Senescence in Disease

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Epigenetic control of cellular

senescence in disease: opportunities

for therapeutic intervention

Stuart P. Atkinson and W. Nicol Keith*

Understanding how senescence is established and maintained is an important

area of study both for normal cell physiology and in tumourigenesis.

Modifications to N-terminal tails of histone proteins, which can lead to

chromatin remodelling, appear to be key to the regulation of the senescence

phenotype. Epigenetic mechanisms such as modification of histone proteinshave been shown to be sufficient to regulate gene expression levels and

specific gene promoters can become epigenetically altered at senescence.

This suggests that epigenetic mechanisms are important in senescence

and further suggests epigenetic deregulation could play an important role in

the bypass of senescence and the acquisition of a tumourigenic phenotype.

Tumour suppressor proteins and cellular senescence are intimately linked and

such proteins are now known to regulate gene expression through chromatin

remodelling, again suggesting a link between chromatin modification and

cellular senescence. Telomere dynamics and the expression of the telomerase

genes are also both implicitly linked to senescence and tumourigenesis, and

epigenetic deregulation of the telomerase gene promoters has been identified

as a possible mechanism for the activation of telomere maintenance

mechanisms in cancer. Recent studies have also suggested that epigenetic

deregulation in stem cells could play an important role in carcinogenesis, and

new models have been suggested for the attainment of tumourigenesis

and bypass of senescence. Overall, proper regulation of the chromatin

environment is suggested to have an important role in the senescence

pathway, such that its deregulation could lead to tumourigenesis.

Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UKBeatson Laboratories, Bearsden, Glasgow, G61 1BD, UK.

*Corresponding author: W. Nicol Keith, Centre for Oncology and Applied Pharmacology,University of Glasgow, Cancer Research UK Beatson Laboratories, Alexander Stone Building,Garscube Estate, Switchback Rd, Bearsden, Glasgow, G61 1BD, UK. Tel: +44 (0)141 330 4811;Fax: +44 (0)141 330 4127; E-mail: [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1 Accession information: DOI: 10.1017/S1462399407000269; Vol. 9; Issue 7; March 2007

&2007 Cambridge University Press

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Cellular senescence provides a barrier toexcessive cellular growth and is triggered bythree main mechanisms. Telomere attritionduring extended cell proliferation leadseventually to cellular senescence (Ref. 1) when

critically short telomeres are recognised asDNA damage, thereby triggering thesenescence arrest. Although this proliferative

barrier provides a limit to the outgrowth of acell, senescence can also be triggered before thislimit in response to cellular damage (such asDNA damage) or stress and oncogenic signalling.As senescent cells do not proliferate, it has beenproposed that cellular senescence is a major

barrier to cancerous transformation (Refs 2, 3)and premature induction of cellular senescencecan therefore be targeted for anti-cancer therapyin tumourigenic cells (Refs 4, 5) (Fig. 1).

The senescence phenotype is associated withmultiple cellular changes including alteredcellular morphology, size and, importantly,expression of numerous genes involved inmany aspects of cell physiology (Refs 6, 7, 8, 9,10). The altered regulation of genes and thecomplex phenotype suggest that epigeneticmechanisms may be involved. Indeed, it is nowapparent that, in addition to multiple geneticpathways, epigenetics plays a major contributingrole in senescence (Refs 11, 12) as well as in the

bypass of senescence leading to tumourigenicity(Refs 13, 14, 15). Therefore, to understand

senescence and tumourigenesis, both geneticand epigenetic mechanisms regulating cellularsenescence must be appreciated.

Epigenetic regulation of gene expressionEpigenetic mechanisms are defined as processesin which changes in gene function can occurwithout a change in the DNA sequence. Suchmechanisms include covalent modifications of histones, DNA methylation, ATP-dependentchromatin remodelling, incorporation of varianthistones, looping and changes in the local

conformation of DNA, and alterations of higher-order chromatin structure. It isunderstood that epigenetic processes such asDNA methylation and post-translationalmodification of histones are fundamental to theregulation of gene expression and therefore tothe regulation of multiple cellular processes.For these reasons it is plausible to suggest thatchanges in the chromatin environment can playa role in the changes in gene expression

involved in senescence and also in the bypassof senescence towards tumourigenesis.

The binding of DNA to histone proteins isrequired for efficient packaging of DNA intothe nucleus of the cell, but this tight binding

creates a barrier for factors that need access toDNA to regulate various cellular processes.Histone proteins bind together to form ahistone octamer comprising two copies each of histones H2A, H2B, H3 and H4 to which DNAwraps round. Each member of the octamer can

be post-translationally modified and onefunction of these modifications is to mediate

On-rampTelomerase

gene expression

O n - r a m p

N o t e l o m e r a s e a c t i v i t y

A L T p a t h w a y

r e c o m b i n a t i o n

Telomerase activity

Immortality

Senescence

E x i t r o u t e

E x i t r o u t e

Normal cell

NeoplasiaImmortality

Routes to immortality

Expert Reviews in Molecular Medicine

© 2007 Cambridge University Press

Figure 1. Routes to immortality. Cellular

senescence (forward and reverse exit routes)

provides a barrier to excessive cellular growth and

so is considered to be a tumour suppressor

mechanism. A normal cell can bypass senescence

and move towards neoplasia through

immortalisation by two means, or two different ‘on-

ramps’. First, cells can maintain their telomeres

through telomerase activation (horizontal on-ramp).

Second, cells can engage the alternative

lengthening of telomeres (ALT) pathway (vertical on-

ramp), which allows telomere elongation through

intra-telomeric recombination processes. The

induction of premature cellular senescence of

neoplastic cells (reverse exit route) may therefore be

a strategy for anti-cancer therapeutics.




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the binding of the histone proteins to DNAand therefore to regulate DNA access andthe transcriptional status of DNA therein.Modifications occur mainly on the histoneN-terminal tail, but also occur on the

histone globular domain, and includeacetylation, methylation, phosphorylation,SUMOylation (for ‘small ubiquitin-relatedmodifier’), ubiquitination and ADP ribosylation.It has been hypothesised that one of the roles of these modifications is to act as a ‘histone code’(Refs 16, 17) in which combinations of histonemodifications can be read as a code andtranslated into an output signal; thus, distinctpatterns of histones may be a signal for genetranscription to be turned on or off.Furthermore, levels of modifications mightfunction to regulate levels of transcription (and

therefore high or low levels of gene expression).In general, silenced or repressed chromatin

(heterochromatin) is characterised by hypo-acetylated histones (histones H3 and H4),methylation of residues Lys9 and Lys27 of histone H3 (K9 H3 and K27 H3) andmethylation of Lys20 of histone H4 (K20 H4).Active, de-repressed chromatin (euchromatin)is characterised by hyper-acetylated histonesand can have methylation of Lys4, Lys36and Lys79 of histone H3 (K4 H3, K36 H3 andK79 H3) (Refs 18, 19). In addition, DNAhypermethylation tends to be associated with

silent chromatin whereas hypomethylationgenerally associates with active chromatin.

Senescence, chromatin and diseaseTheinduction of premature senescence of tumourcells by chemotherapeutics is utilised in anti-cancer therapy. However, it is important torealise that cellular senescence contributes tothe ageing process, can lead to the dysfunctionof tissues such as the heart, lungs and brain(Ref. 20), and is also linked to the pathogenesisof multiple disease states (Table 1) such

as smoking-related lung disease, transplantrejection and liver cirrhosis. Interestingly, it hasalso been demonstrated that senescent cellsmay promote tumourigenesis by providinga permissive tissue microenvironment fortumourigenic cell growth (Refs 21, 22, 23).Therefore, although senescence is an important

barrier to cancer progression, this benefit might be offset by the contribution of cell arrest toother disease states in later life.

In addition, similarly deregulated epigeneticmechanisms have been shown to contributeto ageing, cardiovascular disease, multipleinherited disorders and also tumourigenesis(Table 2). Furthermore, the epigenetic basis of tumourigenicity is an intensely studied area(Refs 13, 14, 24, 25, 26) and it is now wellestablished that the deregulation of epigeneticmodifying enzymes, leading to deregulatedgene expression, plays an important role intumourigenesis (Ref. 14). Such studies have ledto the establishment of an epigenetic model of

cancer to complement, or even replace, thegenetic model for cancer and hypothesises thatepigenetic modifications can play a role intumour initiation and progression (Ref. 26).Another interesting model suggests thatepigenetics is at the very root of tumourigenicity,

by disrupting gene expression at the stem celllevel (Ref. 27). Epigenetic disruption is thereforean important mechanism in the key early eventsin tumourigenesis.

How regulation of the chromatinenvironment affects senescence

Heterochromatin formation and senescenceInitial studies previously established thatchromatin becomes reorganised with increasedpopulation doublings, with large-scale effects

being evident upon senescence (Ref. 28). It hasnow been shown that the formation of heterochromatin at specific foci plays asignificant role in senescence in response tooncogenic insult and telomere attrition, and thatthis senescence arrest as a cellular response to

Table 1. Senescence and disease

Senescence-associateddisease

Refs

Tumourigenesis 21, 22, 23

Wound healing 173Neurodegeneration 174, 175

Alzheimer’s disease 176

Chronic obstructive

pulmonary disease (COPD)

177

Emphysema 178

Kidney failure 179

Cardiovascular disease 180

Chronic inflammation 181, 182

Transplant rejection 183

Osteoarthritis 184

Liver cirrhosis 185

Atherosclerosis 172




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stress might act as a tumour suppressor

mechanism (Refs 2, 3). In addition, it has also been observed that many genes upregulated atsenescence are actually physically clustered(Ref. 29), again indicating that re-organisation of chromatin domains are important in senescence.Therefore, formation of heterochromatin may beviewed as a tumour suppressor mechanism.

Early studies showed that expression of oncogenic Ras induced the accumulation of p16INK4a (also known as cyclin-dependent kinase

inhibitor 2A) and hypophosphorylated pRB

(retinoblastoma protein) with a senescence-likearrest in normal human fibroblasts (Ref. 30). Itwas also noted that this senescence-like arrestwas associated with the formation of distinctheterochromatic structures, which weredesignated senescence-associated heterochromatinfoci (SAHF) (Ref. 30). Importantly, SAHF wereshown to be formed by newly depositedheterochromatin rather than from redistributedheterochromatin, and were concentrated for

Table 2. Chromatin and disease

Disease Gene/epigeneticmechanism

Mechanism of diseasepathogenesis

Refs

Inherited

Angelman syndrome Aberrant genomic imprinting 186Prader– Willi syndrome Aberrant genomic imprinting 186

Beckwith–Wiedemann

syndrome

Aberrant genomic imprinting 186

ICF syndrome DNMT3a Gene mutation 186

Rett syndrome MECP2 Gene mutation 187

Autoimmune

Systemic lupus

erythematosus

Changes in DNA methylation and

histone modifications in lupus cells

188

Cardiovascular

Stress-induced

remodelling

Histone acetylation Gene regulation alterations lead to

remodelling/weakening of the heart

189

Neural

Huntington’s disease Histone acetylation Altered gene expression 190

Alzheimer’s disease HDAC genes Inhibition leading to gene expression

changes may alleviate symptoms

191

Histone acetylation Deregulated acetylation levels

may be threat to neural health

192

Epilepsy Histone acetylation May play role in development

of epilepsy

193

Lung

Lung inflammatory

disease

Histone acetylation Altered expression of inflammatory

genes

194

Spinal

Spinal muscular

atrophy

Histone acetylation Aberrant changes in gene expression 195

OtherDepression HDAC5 Aberrant changes in gene expression 196

Tumourigenesis Histone modification Aberrant changes in gene expression

and possible increased genetic

instability

14, 39,

154, 155

DNA methylation Aberrant changes in gene expression

and increased genetic instability

124, 197

Abbreviations:DNMT3a, DNA (cytosine-5-)-methyltransferase 3 alpha; HDACs/HDAC5, histone deacetylases/histone deacetylase 5; ICF syndrome,centromereinstabilityand facial anomaliessyndrome;MECP2, methylCpGbinding protein 2.




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repressive histone modifications such asmethylation of K9 H3 and heterochromatinprotein 1 (HP1), whereas permissivemodifications, such as methylated K4 H3 andacetylated K9 H3, were excluded (Fig. 2). It was

also established that active transcription was notoccurring at the sites of SAHF formation and thatchromatin features indicative of SAHF formed onE2F-responsive promoters of genes known to bedownregulated at senescence. E2F is atranscription factor important for regulation of genes associated with the cell cycle and was firstidentified as a cellular factor that had the ability

to bind the promoter for the gene encodingadenovirus E2. The genes that gainedheterochromatic features and becamedownregulated included the genes encodingcyclin A and proliferating cell nuclear antigen

(PCNA), and their promoters were bound by pRB,thereby linking pRB binding to the formation of SAHF. It is known that the upregulation of p16INK4a during senescence can inhibit thephosphorylation of pRB, causing it to bind to E2Fto cause a permanent growth arrest by silencinggrowth regulatory genes through altering thechromatin state of the promoter sequences. pRBhas been shown to interact with factors involvedin the formation of heterochromatin, includinghistone deacetylases (HDACs) (Refs 31, 32, 33),the K9 methyltransferase SUV39H1 (Ref. 34) andHP1 proteins (Ref. 35); thus, pRB binding to

regulatory sequences could be sufficient to formSAHF in order to silence gene expression.Importantly, the authors showed in the samestudy that promoters of genes known to beupregulated at senescence gained euchromaticpatterns at their promoters at the same time asSAHF were being formed on other promoters,

Figure 2. The formation of senescence-

associated heterochromatin foci (SAHF) in

response to oncogenic insult. In this model, (a)

an active gene promoter (e.g. cyclin D1) shows

methylation of Lys4 (K4) H3 and acetylation of

H3 and H4, and its chromatin has an open(euchromatic) structure. Under these conditions,

the transcription factor E2F mediates transcription,

whereas retinoblastoma protein (pRB) remains in a

phosphorylated state as a result of low p16INK4a

levels and therefore cannot bind to E2F to repress

transcription. (b) Upon oncogenic insult

(e.g. aberrant Ras signalling), pRB becomes

hypophosphorylated, mediated by increased levels

of p16INK4a. Hypophosphorylated pRB is

sequestered to the promoter along with several

other proteins, such as heterochromatin protein 1

(HP1), histone deacetylase (HDAC) and the Lys9

(K9) H3 methyltransferase SUV39H1, which can

bind pRB. (c) This leads to the silencing of gene

expression by altering the chromatin architecture

towards a heterochromatic state by promoting

K9 H3 methylation and removing/inhibiting

euchromatic modifications, as is observed in SAHF

formation. SAHF formation as a mode of gene

expression regulation in response to oncogenic

insult might lead to the onset of premature

senescence and might therefore inhibit

tumourigenic growth.

P

P

PP RB

RB

HP1

E2F

Openchromatinstructure

Closedchromatinstructure

Oncogenic insulte.g. aberrantRas signalling

Increased p16expression

a

b

c

Geneexpression

Generepression

E2F

SAHF formation

SUV39H1 HDAC

RB

HP1HP1

E2F

SUV39H1 HDAC

H3 and H4acetylation

K4 H3methylation

K9 H3methylation

The formation of senescence-associated heterochromatin foci(SAHF) in response to oncogenicinsult






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suggesting that the formation of SAHFs might bedirected towards certain genes and not anindiscriminate global effect. This indicates thatmodification of the chromatin environment of gene promoters is important for the stable

senescence-like arrest attained. It also suggests amodel in which SAHF formation accompanies thesenescence process and indicates a role for pRB-mediated heterochromatin formation at growthregulatory genes during senescence, leading tothe stable repression of these genes. From this itwas further proposed that failure of certainprocesses in these cells (e.g. mutations in genesencoding p16INK4a or pRB, or silencing events)could lead to bypass of senescence andprogression to cancer as a result of the inhibitionof SAHF formation (Ref. 30).

Several other papers have provided more

evidence of the link between chromatinremodelling and senescence. Braig et al. havedemonstrated that oncogenic Ras could inducea senescence-like arrest in mouse primarylymphocytes by a SUV39H1-dependent and K9H3 methylation-related mechanism (Ref. 36). Inthe same study, the deletion of SUV39H1 inprimary lymphocytes led to the impropermethylation of K9 H3 and the promotion of Ras-driven lymphomagenesis. This study alsocorrelates with other work demonstrating thatloss of methylation of K9 H3 and also K20 H4resulted in genomic instability in a variety of

normal cells and was associated with increasedtumour risk (Ref. 37). Decreased expression of SUV39H1 has also been observed in the earlypremalignant stage of tumour development inthe context of hepatocarcinogenesis induced bymethyl deficiency in rats, whereas loss of tri-methylation of K20 H4 was also observed at

both preneoplastic and tumour stages of livercancer and corresponded to a decrease in theK20 H4 histone methyltransferase, SUV420H2(Ref. 38). Interestingly, loss of tri-methylation of K20 H4, as well as loss of acetylated K16 H4

and DNA hypomethylation, has been shown to be a common epigenetic change in tumourigeniccells (i.e. an ‘epigenetic signature’ of cancer)(Ref. 39). These studies provide evidence thatimproper heterochromatin maintenance mightlead to genomic instability and progression totumourigenicity.

Michaloglou et al. have also demonstratedthat BRAFE600-mediated oncogenic signallingin melanocytes induces a senescence-like

phenotype with the formation of SAHF (Ref. 40).BRAF is a protein kinase and downstreamregulator of Ras, and the V600E mutation in thegene encoding BRAF is an oncogenic mutationobserved frequently in human naevi (moles). A

further study focusing on Ras utilised a mousemodel with a conditional oncogenic K-rasV12allele activated by the Cre recombinase (Ref. 41).Upon activation of oncogenic Ras, the micedeveloped multiple lung adenomas (pre-malignant) and a few lung adenocarcinomas(malignant), and markers for senescence wereevident in the adenomas but not in thecarcinomas. Interestingly, HP1g was stronglypositive in the adenomas, whereas thecarcinomas were negative. As the HP1 proteinscan bind methylated K9 H3 to mediate genesilencing, this study further reinforces the role

of epigenetics in senescence. The formation of SAHF has also been shown in response tooverexpression of E2F3 (Ref. 42) and repressionof the E1A (adenovirus 5 early region 1Aprotein)-associated protein p400 (Ref. 43). In thefirst study, which analysed whether deregulatedE2F activity would be sufficient to mimic loss of pRB, short-term deregulation of E2F3 led tohyperplasia of quiescent melanotrophs but didnot lead to tumourigenicity due to the inductionof senescence with the appearance of SAHF(Ref. 42). p400, a protein belonging to the SWI2/SNF2 family of chromatin-remodelling proteins

and postulated to be a transforming agent, wasstudied in relation to the p53/p21 senescencepathway (Ref. 43). When p400 expression wasrepressed in normal human fibroblasts and inhuman fibroblasts transduced with humantelomerase reverse transcriptase (hTERT),senescence was induced along with theappearance of SAHF, suggesting that p400expression could repress p53 induction of p21expression, possibly allowing the bypass of thesenescence arrest.

Further studies into the formation of SAHF

have also indicated roles for the chromatinregulators histone regulating A (HIRA) andanti-silencing factor 1 (ASF1), which wereshown to be rate limiting for the formation of SAHF and were required for senescence(Ref. 44), whereas the high-mobility group A(HMGA) proteins have been shown to be majorstructural components of SAHFs (Ref. 45).HMGA proteins are usually associated withthe formation of chromatin environments




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permissive for gene transcription, but have beenshown to be vital for SAHF formation and generepression in Ras-induced senescence of normalhuman fibroblasts (Ref. 45). Also, similar to therole of pRB, the growth regulatory protein

prohibitin, which has previously been linked tothe regulation of senescence (Ref. 46), has also been shown to localise to SAHFs induced byDNA-damaging agents (Ref. 47). It was shownthat prohibitin plays an integral role in thestability of the senescent phenotype bymaintaining the repression of E2F-responsivepromoters through the maintenance of arepressive chromatin environment by interactionwith HP1g. Reduction in the cellular levels of prohibitin led to a reduction in the numberof SAHFs and impaired the ability of the cells toundergo senescence, further indicating an

integral role for epigenetics in cellular senescence.The formation of SAHF in senescence arrest has

also been studied in vivo in ageing primates(Ref. 48). Increases in the presence of telomere-dysfunction-induced DNA damage foci werecorrelated to increasing age in dermal fibroblastsand, importantly, this was further correlated toSAHF formation. This study indicates thattelomere dysfunction, occurring as a result of telomere attrition with increased cellularproliferation, also induces SAHF formation.Whether this occurs in human cells with ageingis as yet unknown, but this study does indicate

that SAHF formation at senescence is not only aresponse to oncogenic stress, but might also be acommon mechanism in cellular senescence.

In summary, multiple studies have now shownthe strong correlation between the formationof heterochromatin and the attainment andmaintenance of the senescence phenotype.Repression of proliferation-associated genes bythe formation of heterochromatin serves to

block cellular division and so acts as a potenttumour suppressor mechanism. Furthermore,improper regulation of heterochromatin is

associated with deregulated cellular proliferationand tumourigenesis.

Epigenetic modifications by thesenescence mediators p53 and INGSimilar to pRB, other tumour suppressor proteinshave also been shown to interact with histone-modifying enzymes to regulate transcription(Ref. 49). p53 is an important mediator of senescence and one of its main transcriptional

targets is p21. It is known that p53 binds to andactivates the p21 promoter and is mediated, inpart, by increased promoter-proximal histoneacetylation (Ref. 50) through the recruitmentof histone acetyltransferases (HATs) (Ref. 51)

and the displacement of HDAC1 (Ref. 52).Interactions between p53 and the HAT p300 andprotein arginine methyltransferases, which canalso mediate transcriptional activation by post-translational modifications of histones, have also

been reported to be required for p53-dependentgene activation (Ref. 53). However, p53 alsointeracts with histone deacetylases to mediategene repression as it has been shown that p53

binding to promoters was concomitant withincreases in levels of K9 methylation and loss of acetylation of K9 H3 (Refs 54, 55, 56). Given thatepigenetics plays a key role in transcriptional

control by p53, it is important to note thatderegulation of epigenetic mechanisms couldaffect p53-mediated senescence or apoptosis.

Theinhibitor of growth (ING) familyof proteinsare also important regulators of cellularsenescence through the regulation of geneexpression (Ref. 57). Like p53, HATs and HDACshave both been shown to interact with the INGproteins to mediate chromatin-remodellingactivities and gene expression (Ref. 58). Studieshave shown a direct interaction of an ING familymember with chromatin upon senescence,suggesting that ING proteins might be important

mediators of senescence through epigeneticcontrol of gene expression (Refs 59, 60).Furthermore, it has been shown that ING2 can

bind di- and tri-methylated K4 H3 to mediategene repression through histone deacetylation

by associated factors in the mSin3a–HDACrepressor complex (Ref. 61), allowing subsequentrepressive lysine methylation required for theformation of heterochromatin. This reversal of chromatic states (from euchromatic to hetero-chromatic) is an important mechanism to repressproliferation-associated genes at senescence, and

one method to realise such a reversal could be the sequestering to chromatin of ING2-mediated histone deacetylation by a modificationassociated with active gene expression(methylation of K4 H3).

Reversing the irreversible: a newepigenetic mechanism uncoveredMethylation of K9 H3 plays an important role inthe maintenance of gene repression and in




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SAHF-mediated gene repression at senescence.Methylation of K9 H3 was generally understoodto be a stable, long-lasting modification, allowingfor effective gene silencing. However, multiplestudies have now shown that methylation of K9

H3 is readily reversible (Refs 62, 63, 64, 65, 66)and it has been suggested that enzymes thatmediate K9 H3 demethylation could also affecttumourigenesis (Ref. 65). GASC1 (for ‘geneamplified in squamous cell carcinoma 1’; alsoknown as JMJD2C, for ‘jumonji domaincontaining 2 family member C’) is one suchprotein found with the ability to demethylate di-and tri-methylated K9 H3 (Ref. 65). This,coupled with previous findings that GASC1 wasamplified in several carcinoma cell lines and thatinhibition of the protein led to a decrease incellular proliferation, led to the suggestion

that GASC1 could act as an oncogene.Hypothetically, overexpression of GASC1 orother K9 H3 demethylases could lead to thedissolution of SAHF and to the bypass of senescence by the de-repression of proliferation-associated genes. In such a model, the formationof SAHF in response to a senescence-inducingsignal would ‘force’ cells to undergo prematuresenescence and stop possible tumourigenicity.However, if SAHF formation was inhibited bythe overexpression or aberrant localisation of aK9 H3 demethylase, this could allow cells tomove preferentially towards tumourigenesis and

bypass senescence (Fig. 3).

Polycomb group proteins, stem cellsand senescenceAlthough it has been shown that the gain of heterochromatin by increased levels of repressive histone modifications is linked tosenescence, the gain of repressive histone-modifying activity by Polycomb group (PcG)proteins and gene silencing has also beenobserved to allow extension of lifespan and isinvolved in tumourigenesis. There are two

distinct PcG complexes, Polycomb repressivecomplex 1 and 2 (PRC1 and PRC2); thesemultiprotein complexes can repress target geneexpression by catalysing the addition of repressive modifications of histones, such asK27 H3 methylation, and allowing thecompaction and maintenance of chromatin intoa heterochromatic structure.

Loss of function of the PcG protein BMI1 (for‘B-cell-specific Moloney murine leukaemia

virus integration site 1’) was shown to impairthe progression of mouse embryonic fibroblastsinto S-phase; indeed, these cells underwentpremature senescence, thereby linkingtranscriptional repression by PcG proteins with

cell-cycle control and senescence (Ref. 67).Additionally, BMI1 has been shown to bedownregulated in senescing human fibroblasts(Ref. 68), whereas overexpression has beenobserved in tumours and tumour cell lines(Refs 69, 70, 71, 72) and BMI1 is required for theshort-term survival of cancer cells (Ref. 73).

The role of senescence-associatedheterochromatin foci (SAHF) insenescence and tumourigenesis



‘Normal’cell

SAHF formation

Senescence signala

‘Normal’cell

SAHF formation

Senescence signalb

Senescence Tumourigenesis

Figure 3. The role of senescence-associated

heterochromatin foci (SAHF) in senescence and

tumourigenesis. (a) Formation of SAHF after

oncogenic stress in a ‘normal’ cell might promote

the induction of senescence over tumourigenesis

by the repression of specific genes. (b) However,

if SAHF formation is inhibited, potentially by

the reversal of Lys9 (K9) H3 methylation,

tumourigenesis may be promoted owing to

‘blockage’ of the route towards senescence.




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Interestingly, additional studies have shown thatoverexpression of BMI1 allows immortalisationand transformation of some human cells(Refs 67, 74, 75). Importantly, the INK4a locusfrom which the cyclin-dependent kinase

inhibitors p16INK4a

and p14ARF

are transcribedare crucial downstream targets for BMI1 withregard to its effects on cell proliferationand senescence (Ref. 68). Therefore, throughinhibiting the expression of these cyclin-dependent kinase inhibitors, BMI1 can lead tothe promotion of cell-cycle progression.

EZH2 (for ‘enhancer of zeste homolog 2’),another PcG protein, has been shown tomethylate K27 H3 (Ref. 76) and is alsooverexpressed in some cancers, again linking theformation of heterochromatin to the extension of lifespan (Refs 77, 78, 79). EZH2 expression is

also known to be repressed by pRB andactivated p53, suggesting a possible mechanismfor EZH2 overexpression in cancers (Ref. 79)and is further observed to be specificallydownregulated in senescent cells (Ref. 80). Lossof K27 H3 histone methyltransferase activity atsenescence might correspond to loss of heterochromatin and activation of genes suchas those encoding p16INK4a and p14ARF, leadingto cellular senescence (Ref. 81). By contrast,overexpression of EZH2 in cancers might lead tothe proper maintenance of heterochromatin atthe INK4a locus, allowing the elongation of

lifespan in cancer cells by bypassing senescencethrough the repression of expression of genessuch as those encoding p16INK4a and p14ARF.

Complementing these reports have been recentstudies on epigenetics in embryonic stem cells(ESCs) and how epigenetics affects pluripotency;these studies have suggested another route bywhich a normal cell might bypass senescence andgain tumourigenic properties. The chromatinenvironment of ESCs has been shown to behyperdynamic and therefore in a highlypermissive state for transcription. However, as a

cell differentiates along various lineages, specificgenes will become repressed, whereas others arefurther activated, leading to a less dynamic andtherefore less permissive chromatin environment(Refs 82, 83, 84). PRC1 and PRC2 have now

been shown to be integral in maintainingpluripotency by initiating and maintaining thesilencing of genes involved in differentiation bymarking such genes with K27 H3 methylation(Refs 85, 86, 87, 88). Therefore, if any of the

components of PRC1 and PRC2 becomeoverexpressed in a normal cell, genes involved indifferentiation could become epigeneticallysilenced and perhaps allow the reversion of anormal cell towards a more pluripotent state

(Fig. 4). This could allow pluripotent stem-likecells to arise in normal tissues and nucleatecancer growth. Supporting this, as mentionedpreviously, it has been shown that genes suchas those encoding BMI1 and EZH2 areoverexpressed in some cancers (Refs 69, 70, 71, 72,77, 78, 79).

These studies firmly establish a link betweenPcG group proteins, senescence and themodulation of the chromatin environment.Supporting this hypothesis is a recent study of PcG group proteins in prostate cancer (Ref. 89).This study showed that increased expression of

BMI1 and EZH2, with increases in K27 H3methylation and gene silencing, were linked toincreased malignant potential and metastasis inprostate cancer and were also linked to failurein therapy of prostate cancer. Prior work bythe same group also identified an 11-genesignature that was common between normalstem cells with normal self-renewal capacityand malignant tumour cells (Ref. 90). Thesepapers suggest that PcG-mediated genesilencing can mediate increased malignancyperhaps by bestowing stem cell properties topreviously non-metastatic cancer cells with low

malignant potential; this could occur throughthe repression of multiple developmental anddifferentiation pathways by epigenetic changesto gene expression. Thus, PcG-mediatedpathways are now known to be involved in bothnormal stem cell function and highly malignanttumour cells. This reinforces the concept thatregulation of the chromatin environment andgene regulation by the PcG pathway is veryimportant to proper stem cell function and toprevent highly malignant metastatic tumoursfrom arising.

Support for the ‘reversion’ theory has beenshown in a study of committed progenitor cellsexpressing the mixed lineage leukaemia (MLL)–AF9 fusion protein known to impart leukaemiastem cell (LSC) properties (Ref. 91). These cellsgained the expression of a subset of geneshighly expressed in haematopoietic stem cells(HSCs) and also gained self-renewal properties.The MLL protein is a common fusion partnerin leukaemias and is a trithorax group




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(Trx-G) member involved in the transcriptionalactivation of gene expression; MLL is known

to mediate methylation of K4 H3 (Ref. 92).This study is interesting as it links the action of an epigenetic modifier with the reversion of amore committed cell towards a more primitivestem cell type and tumourigenesis.

These studies also raise questions against therising popularity of the stem cell theory of cancer (Ref. 93), by suggesting that somecancers might arise from the gain of pluripotency by deregulation of the chromatinenvironment in normal somatic cells. However,lending support to the cancer stem cellhypothesis is the premise that epigeneticdisruption of stem cells might be a commonunifying theme in cancer aetiology (Ref. 27),which is an attractive proposition. This so-called ‘epigenetic progenitor model’ might helpto explain many different aspects about cancer,

including initiation, progression, and geneticand epigenetic instability. To understand

Figure 4. Attainment of pluripotency in normal

cells: a possible path towards the cancer stem

cell. (a) In embryonic stem cells (ESCs), Polycomb

group (PcG) complexes [Polycomb repressive

complex 1 and 2 (PRC1 and PRC2)] function to

repress differentiation-associated genes by

repressive histone modifications [Lys9 (K9) H3 and

Lys27 (K27) H3 methylation], whereas pluripotency

genes are expressed with the presence of

permissive histone modifications [acetylation of

histones H3 and H4 and methylation of Lys4 (K4)H3]. This allows for the self-renewal capacity

characteristic of stem cells. (b) Loss of the PcG-

mediated repressive modifications leads to the

expression of lineage-specific genes upon

differentiation of ESCs, leading to the cell

becoming more committed to a certain lineage.

Pluripotency genes also become repressed and

therefore these cells lose some ESC

characteristics. (c) Overexpression of PcG proteins

is frequently observed in cancer cells and it is

possible that overexpression of PcG proteins in

normal cells could allow the silencing of lineage-

specific genes involved in differentiation by

repressive histone modifications. (d) This might

also allow the cell to re-express pluripotency-

associated genes and revert back to a pluripotent

state, undergoing deregulated cell proliferation. This

may be an epigenetic mechanism by which normal

cells can bypass senescence and grow without

limit, allowing sustained cell proliferation and self-

renewal. Therefore, this could represent a new

mechanism by which cancer stem cells can arise

and nucleate cancer growth.

Attainment of pluripotency in normalcells: a possible path towards thecancer stem cell



Cancerstemcells?

a

b

c

d

Embryonic stem cells

Lineage-specified cells

Deregulated lineage-specified cells

Pluripotent cancer cell

Differentiation

Tumourigenic event

Attainment ofpluripotency

K27 H3methylation

K9 H3methylation

Pluripotencygenes

Differentiationgenes


K4 H3methylation

PRC1/ PRC2

Normal

cells




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whether one of these models is predominant orwhether they work in conjunction to fostertumourigenesis is an important goal.

Histone acetylation and senescence

As previously mentioned, histone acetylationmight also have an important role in mediatingthe senescence phenotype. The HATs p300and CREB-binding protein (CBP) were bothstudied in senescing human melanocytes and,interestingly, levels of both proteins wereobserved to decrease as these cells underwentreplicative senescence (Refs 94, 95). Thedecrease in the levels of these HATs led todramatic decreases in total histone H4acetylation levels. Specifically, the promoter forthe gene encoding cyclin E was shown to loseH4 acetylation and become repressed upon

senescence, whereas cyclin E was highlyexpressed and H4 acetylation was high inproliferating cells. This parallels previous studiesof the cyclin E promoter during senescence,which showed that pRB and HDACs bound tothe promoter to repress transcription, linking thesenescent phenotype to the pRB/p16INK4a

pathway and the formation of SAHF (Ref. 30). Asmentioned previously, p53 also interacts withenzymes that control histone acetylation,indicating a role for two main senescencemediatorsin the modulationof histoneacetylation.

However, promotion of histone acetylation bythe use of HDAC inhibitors can induce asenescence-like phenotype in human fibroblasts(Refs 96, 97, 98). Furthermore, in senescingnormal human fibroblasts, HDAC1 becomesdownregulated (Ref. 98), indicating that overallthe promotion of acetylation leads to activationof the senescence phenotype. Interestingly,histone deacetylation has also been shown tohave a role in the extension of cellular lifespan.The yeast protein Sir2 (for ‘silent mating typeinformation regulation 2’), identified as aNADþ-dependent HDAC involved in

ribosomal DNA silencing, contributes to thereplicative lifespan of yeast (Refs 99, 100).Enhanced activity of the gene encoding Sir2was also found to be associated with increasedlongevity in Caenorhabditis elegans (Ref. 101),Drosophila (Ref. 102) and rodents (Ref. 103).Drosophila Sir2 has also been shown to beinvolved in epigenetic gene silencing by thePcG proteins and is physically associated with acomplex containing the Drosophila homologue

of EZH2. This further implicates Sir2 in thecontrol of senescence (Ref. 104) and suggests arole for Sir proteins in PcG-mediated generepression that might affect cellular proliferationand lifespan as discussed above. A human

homologue of yeast Sir2, SIRT1, has also beenassociated with longevity and is believed to actprimarily by inhibiting cellular senescence(Refs 105, 106, 107) and overexpression has beenobserved in leukaemia cells (Ref. 108). SIRT1 andother Sir2 homologues are able to deacetylatetranscription factors and p53, and also have K16H4 deacetylase activity (Ref. 109). Recently, aSIRT1 inhibitor, Sirtinol, was shown to induce asenescence-like growth arrest in human cancercells (Ref. 110) in agreement with previous dataindicating that the use of HDAC inhibitors couldlead to induction of the senescence phenotype.

Additionally, loss of histone acetylation has been linked to tumourigenesis throughdecreased K16 H4 acetylation, as well asdecreased tri-methylation of K20 H4 and DNAhypomethylation, as demonstrated in cancercells and tissues (Ref. 39).

These studies suggest that theincreases in globalhistoneacetylation can lead to senescence, perhapsmediated by the re-expression of genes previouslysilenced by heterochromatin that, when activated,promote senescence. By contrast, de-acetylationmight serve to repress genes that contribute tothe senescence phenotype. Again, this shows

that regulation of the chromatin environment iscrucial for proper cellular control.

DNA methylation and senescenceGenerally, histone modifications and DNAmethylation are often discussed separately inrelation to their effects on gene expression,although it is now known that they are in factintimately linked (Refs 111, 112, 113, 114), withmultiple interactions now known betweenproteins associated with histone modificationsand those associated with DNA methylation.

Generally, DNA methylation of CpG islands(regions of adjacent cytosine and guanineresidues linked by phosphodiester bonds) atgene promoters is correlated with generepression, whereas expression is linked to thelack of methylation. Global DNA methylationhas been shown to decrease with age (Ref. 115)and this loss had been hypothesised to functionas a counting mechanism for senescence, suchthat senescence would occur when a cell loses a




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certain degree of DNA methylation (Refs 115,116). Treatment with 5-azadeoxycytidine(5-azadC), a DNA demethylating agent, has

been shown to reduce the replicative lifespan of normal human fibroblasts, supporting the DNA

methylation counting mechanism hypothesis(Refs 117, 118). Also in agreement with thishypothesis are the findings that DNAmethyltransferase activity is elevated in cancercells in vitro, in tumours in vivo (Ref. 119) andin transformed cells (Refs 120, 121), whereasDNA methyltransferase activity decreases inserially passaged normal human fibroblasts(Ref. 122). Therefore, the loss of DNAmethylation with age might eventually havedeleterious consequences, such as deregulationof gene expression. Consistent with this view, ithas been hypothesised that DNA methylation

might repress a set of growth inhibitory genes(Refs 24, 123) and, as the number of cellpopulation doublings increases and DNAmethylation decreases, the repression of suchgrowth inhibitory genes might become lessstringent, leading to their eventual expressionand contributing to senescence. However,it has also been shown that a striking feature of some tumours (both benign and malignant) isthat global DNA methylation is reduced(Refs 124, 125). This loss of DNA methylationis also linked to chromosomal instability,which can contribute to tumourigenesis

(Refs 126, 127). Thus, paradoxically, it has beenshown that the loss of DNA methylation cancontribute to senescence and tumourigenicity.However, it is acknowledged that gene-specificmethylation is variable in cancer and that manytumour suppressor genes show promoter-specific DNA hypermethylation leading togene silencing, including the genes encodingpRB (Ref. 128) and p16INK4a (Ref. 129), whereasthe genome is hypomethylated overall.Interestingly, recent data have shown thatp16INK4a is crucially involved in DNA

methylation, leading to gene silencing and breasttumourigenesis (Ref. 130). Loss of p16INK4a leadsto the increased expression of key PcG proteins,resulting in a specific pattern of DNAhypermethylation and importantly the silencingof the gene encoding HOXA9, a transcriptionfactor that is epigenetically silenced in breasttumourigenesis. Therefore, it is likely thatglobal loss of DNA methylation leading tochromosomal instability could trigger cellular

senescence and that promoter-specific alterationsin key tumour suppressor genes might allow the

bypass of senescence and lead to tumourigenicity.

Senescence and DNA damage detection

by histone modificationHistone modifications can also play a role in thedetection and repair of DNA damage that, aspreviously mentioned, can lead to prematuresenescence. DNA damage can be incurredthrough the actions of various types of radiation(Ref. 131), DNA-damaging drugs (many of which are used as anti-tumourigenic therapies)(Ref. 132) and oxidative stress (Ref. 133), andhistone phosphorylation is now know to play arole in mediating cellular responses to this DNAdamage. Signalling kinases, notably ATM (for‘ataxia telangiectasia mutated’) and ATR (for

‘ataxia telangiectasia and Rad3 related’), arerecruited to the site of damage and are activated,leading to phosphorylation of histone H2AXmolecules (g-H2AX) (Ref. 134). Histone H2AX isa variant of the H2A histone that makes up partof the histone octamer. The phosphorylation of histone H2AX facilitates the recruitment of checkpoint and DNA repair factors includingp53-binding protein 1 (153BP1), and promotesthe activation of checkpoint kinase 1 (Chk1) andChk2, which can activate p53 and therebymediate cellular senescence. Furthermore, it isknown that the uncapping of telomeres can leadto senescence through a DNA damage responsemediated by a similar mechanism (Refs 135, 136).Phosphorylated histone H2AX also serves torecruit histone modifiers and ATP-dependentchromatin remodellers to these sites of DNAdamage and aid the repair of these sites (Ref. 137).

Two recent studies have uncovered a link between histone lysine methylation and theDNA damage checkpoint and double-strand

break (DSB) repair proteins 53BP1 in mammalsand Schizosaccharomyces pombe putative homologCrb2 (for ‘Crumbs-like protein 2’): the

relocalisation of 53BP1 and Crb2 to DNA DSBshas been found to require histone methylation(Refs 138, 139). It was proposed that 53BP1senses DSBs through changes in higher-orderchromatin structure that occur upon exposureof the 53BP1 binding site [methylated Lys79 H3(K79 H3), which lies in the globular domain of histone H3] (Ref. 138). In S. pombe, a similarmechanism occurs with Crb2 binding tomethylated K20 (Ref. 139). However, recent




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studies have shown that in fact 53BP1 and Crb2 bind to the same modified lysine residue, di-methylated K20 H4 (Ref. 140).

Epigenetic regulation of telomerase

gene expression and senescenceTelomerase and telomeres in senescenceIn adult humans, most normalcells have lowor nodetectable telomerase activity (Ref. 141). The lackof telomerase activity, or other telomeremaintenance mechanisms, leads to the loss of around 50–100 bp of telomeric sequence at eachcell division (Refs 142, 143). The loss of telomeric tract during cell division presents alimit to the maximum number of divisions acell can undertake before entering senescenceand therefore provides a limit to its lifespan(Ref. 144). Cellular senescence as a response to

telomere attrition is thought to have evolved asa tumour suppressive barrier against excessiveclonal expansion. However, the detection of telomerase activity in an estimated 85% of malignant tumours, together with the stronglinks between telomerase activity and malignantcells (Refs 141, 145), indicates the importance of telomerase activity in tumourigenesis and alsosuggests that telomerase activation is the mostcommon mechanism for telomere maintenancein cancer cells. However, several immortal celllines and tumours lack telomerase activity, yethave the ability to elongate their telomeres.Such non-telomerase-dependent mechanisms of telomere maintenance include the alternativelengthening of telomeres (ALT) pathway(Ref. 146) (Fig. 1).

In cells where ALT is utilised, telomere length ismaintained by a recombination-based DNAreplication mechanism (Ref. 147) and leads toheterogeneity in telomere lengths (Ref. 148).Also associated with use of the ALT mechanismis the presence of ALT-associated promyelocyticleukemia protein (PML) bodies, called APBs(Ref. 149), which contain the protein PML,

telomeric DNA, the telomeric-binding proteinstelomere repeat factor 1 (TRF1) and TRF2, andproteins involved in DNA recombination andreplication. The utilisation of ALT in tumourcells is much rarer than the utilisation of telomerase activity (Ref. 148) and it has beenhypothesised that ALT might normally occurunder selective pressure when expression of telomerase is prevented. ALT is commonlyactivated in tumours of mesenchymal origin,

including osteosarcomas, soft tissue sarcomasand glioblastoma multiforme (Refs 150, 151,152, 153). Furthermore, in normal mesenchymaltissues, telomerase activity is more tightlyrepressed that in epithelial cells, possibly leading

to the selective pressure for ALT activation overtelomerase activity in these cell types.

Epigenetic regulation of telomeremaintenance mechanismsExpression of the human telomerase RNAcomponent (hTR) and hTERT are understood to

be the minimal requirements for telomeraseactivity. Studies have shown that specificepigenetic modifications at the hTR and hTERTpromoters correlate with expression orrepression of hTR and hTERT (Refs 154, 155).Lack of expression of hTR in ALT cell lines, and

hTERT in ALT and normal cell lines, isassociated with a repressive chromatinenvironment at the appropriate gene promoter.Conversely, expression of hTR in ALT andtelomerase-positive cell lines, and hTERTexpression in telomerase-positive cell lines, isassociated with a more permissive chromatinenvironment at the appropriate gene promoter.Moreover, in the pre-senescent normal fibroblastcell line WI38, which has no detectable hTERTexpression and low hTR expression, thetelomerase gene promoters contain no greatquantities of either positive or negativechromatin modifications, and therefore thechromatin environment of the promoter remainsin a neutral chromatin configuration (Fig. 5a).

Furthermore, upon comparison of the hTERTpromoter chromatin environment in ALT celllines (including a cell transformed from theparental WI38 cell line WI38-SV40) withthe normal WI38 cell line, an increase in theamounts of repressive modifications is evidentand links the heavy repression of hTERTexpression by the chromatin environment to theALT phenotype. In the telomerase-positive

tumour cell lines, permissive modifications aregained at the hTERT promoter relative to theWI38 cell line. Similarly, heavy repression of hTR is evident in some ALT cell lines with nohTR expression, whereas high levels of permissive chromatin modifications are linkedto hTR expression. Additionally, treatment of normal and ALT cells with 5-azadC incombination with the HDAC inhibitortrichostatin A (TSA) caused chromatin




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remodelling of both promoters and led to theformation of a more permissive chromatinenvironment, as well as reactivation orincreased expression of hTR and hTERTexpression. This suggests a model by which

gain of permissive modifications at thetelomerase gene promoters leads to telomeraseactivity by the expression of hTR and hTERT,whereas the gain of repressive modificationsleads to activation of the ALT mechanism(Fig. 5b). Therefore, epigenetic deregulation of

Figure 5. Epigenetic regulation of telomere

maintenance mechanisms. (a) Histograms

showing a simplistic view of what we know about

the relationship between the relative levels of

repressive and permissive chromatin modifications

at the telomerase gene promoters in telomerase-

expressing cancer cells, pre-senescent normalcells and cells that maintain their telomeres by the

alternative lengthening of telomeres (ALT)

mechanism. The presence of permissive histone

modifications at the telomerase gene promoters is

linked to telomerase activity, whereas the presence

of repressive histone modifications is linked to the

activation of ALT. Both of these scenarios are in

contrast to the chromatin environment of pre-

senescent normal cells, in which permissive and

repressive modifications are at a minimum. This

suggests a model by which epigenetic deregulation

of the telomerase gene promoters could lead to

aberrant telomere maintenance. (b) Model depicting

how chromatin modifications might influencetelomere maintenance mechanisms. In a ‘normal’

cell that does not have telomerase activity and has

no or low levels of expression of the telomerase

genes, few chromatin modifications are present at

the gene promoters. The choice of telomere

maintenance mechanism may be influenced

through epigenetic mechanisms and changes

in histone modifications, possibly during

tumourigenesis. In the left-hand pathway shown,

telomerase gene expression is linked to the

accumulation of permissive histone modifications

[acetylation of histones H3 and H4 and methylation

of Lys4 (K4) H3], which leads to telomerase activity

and the elongation/maintenance of telomerelength. However, in the right-hand pathway shown,

heavy repression of the telomerase gene promoters

by the accumulation of repressive histone

modifications [methylation of Lys20 (K20) H4 and/or methylation of Lys9 (K9) H3] could lead to

utilisation of the ALT mechanism rather than

telomerase activity for telomere elongation/maintenance owing to the repression of telomerase

gene expression.

Epigenetic regulation of telomeremaintenance mechanisms



Telomeraseactivity

Pre-senescentnormal cell

Normal

Epigeneticmechanisms

Telomeraseactivity

Cancer ALT

Telomerase generepression

ALT

Permissivemodifications

Repressivemodifications

Telomerase generepression

Telomerase geneexpression

b

a

K20 H4methylation K9 H3methylation


K4 H3methylation




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the telomerase gene promoters might allow theactivation of a telomere maintenancemechanism, allowing bypass of senescence.

Epigenetic deregulation and

tumourigenicity in adult stem cellsFurther studies into the epigenetic regulation of the telomerase gene promoters utilised humanmesenchymal stem cells (hMSCs) (Ref. 156).These cells do not show telomerase activity buthave been shown to be the target for neoplastictransformation after transduction with hTERT(Ref. 155). However, by supplying hTERT, themolecular events required to upregulate hTERTexpression in cancer stem cell development aremissed. Therefore, hMSCs are ideal for theidentification of molecular mechanismsregulating telomerase gene expression in stem

cells.It has now been shown that repression of

hTERT expression in hMSCs is a result of promoter-specific histone hypo-acetylationcoupled with low RNA polymerase II (RNAPII)and transcription factor IIB (TFIIB) tracking.This repression can be overcome by treatmentof hMSCs with TSA, concomitant with increasesin promoter-specific histone acetylation andincreases in RNAPII and TFIIB tracking. hTRexpression is also increased in TSA-treatedhMSCs, concomitant with changes in RNAPIIand TFIIB dynamics. These data suggestthat deregulation of chromatin modificationsat the hTERT and hTR promoter mightfacilitate tumourigenesis in hMSCs and supportsthe hypothesis that tumourigenesis can arise inepigenetically deregulated stem cell populations.

Epigenetic regulation of telomere lengthIt has been shown that epigenetic modificationscan also regulate telomere length through themodulation of the chromatin environment of the telomeres themselves. Telomeres are rich inheterochromatin features (Ref. 157) and

disruption of this heterochromatin structure has been shown to lead to altered telomere lengthin mice, established by knockout of theSUV39H1 and SUV39H2 histone methyltransferases in mice (Ref. 158) and by theknockout of either DNA methyltransferase 1(DNMT1) or both DNMT-3a and -3b in mouseESCs (Ref. 159). In the latter study, the lack of DNMTs led to increased intra-telomericrecombination and increased the levels of other

markers of the ALT telomere maintenancemechanism, further suggesting the role forepigenetics in the choice of telomeremaintenance mechanism used in cancer cells.This also shows that epigenetic deregulation can

lead to immortalisation events and mightfacilitate tumourigenesis.

Therapeutic aspects of chromatin-mediated senescence

The intimate links between senescence andchromatin that have been uncovered mayprovide therapeutic targets for anti-cancertreatment. As induction of prematuresenescence in tumour cells is targeted as ananti-cancer therapy (Refs 4, 5), one therapeuticavenue may be to induce premature cellularsenescence through control of the enzymes that

catalyse epigenetic modifications. Intimateknowledge of gene expression changes insenescence might allow targeted upregulationor downregulation of such genes by epigenetictherapies. Also, epigenetic therapy might allowthe reversal of senescence in certain cell typesassociated with senescence-related disease.However, it must be remembered that cellularsenescence also plays roles in multiple otherdisease states and might potentially nurturetumourigenic growth. Overall, it can beappreciated that this mode of therapy wouldrequire strict control.

Current epigenetic therapies include HDACand DNMT inhibitors (Table 3), which promotere-expression of silenced genes and are widelyused in cancer therapies to facilitate there-expression of tumour suppressor genes.However useful these drugs are, other more-targeted means of enzyme function ormodulating gene expression would beadvantageous. As these drugs are not targetedthey will have pan-genomic effects and couldnurture tumour growth by aberrant activation of growth-promoting genes or cancer-associated

genes, further indicating a requirement for strictcontrol for such epigenetic therapies. The use of therapeutic RNA interference (RNAi) can allowgreater targeting of specific enzymes involved inepigenetic processes, whereas changes in specificgene expression by the use of engineeredtranscription factors is an exciting newmechanism of gene control (Ref. 160). By fusingepigenetic modifiers to engineered transcriptionfactors that can bind to specific gene promoters,




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gene expression can be altered through epigeneticmechanisms. However, the ultimate therapeuticaim is to find small molecules that could besuccessfully moved into the clinical arena. Thisapproach has recently been successfully used inone study that screened for compounds thatwere able to modulate cellular senescence(Ref. 161) and could allow further detailedanalysis of the senescence pathway andepigenetic mechanisms important to it.

Epigenetic therapy might also allow the

targeting of cancer stem cells as current cancertherapeutics may not efficiently target such cellsdue to their unique cellular physiology. Recentstudies demonstrating the importance of epigenetic silencers in the maintenance of pluripotency and a proposed role intumourigenesis suggests that it may be possibleto target such cells more efficiently throughtargeting epigenetic silencers. Furthermore,studies have shown that the PcG proteins may

be regulated by signalling pathways unique topluripotent cells and targeting of thesepathways might allow modulation of epigeneticsilencing and hence pluripotency, and thereforemay have direct relevance to the treatment of cancer stem cells (Refs 162, 163, 164). Molecularsignalling pathways such as PTEN (for‘phosphate and tensin homologue’), Wnt,Hedgehog and Notch have been identified astargets for therapies aimed at cancer stem cellsand therefore targeted gene expression changes

of members of these pathways by epigenetictherapies may allow for selectivity (Ref. 165).However, effects on normal stem cells mightalso become compromised. Studies havesuggested other signalling pathways that couldaffect epigenetic modifications and thereforeprovide suitable therapeutic targets. A recentstudy into the Janus kinases/signal transducersand activators of transcription (JAK/STAT)pathway in Drosophila, a pathway whose

Table 3. Current drugs against epigenetic modifiers

Epigenetic therapy Mode of action

S-adenosylhomocysteine Inhibits S-adenosyl-L-methionine (SAM)-dependent

methyltransferases

5-azacytidine (azacytidine) DNA methyltransferase (DNMT) inhibitor (nucleosideanalogue)

5-aza-20-deoxycytidine (decitabine) DNMT inhibitor (nucleoside analogue)

1-( b-D-ribofuranosyl)-1,2-

dihydropyrimidin-2-one (zebularine)

DNMT inhibitor (nucleoside analogue)

(-)Epigallocatechin-3 gallate (EGCG) DNMT inhibitor (nucleoside analogue)

Procaine DNMT inhibitor (nucleoside analogue)

Procainamide DNMT inhibitor (nucleoside analogue)

Hydralazine DNMT inhibitor (nucleoside analogue)

MG98 DNMT1 inhibitor (second-generation phosphorothioate

antisense oligodeoxynucleotide)

Trichostatin A (TSA) Hydroxamate histone deacetylase (HDAC) inhibitor

Suberoylanilide hydroxamic acid (SAHA) Hydroxamate HDAC inhibitor

m-Carboxycinnamic acid bishydroxamide

(CBHA)

Hydroxamate HDAC inhibitor

LAQ824 Hydroxamate HDAC inhibitor

PXD101 Hydroxamate HDAC inhibitor

Depsipeptide Cyclic peptide HDAC inhibitor

Apicidin Cyclic peptide HDAC inhibitor

Cyclic hydroxamic-acid-containing

peptides (CHAPs)

Cyclic peptide HDAC inhibitor

Valproic acid Aliphatic acid HDAC inhibitor

Phenylbutyrate Aliphatic acid HDAC inhibitor

MS-275 Benzamide HDAC inhibitor

CI-994 Benzamide HDAC inhibitor

Trifluoromethyl ketones Electrophilic ketone HDAC inhibitor

Alpha-ketoamides Electrophilic ketone HDAC inhibitor




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deregulation has been implicated in human cancer(Ref. 166), has shown that over-activation of JAKkinase disrupted heterochromatin formation andchanged the expression of genes through STAT-independent routes. Further understanding of

pathways that control epigenetic modifiers couldrepresent a wealth of opportunity for discovery of therapeutic targets.

Data establishing epigenetic processes asimportant regulators of senescence andtumourigenesis also suggest that such epigeneticmodifications may be useful as prognosticmarkers. Expression of genes involved inepigenetic mechanisms have been shown to beassociated with therapy failure in prostate cancer(Ref. 89) and the presence of certain patterns of global histone modifications have been linked torecurrence of prostate cancer (Ref. 167). Loss of

lysine acetylation and methylation on histone H4has also been identified as an epigeneticsignature of human cancer (Ref. 39), whereasother studies have identified the loss of repressive lysine methylation and the enzymesthat catalyse the addition of these modificationsas early markers of tumourigenesis (Refs 37, 38).Further levels of methylation status of K27 H3might allow the differentiation between weaklyand strongly malignant tumour cells (Ref. 89).Therefore, analysis and detection of suchmodifications and enzymes could be importantin early cancer detection and might also allow

tailored epigenetic therapies. Furthermore, aschemotherapeutics often promote prematurecellular senescence as an anti-cancer therapy andsenescence-associated chromatin changes arenow understood, such changes might be usedas markers of response to chemotherapeutictherapies (Ref. 168).

Conclusions and summary It is now accepted that epigenetic mechanisms arevital regulators of gene expression and thederegulation of epigenetic mechanisms has been

linked to cancer in multiple studies. Pathwaysand mechanisms important to the senescencephenotype have all been shown to regulate or beregulated by epigenetic mechanisms, underlyingthe importance of this level of control.Epigenetics provides the perfect regulatorymechanism for dynamic changes in globalgene expression required for multiple cellularprocesses such as senescence. Deregulation of epigenetic modifications may therefore promote

tumourigenesis through the disruption of thesenescence phenotype and the deregulation of gene expression.

How does the research now proceed? The roleepigenetics has to play in cellular processes such

as senescence, tumourigenesis and stem cellphenotypes has only recently been uncoveredin any great detail. In addition, epigeneticregulation is key to many other cellularprocesses, whereas deregulation is an importantfactor in numerous disease states. Furtheranalysis of global states of chromatin indifferent cell types and comparisons betweennormal and diseased tissues might allowgreater understanding of the causes of thedisease and also potentially lead to thediscovery of therapeutic targets. Such studieshave already begun, as mentioned, in ESCs

(Refs 85, 86, 87, 88) and also in cancer,including the exciting prospect of the humanepigenome project, an international projectaiming to identify all the chemical changes andrelationships among chromatin constituents thatprovide function to the DNA code (Refs 39, 167,169, 170, 171). Such studies will allow us tounderstand how chromatin modificationsmight control distinct cellular processes andwhat enzymes mediate such changes. Fullunderstanding of the signalling pathways thatmodulate enzymatic control is also animportant step to begin to uncover how the

deregulation of these pathways affects theepigenetic control of gene expression andcellular functions, and also potentially to enablethe discovery of novel therapeutic targets.

Acknowledgements and fundingResearch in the authors’ laboratory is supported

by Cancer Research UK, University of Glasgowand European Community grant LSHC-CT-2004-502943. The authors thank the anonymouspeer reviewers for their invaluable comments.

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Further reading, resources and contacts

The Cancer Research UK Laboratories in Glasgow, UK, houses nearly 20 research groups working towards

understanding cancer growth and its treatment:

http://www.beatson.gla.ac.uk /

Antibody and protocol resources

Both Upstate and Abcam are providers of ChIP-grade antibodies and are widely used in the chromatin field

http://www.abcam.com

http://www.upstate.com

Upstate also hosts a new informational resource that details significant developments within the field of

chromatin research:

http://www.histone.com

Chromatin resources

The Chromatin Structure and Function website has excellent links to researchers, journals, search tools and

other related topics. It also includes a mailing list for recent chromatin papers from all the major journals:

http://www.chromatin.us/chrom.html

The Human Epigenome Project homepage:

http://www.epigenome.org/

Chromatin.com keeps up to date with allthe recent high-impact journal articles in thefield of chromatin andalso

with chromatin-related meetings:

http://www.chromatin.com

Other useful resources

The biopharmaceutical company Geron is involved in drug discovery for regenerative medicine and oncology:

http://www.geron.com/

Various laboratory homepages with excellent links on senescence, telomerase and telomeres, further resources

and journal articles:

Jerry Shay’s and Woodring Wright’s laboratory homepage:

http://www4.utsouthwestern.edu/cellbio/shay-wright/index.html

Igor Roninson’s laboratory homepage

http://www.ordwayresearch.org/profile_roninson.html

Judith Campisi’s laboratory homepage

http://www.lbl.gov/lifesciences/CMB/Campisi.html




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Features associated with this article

Figures

Figure 1. Routes to immortality.

Figure 2. The formation of senescence-associated heterochromatin foci (SAHF) in response to oncogenic insult.

Figure 3. The role of senescence-associated heterochromatin foci (SAHF) in senescence and tumourigenesis.Figure 4. Attainment of pluripotency in normal cells: a possible path towards the cancer stem cell.

Figure 5. Epigenetic regulation of telomere maintenance mechanisms.

Tables

Table 1. Senescence and disease.

Table 2. Chromatin and disease.

Table 3. Current drugs against epigenetic modifiers.

Citation details for this article

Stuart P. Atkinson and W. Nicol Keith (2007) Epigenetic control of cellular senescence in disease: opportunities

for therapeutic intervention. Expert Rev. Mol. Med. Vol. 9, Issue 7, March 2007, DOI: 10.1017/ S1462399407000269


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Senescence in Disease