 [103,104] Indeed,
targeting the Ras signaling pathway by drugs such as
methotrexate and inhibitors of ERK⁄ mitogen activated
protein kinase (MAPK) decreases DNA methylation in
malignant hematologic diseases and colon cancer cells indicating a causal relationship between Ras signaling and DNA methylation [105–108]
Ras activation would affect the DNA-methylation state and chromatin dynamics in the other direction as well Expression of v-H-Ras in mouse embryonal P19 cells resulted in genome-wide demethylation of certain genes, including a skeletal muscle-specific gene, adrenal cortex (c21)-specific gene, ubiquitous genes and exo-genously introduced sequences [100,109] Hence, DNA demethylase might be a potential downstream effector
of Ras signaling [1,2] Also, stimulation of the Ras– MAPK pathway leads to chromatin modification by histone H3 serine 10 and 28 phosphorylation in an acetylation-dependent and -independent fashion [55]
In summary, activation of the Ras-signaling pathway would trigger the methylation aberrations and histone modifications that are a hallmark of cancer: regional DNA hypermethylation and global hypomethylation
An attractive hypothesis is that K-Ras or H-Ras sig-nals originating from the membrane at different lipid raft-anchored Ras pools would have distinct effects on DNA methylation and demethylation [1,2,91,92], and histone 3 phosphorylation and acetylation machineries [55] Interestingly, this relationship between lipid rafts,
Fig 2 Epigenetic silencing of repair genes affects genetics O6-methylguanine DNA-methyltransferase (MGMT) gene silencing through promoter methylation demonstrates how the loss of MGMT expression results
in G to A transitions of the K-Ras oncogene (the most frequently mutated isoform of Ras), and of p53 [1,5,6,41,99] MGMT, a DNA repair protein, removes mutagenic and cytotoxic adducts from the O6 position of guanine [41,183] O6-methylguanine often mispairs with thymine during replication, and it results in conversion from a guanine– cytosine (GC) pair to an adenine–thymine (AT) pair if the adduct is not removed.
Trang 7Ras and epigenetic states might be bilateral as well
because many lipid raft component encoding genes are
known to be regulated by DNA methylation
[2,91,110]
FAS, FAS ligand, FAS-associated death
domain and lipid raft-mediated FAS
signaling
FAS-triggered apoptosis is another critical process
which is an effector of Ras signaling and is tightly
associated with epigenetic deregulation The 36 kDa
cell surface cytokine receptor, FAS (TNFRSF6⁄ FAS ⁄
APT1⁄ APO1 ⁄ CD95, OMIM 134637) contains a
16-amino acid signal sequence followed by a mature
protein of 319 amino acids that contains a solo
trans-membrane domain and two specialized functional
domains; a FAS death domain and a FAS ligand
(FASL) binding domain [111] FAS-associated death
domain (FADD, OMIM 602457) protein is the
univer-sal adaptor-protein for apoptosis This FADD
medi-ates signaling of all known death domain-containing
members of the tumor necrosis factor (TNF) receptor
superfamily [112] The FADD gene contains two exons
and spans 3.6 kb [113] Northern blot analysis
revealed that FADD was expressed as a 1.6-kb mRNA
in many fetal and adult tissues [114] The death
domain of FADD is 25–30% identical to those of
FAS and the TNF receptor, TNFR1 (OMIM 191190)
Natural ligands, cognate agonist antibodies and
inter-actions of FADD with FAS at their respective death
domains trigger apoptosis through FAS and TNFRI
High expression of FADD in mammalian cells induces
apoptosis, which can be blocked by Crma, a Pox-virus
gene product that also blocks FAS-induced apoptosis
[115,116]
The FAS protein shows structural homology with a
number of cell surface receptors, including TNFR1
and the low-affinity nerve growth factor receptor It
has been shown that following activation of T cells,
the FAS receptor is rapidly induced The interaction
between FAS and FASL induces cell death that occurs
in a cell-autonomous manner, similar to the classic
apoptotic sequence [117,118] FAS activates caspase 3
by inducing the cleavage of the caspase zymogen to its
active subunits and by stimulating the denitrosylation
of its active site thiol [119]
Myc-induced apoptosis requires interaction between
FAS and FASL on the cell surface [120] Hueber et al
established the dependence of Myc on FAS signaling
for its potent cell killing activity [120,121] The
path-way leading to apoptosis by FAS cross-linking with
FASL results in the formation of a death-inducing
signaling complex (DISC) composed of FAS, the signal adaptor protein FADD, and procaspase 8 and 10, and the caspase 8⁄ 10 regulator C-FLIP [91,122,123] Yeh
et al.[115] proposed that the interaction of FADD and FAS through their C-terminal death domains unmasks the N-terminal effector domain of FADD, allowing it
to recruit caspase 8 (CASP-8; 601763) to the FAS sig-naling complex This results in activation of a cysteine protease cascade, which leads to cell death Apoptosis triggered by infection, radiation or chemotherapeutic drugs is also mediated by FAS This process involves modification, placement in membrane, aggregation in lipid rafts and internalization of the FAS–DISC com-plex [91,124–130] Internalization of FAS with either lipid rafts or an endosomal compartment may deter-mine which signaling pathways are involved When internalization of FAS is blocked, the receptor cannot induce apoptosis and instead remains fully engaged, most probably in activating nonapoptotic⁄ proliferative pathways [112,115,125,127,131]
Inter-relation of genetic and epigenetic alterations in cancer
It is now evident, as discussed above, that changes occurring in cancer cells, including chromosomal instability, an increased propensity for mutation, acti-vation of oncogenes, silencing of tumor suppressor genes and inactivation of DNA repair systems are a result of both genetic and epigenetic abnormalities The correlation between the status of -CpG-island hypermethylation and⁄ or mutations in critical genes shows that, for virtually every tumor type, both gene-specific hypermethylation and distinct genetic altera-tions over time are major driving forces in neoplastic development But naturally occurring mutations of specific genes in somatic cells are infrequent, because under normal conditions maintenance of genomic integrity is guarded by a complex array of DNA monitoring and repair enzymes Karyotypic order is also guaranteed by other molecular guards, such as cell-cycle check-points that operate at critical times during mitotic division Together, these systems ensure that mutations are rare events, so rare indeed that the multiple mutations known to be present in tumor cells, which are necessary for cancer progres-sion, are low probability events within a normal human life span However, during oncogenesis epige-netic silencing of genes encoding DNA repair proteins (for example, MGMT) may cause retention of mutants as well as encourage neo-mutants [1–6,41] The FAS apoptotic pathway is one of the most promising targets of this process
Trang 8FAS mutations in cancer and other
diseases
Several lines of evidence have highlighted that perhaps
all tumor cells express FAS, but in many cases the
gene is mutated encoding a nonfunctional protein
Some cancers such as papillary thyroid carcinoma,
however, do express functional FAS [132] Analysis of
the entire FAS coding region in micro-dissected biopsy
samples from 21 burn scar-related squamous cell
carci-nomas revealed somatic point mutations in all of the
splice sites from three patients [133] The mutations
were located in all three domains of the protein: the
death domain, ligand-binding domain and
transmem-brane domain of the FAS gene Analyses of the other
FAS alleles in tumors carrying the N239D and C162R
mutations indicated loss of heterozygosity, and
expres-sion of FAS was confirmed in all tumors with FAS
mutations [91,133]
In contrast to the situation in burn scar-related
squamous cell carcinoma, no mutations were detected
in 50 cases of conventional squamous cell carcinoma
[133] This difference in mutation of the FAS gene is
interesting because burn scar-related squamous cell
carcinoma is usually more aggressive than
conven-tional squamous cell carcinoma It was therefore
sug-gested that somatic mutations in the FAS gene may
contribute to the development and⁄ or progression of
burn scar-related squamous cell carcinoma The
fol-lowing mutations in the gene encoding FAS were
iden-tified: a 957A-to-G transition resulting in an N239D
substitution in the FAS death domain; a 547A-to-G
transition resulting in an N102S substitution in the
FAS ligand-binding domain; a 726T-to-C transition
resulting in a C162R substitution in the FAS
trans-membrane domain [133] Zhang et al [134] genotyped
1000 Han Chinese lung cancer (211980) patients
and 1270 controls for two functional polymorphisms
in the promoter regions of the FAS and FASL
genes, -1377G-to-A (134637.0021) and -844T-to-C
(134638.0002), respectively Compared with
noncarri-ers, there was an increased risk of developing lung
cancer for carriers of either the FAS -1377AA or the
FASL -844CC genotype; carriers of both homozygous
genotypes had a more than fourfold increased risk
[134] Their results further support the concept that
the inactivation of FAS- and FASL-triggered
apopto-sis pathway plays an important role in human
carcino-genesis [91,135]
A heterozygous mutation in the FAS gene in five
unrelated children (134637.0001–134637.0005), with a
rare autoimmune lymphoproliferative (lpr) syndrome
was identified by Fisher et al [136] The disease is
characterized by massive nonmalignant lymphadenopa-thy, heightened autoimmunity and expanded popula-tions of TCR-CD3(+)CD4())CD8()) lymphocytes, resulting from defective FAS-mediated T-lymphocyte apoptosis While delineating the prognostic markers for the disorder, Sneller et al [137] further analyzed one of the patients studied by Fisher et al., and pointed out its resemblance to autosomal recessive lpr⁄ gld (generalized lymphoproliferative disorder) mouse The lpr and gld mice bear mutated genes for FAS and FASL, respectively The murine autosomal recessive lpr phenotype is characterized by lymphade-nopathy, hypergammaglobulinemia, multiple autoanti-bodies and the accumulation of large numbers of nonmalignant CD4, CD8 and T cells Affected mice usually develop a systemic lupus erythematosus-like autoimmune disease, and a defect in the negative selection of self-reactive T lymphocytes in the thymus The mouse lpr phenotype is identical to the phenotype displayed by human patients bearing mutated FAS [138,139]
Epigenetic downregulation of apoptosis – the role of the Ras signaling pathway
In addition to genetic mutations in FAS as discussed above, FAS is silenced by epigenetic mechanisms in several cancers Several lines of evidence suggest that the trigger for methylation of the FAS gene is activa-tion of the Ras fi Raf fi MEK fi ERK fi Elk signaling pathway [1,91,98] Methylation of the FAS gene is associated with loss of FAS expression in anti-gen-specific cytotoxic T cells [140] There is evidence for involvement of DNA methylation in silencing of FAS–FASL signaling and loss of apoptosis [141] Silencing of FASL and TRAIL-R1, TRAIL-R2 and Caspase-8 expression by DNA methylation has been linked to resistance of small cell lung cancer cells to FASL and TRAIL induced apoptosis [142] FAS pro-moter methylation in prostatic and bladder carcinomas and respective cell lines correlates with downregulation
of FAS expression [143]
H-Ras is linked to the silencing of FAS-triggered apoptosis through DNA methylation Peli et al [144] reported that oncogenic H-Ras downregulated FAS
by DNA methylation It was suggested that the phosphatidylinositol 3-kinase pathway was involved in mediating this effect of RAS The involvement of phosphatidylinositol 3-kinase points to the possibility that some of the known anti-apoptotic effects of PKB⁄ Akt kinase may be mediated, at least in part, by the downregulation of FAS expression through DNA
Trang 9methylation [144] It remains to be seen whether this
effect of the Ras signaling pathway on DNA
methyla-tion is brought about by increase of DNMT1 as
pre-viously reported [102] or through activation of
factor(s) that recruits DNMT to specific targets such
as apoptosis
The epigenetic downregulation of apoptosis
path-ways involves additional genes in various types of
tumors In neuroblastomas and neurobalstoma cell
lines, which are resistant to apoptosis induced by
TRAIL, CASP-8 and the FLIP gene, and in tissues
adjacent to tumors the CASP-8 gene is
hypermethyla-ted [145] The FLIP protein is a negative regulator of
CASP-8, and the methylation of CASP-8 and FLIP
genes is somewhat correlated [91,125,145]
Mechanisms of silencing of FAS in
response to Ras activation
How does Ras activation cause methylation and
epige-netic silencing of FAS and other apoptosis-related
genes? Activated Ras epigenetically silences FAS
expression in mouse NIH3T3 cells [143], and in human
K-Ras transformed cell line, HEC1A [98]
Twenty-eight ‘Ras epigenetic silencing effector’ (RESE) genes
were discovered in a genome-wide functional screen
[98] Nine RESEs were found to be bound to different
regions of the FAS promoter in K-Ras-transformed
NIH3T3 cells [98], whereas in nontransfected NIH3T3
cells only one RESE (NPM2) was associated with the
FAS promoter It was therefore proposed that these
nine RESEs were recruited to specific regions of FAS
promoter in response to expression of oncogenic
K-Ras and are involved in the recruitment of DNMT1
and other chromatin modifiers to the promoter,
result-ing in DNA methylation and epigenetic silencresult-ing In
support of this hypothesis, knockdown of any of the
28 RESEs in K-Ras-transformed NIH3T3 cells
resulted in an absence of DNMT1 on the FAS
promoter, demethylation of the FAS promoter and
induction of FAS expression
What are the biochemical and cellular functions of
other RESEs? Among the 28 RESE proteins
discov-ered using a functional genomics approach there
are transcriptional activators and repressors (CTCF,
EID1, E2F1, RCOR2 and TRIM66⁄ TIF1D),
sequence-specific DNA-binding proteins (SOX14,
ZCCHC4 and ZFP345B), histone methyltransferases
(DOT1L, EZH2 and SMYD1), histone deacetylase
(HDAC9), histone chaperones (ASF1A and NPM2),
DNMT1 and several Polycomb group proteins (BMI1,
EED and EZH2) Several recent studies have linked
Polycomb proteins to abnormal DNA methylation and
gene silencing [1,146–148] It is surprising that one of the nuclear RESEs is BAZ2A⁄ TIP5, previously known
to be involved only in repression of RNA polymer-ase I-directed ribosomal gene transcription [149] A number of RESEs were substantially upregulated at the transcriptional or post-transcriptional level in K-Ras-transformed NIH3T3 cells compared with non-transformed NIH3T3 cells, explaining at least in part, how K-Ras activates this silencing pathway (Fig 3A) Treatment of K-Ras NIH3T3 cells with the demethy-lating drug 5-aza-CdR resulted in FAS re-expression, supporting the hypothesis that FAS is regulated by DNA methylation in Ras-transformed cells [98]
Epigenetic inactivation of the RAS effector homolog RASSF1
Genes encoding human RAS effector homolog, RASSF1 (OMIM, 605082) family proteins, along with several putative tumor-suppressor genes are located at chromosome 3p21 [83,150–152] RASSF1 produce eight transcripts, A–H, derived from alternative splic-ing and promoter usage [152–154] The RASSF1 gene contributes to the spatiotemporal regulation of mitosis through a number of regulatory mechanisms that cooperate to restrict the activity of APC⁄ C to a spe-cific period in the cell cycle [153–155] Mechanistic roles for RASSF1A in inducing apoptosis in cancer cells and solid tumors are emerging [156–158] RASSF1A function was missing in a variety of solid tumors and cancer cell lines, including small cell lung cancer and prostate [150–156] DNA methylation of the CpG island promoter sequence of RASSF1A was implicated in its silencing [16,155] RASSF1A is the most frequently methylated gene in both primary tumors and cell lines and in a group of nine genes mapped in 175 primary pediatric tumors and 23 tumor cell lines RASSF1A methylation was tumor specific and absent in adjacent nonmalignant tissues [157] RASSF1A gene silencing is also associated with aber-rant methylation and histone deacetylation in a variety
of other cancers [158–163] RASSF2 methylation and inactivation is a consequence as well of K-Ras-induced oncogenic transformation [164] Apart from Ras-regu-lated methylation of RASSF1A, Ras and RASSF1A have direct physical interaction in cellular physiology [155,160]
Lipid raft facilitated Ras signaling and chromatin modification
Clustering of raft-associated receptors, like epidermal growth factor receptor, facilitates the early step of