Oncogene (2011) 30, 2901–2911
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
www.nature.com/onc
REVIEW
Transcriptional regulation of cellular senescence
F Lanigan1, JG Geraghty3 and AP Bracken1,2
1
Smurfit Genetics Department, The Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland; 2The Adelaide and Meath
Hospital, including The National Children’s Hospital, Dublin, Ireland and 3Department of Breast and Endocrine Surgery, St Vincent’s
Hospital, University College Dublin, Dublin, Ireland
Cellular senescence is an irreversible arrest of proliferation. It is activated when a cell encounters stress such as
DNA damage, telomere shortening or oncogene activation. Like apoptosis, it impedes tumour progression and
acts as a barrier that pre-neoplastic cells must overcome
during their evolution toward the full tumourigenic state.
This review focuses on the role of transcriptional
regulators in the control of cellular senescence, explores
how their function is perturbed in cancer and discusses the
potential to harness this knowledge for future cancer
therapies.
Oncogene (2011) 30, 2901–2911; doi:10.1038/onc.2011.34;
published online 7 March 2011
Keywords:
therapy
cellular senescence; transcription; cancer
Introduction
The initial discovery of cellular senescence dates back to
the demonstration that primary human diploid fibroblasts, when cultured in vitro, have a finite capacity for
growth, followed by an irreversible arrest of the cell cycle
(Hayflick and Moorhead, 1961). This phenomenon,
known as ‘replicative senescence’ because it correlates
with telomere attrition (Harley et al., 1990), has since
been observed in multiple cell types from several different
organisms (Collado and Serrano, 2010). The definition of
cellular senescence has expanded to include the phenotypically similar growth arrest caused by various other
sources of cellular stress, including DNA damage and
oxidative stress (Serrano and Blasco, 2001). Another
form, called oncogene-induced cellular senescence, was
first described in mouse and human primary cultured
fibroblasts overexpressing RasV12 (Serrano et al., 1997).
These cells underwent a growth arrest, accompanied by
morphological changes typically associated with cellular
senescence, including a large, flattened cellular morphology, and positive staining for senescence-associated bgalactosidase (Kuilman et al., 2010).
Correspondence: Dr AP Bracken, Smurfit Genetics Department, The
Smurfit Institute of Genetics, Trinity College Dublin, Lincoln Place,
Dublin 2, Ireland.
E-mail: [email protected]
Received 28 October 2010; revised and accepted 19 January 2011;
published online 7 March 2011
The in vivo relevance of cellular senescence as a barrier
to tumour progression was first shown in a transgenic
model of the E2F3 transcription factor (Lazzerini
Denchi et al., 2005). When ectopically expressed in the
pituitary gland, E2F3 led to initial hyperplasia followed
by the induction of cellular senescence. The subsequent
discovery of senescent cells in growth-arrested human
benign naevi, or moles, which frequently possess
oncogenic mutations in the protein kinase BRAF, but
rarely develop into malignant melanomas, was another
striking example (Michaloglou et al., 2005). A concurrent study, in which RasV12 was conditionally
expressed in the mouse lung, also showed the relevance
of cellular senescence in vivo (Collado et al., 2005). These
mice developed multiple pre-neoplastic lung adenomas,
and displayed infrequent progression to neoplastic
adenomas. Significantly, while the pre-malignant cells
displayed markers of cellular senescence, these markers
were absent in the malignant adenocarcinoma cells.
Several studies have gone on to firmly establish that the
cellular senescence response is a key tumour suppressive
mechanism that acts to block the progression of preneoplastic lesions to full blown prostate (Chen et al.,
2005; Majumder et al., 2008), colon (Bartkova et al.,
2006; Kuilman et al., 2008; Fujita et al., 2009; Bennecke
et al., 2010), lymph (Braig et al., 2005), and breast
(Swarbrick et al., 2008) cancers. Taken together, these
studies have established that cellular senescence is, like
apoptosis, an inherent cellular defense that acts as a
barrier to tumour progression.
This review will describe the key molecular pathways
that control cellular senescence, focusing on recent
findings with regard to the transcriptional regulation of
these pathways. We will discuss how these transcriptional regulators are perturbed in human cancer and
how molecular insights into their function can be best
exploited for future cancer therapies.
Molecular pathways controlling cellular senescence
The retinoblastoma protein (pRb) and p53 tumour
suppressor pathways represent the core signalling pathways
involved in the induction of cellular senescence (Lowe and
Sherr, 2003; Narita and Lowe, 2005; Collado and Serrano,
2010). Consistent with this, members of these pathways are
well-known oncogenes and tumour suppressors and are
frequently genetically disrupted in cancer (Figure 1).
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Figure 1 Molecular mechanisms of cellular senescence. The p53
and pRb pathways are central to cellular senescence control.
Recent evidence suggests that multiple types of cellular stress can
activate the p53 pathway by common or at least partially
overlapping mechanisms. Telomere attrition, radiation, cytotoxic
drugs and oncogene-induced DNA replication stress have all been
shown to induce the DNA damage response (DDR), resulting in
the activation of the checkpoint kinases ATM/ATR and CHK1/2,
and leading to p53 accumulation. p53 can also be activated
downstream of p14ARF, which binds to the E3 ubiquitin ligase,
MDM2, preventing the degradation of p53. MDM2 can directly
bind to p53 and control its transcriptional function, as well as
regulate degradation of p53 through ubiquitination. This stabilisation and accumulation of p53 allows the activation of downstream
genes such as p21CIP1, and the induction of cellular senescence or
apoptosis, depending on the cellular context. The p16INK4a tumour
suppressor, transcriptionally upregulated in stressed cells, inhibits
cyclin D-dependent kinases, thereby preventing the phosphorylation and inactivation of the retinoblastoma protein, pRb. This
promotes the repressive association between pRb and the E2F1-3
pro-proliferative transcriptional activators, preventing progression
through the cell cycle, and permitting the formation of a repressive
or ‘closed’ chromatin structure around E2F target gene promoters,
known as SAHF. The mechanisms of transcriptional regulation of
p16INK4a and p14ARF in senescent human cells are not yet fully
understood (red, tumour suppressors; green, oncogenes).
The tumour suppressive p53 transcription factor is
required for cellular senescence, and mutation or
deletion of the TP53 gene occurs in more than half of
human cancers (Hollstein et al., 1994). Wild-type p53
accumulates in response to cellular stresses, and
activates a specific program of target genes to restrict
the growth of abnormal or damaged cells by inducing
one of three outcomes: apoptosis, a transient cell cycle
arrest or a permanent cell cycle arrest (cellular
senescence). The outcome reached is thought to depend
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on the type and level of stress signal involved, and the
cellular context (Riley et al., 2008; Vousden and Prives,
2009). Transcriptional target genes of p53 include the
cyclin-dependent kinase (CDK) inhibitor p21, the proapoptotic genes BAX and APAF1, and the E3 ubiquitin
ligase MDM2 (Vousden and Lu, 2002; Riley et al.,
2008). MDM2 negatively regulates the levels of p53,
forming a regulatory feedback loop (Haupt et al., 1997;
Kubbutat et al., 1997). The upstream p14Arf protein
(p19Arf in mice) in turn prevents the degradation of p53
(Sherr, 2006). It is not fully understood how p53 plays a
role in cellular senescence, but its ability to transactivate
the p21 promoter, leading to the accumulation of p21
protein, is well established as a means by which p53 can
induce growth arrest (Levine, 1997; Vousden and Prives,
2009). The p21 protein primarily acts by the inhibition
of cyclin/CDK complexes upstream of pRb. This
inhibition leads to a reduction in pRb phosphorylation,
increased association of pRb with E2F1-3 and repression of E2F target genes. Interestingly, in mouse
fibroblasts, the loss of p21 does not recapitulate p53
loss, and these cells still undergo cellular senescence
(Pantoja and Serrano, 1999). In contrast, in human
fibroblasts, p21 loss was reported to lead to immortalisation (Brown et al., 1997). However, it was not clear if
p53 was functional in the cells used in this study.
Therefore, it seems likely that p53 has additional target
genes that are important in cellular senescence control.
Two potential candidates are the direct p53 target genes
PAI-1 and miR-34 (Kortlever et al., 2006; He et al.,
2007). Supporting this role, the suppression of PAI-1
confers bypass of senescence, whereas ectopic expression
of either PAI-1 or miR34 leads to the induction of
cellular senescence.
The RB1 gene (pRb protein) was initially identified as
a tumour suppressor owing to its inactivation in a
childhood eye tumour called retinoblastoma (Burkhart
and Sage, 2008). Its tumour suppressor activity is mainly
attributed to its ability to bind and inactivate the E2F
family of transcription factors, which transactivate
genes encoding cell cycle proteins and DNA replication
factors required for cell growth (Bracken et al., 2004;
Blais and Dynlacht, 2007). pRb is a member of the
pocket protein family along with the related proteins
p107 and p130 (Classon and Harlow, 2002). The pocket
proteins are substrates for cyclin/CDK complexes,
which phosphorylate them, releasing the E2F transcription factors, and allowing progression through the cell
cycle (Classon et al., 2000). The CDKs are themselves
inhibited by the CDK inhibitors p16INK4a and p15INK4b,
both of which are upregulated during cellular senescence
(Collado and Serrano, 2006), leading to reduced
phosphorylation of pRb, and E2F inactivation. This
results in the accumulation of heterochromatin around
E2F-responsive promoters in senescent cells, stably
silencing E2F-regulated genes and forming what are
known as senescence-associated heterochromatic foci
(SAHF) (Narita et al., 2003).
It is likely that pRb possesses more tumour suppressive activity than the other pocket proteins, as mutations
affecting p107 and p130 are very rarely observed in
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human cancers (Burkhart and Sage, 2008). Indeed, pRb
seems to play a non-redundant role in tumour suppression by permanent repression of E2F target genes during
cellular senescence, but not quiescence, meaning that
loss of pRb, but not p107 or p130, results in a defective
senescence response (Sage et al., 2003; Chicas et al.,
2010).
The INK4A-ARF locus
Strikingly, both the p16INK4a and p14Arf proteins are
encoded by the same alternatively spliced gene locus,
called the CDKN2A or INK4A-ARF locus (Quelle et al.,
1995; Gil and Peters, 2006). Although it is unclear as to
how exactly the unusual gene arrangement of INK4AARF came to be, its design means that, by mutating or
silencing this locus, cells can simultaneously disrupt the
Rb and p53 pathways, thus bypassing cellular senescence. It is therefore not surprising that this is one of the
most commonly mutated, deleted and epigenetically
silenced genomic regions in human tumours (Gil and
Peters, 2006; Esteller, 2008).
To date, many of our insights into the role of the
INK4A-ARF locus in cellular senescence have been
derived from mouse models or mouse primary culture
lines. However, the main difficulty in using mice to
define cellular senescence pathways in tumourigenesis is
that, although in mice, the bypass of cellular senescence
appears to occur primarily through loss of the Arf-p53
signalling axis, the INK4A-pRb pathway seems to be
more important in human cells. This is supported by
epidemiological data showing high levels of mutations
affecting p16INK4A as opposed to p14ARF in human
tumours (Gil and Peters, 2006). Voorhoeve and Agami
(2003) elegantly addressed this question in vitro in
primary human fibroblasts using small interfering
RNAs targeted to p16INK4A and p14Arf separately. They
found that loss of p16INK4a and p53 can synergise to
promote transformation, whereas loss of p14Arf, while
enhancing the rate of proliferation, does not replace loss
of p53 in a transformation assay. Taken together, these
data suggest that p16INK4a is the major tumour suppressor of the INK4A-ARF locus in human cells, acting in
concert with p53 to protect from oncogenic transformation. However, recent reports have also proposed a more
central role for mouse p16Ink4a in some situations,
including the progression of colorectal cancers and
melanoma (Bennecke et al., 2010; Carragher et al., 2010;
Monahan et al., 2010), Interestingly, no evidence to date
supports the notion that in human cells, like in mouse
cells, p14ARF is transcriptionally activated in response to
stress (Wei et al., 2001; Voorhoeve and Agami, 2003).
Collectively, the data described above highlights the
relevance of the INK4A gene in senescence induction.
The major question is, therefore, how is this gene
activated when cells encounter stress? It is clear that
signals such as DNA damage, oxidative stress, cytotoxic
drugs and oncogene activation lead to the activation
of INK4A transcription and the accumulation of the
p16INK4a protein. However, although the various
mechanisms that lead to p53 activation are now
relatively well defined (Figure 1), we do not yet fully
understand how INK4A transcription is activated
during cellular senescence.
Transcriptional regulation of the INK4A-ARF locus
Polycomb group proteins (PRC1 and PRC2)
In 1999, seminal work by Jacobs et al. first showed that
polycomb group proteins are critical for the transcriptional repression of the INK4A-ARF locus. Indeed,
mouse embryonic fibroblasts deficient for the polycomb
group protein BMI1 underwent premature cellular
senescence, due to the derepression of both the INK4A
and ARF genes (Jacobs et al., 1999). The polycomb
group proteins are chromatin remodellers that function
to repress gene expression by shaping chromatin
structure (Sparmann and van Lohuizen, 2006; Bracken
and Helin, 2009). They combine to form the polycomb
repressive complexes PRC1, composed of a PC (Polycomb) protein (for example, CBX7/CBX8), a PSC
(Posterior sex combs) protein (for example, BMI1/
MEL18), a RING (Really Interesting New Gene)
protein, a PH (Polyhomeotic) protein and an SCML
(Sex combs on mid-leg) protein; and PRC2, composed
of EED, SUZ12 and EZH2 in mammals (Muller and
Verrijzer, 2009; Simon and Kingston, 2009).
The PRC2 component EZH2 catalyses the trimethylation of lysine 27 on histone H3 (H3K27me3), a
mark involved in the recruitment of the PRC1 complex
by recognition of the H3K27 mark by the chromobox
domain in the CBX protein (Figure 2a). This facilitates
gene silencing, most likely through chromatin remodelling (Bracken and Helin, 2009). In addition to BMI1,
two additional PRC1 components, CBX7 and CBX8,
have been shown to extend the lifespan of cells by
repression of the INK4A-ARF locus (Gil et al., 2004;
Dietrich et al., 2007). Conversely, knockdown of the
polycomb components CBX7, CBX8, BMI1 or MEL18
results in INK4A derepression and proliferative arrest.
(Jacobs et al., 1999; Itahana et al., 2003; Gil et al., 2004;
Dietrich et al., 2007; Maertens et al., 2009). Interestingly, polycomb group proteins are also essential for
stem cell self-renewal in both mice and humans (Lessard
and Sauvageau, 2003; Molofsky et al., 2003; Park et al.,
2003; Cales et al., 2008), and this is in part due to their
ability to bind and repress the INK4A-ARF locus.
Polycombs regulate INK4A-ARF, by directly binding
to and ‘blanketing’ the complete INK4A-ARF locus, an
association that is necessary for maintaining transcriptional repression in unstressed cells (Bracken et al., 2007;
Dietrich et al., 2007; Kotake et al., 2007). Recently, a
long non-coding RNA (ncRNA) called ANRIL was
shown to be required, in addition to the H3K27me3
mark, for the recruitment of the PRC1 complex to the
INK4A-ARF locus (Yap et al., 2010). ANRIL is one
example of a long ncRNA from a family that are
emerging as recruiters of chromatin remodelling complexes to target genes (Guttman et al., 2009; Mercer
et al., 2009). ANRIL is transcribed in cis from within the
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Transcriptional regulation of cellular senescence
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Figure 2 Transcriptional regulation of the INK4A locus. Polycomb group proteins directly bind to and regulate the INK4A locus,
facilitating repression of the INK4A gene in unstressed cells. Although the mechanism by which polycombs are recruited to specific
regions of DNA is unclear, we know that, once recruited, these proteins act to stably silence gene expression by altering chromatin
structure. Some examples of emerging mechanisms by which polycomb group proteins are recruited to and maintained at the INK4A
locus in unstressed cells are outlined: (a) EZH2 catalyses the H3K27me3 epigenetic mark, which promotes recruitment of the PRC1
complex; (b) recruitment of the PRC1 complex is also partly dependent on the association of the CBX7 component with the long
ncRNA ANRIL; and (c) the transcription factor Zfp277 binds directly to the BMI1 component of the PRC1 complex and is partially
responsible for its recruitment to the INK4A locus. In senescent cells, polycomb proteins are lost or displaced from the INK4A locus by
a number of established mechanisms, including (d) EZH2 downregulation resulting in the loss of H3K27 methylation, (e) the histone
demethylase JMJD3, which actively removes the H3K27me3 mark and (f) the SWI/SNF chromatin remodelling complex, which is
proposed to displace polycombs from the INK4A locus. We also propose a novel mechanism (g) by which polycomb proteins may be
displaced by transcriptional activators. The mechanisms described here, while referring to the INK4A promoter, may also be relevant
to the p14ARF and p15INK4b promoters.
INK4B-ARF-INK4A locus, binds to CBX7, and this, in
addition to the H3K27me3 epigenetic mark, contributes
to its binding to the locus (Yap et al., 2010). Indeed,
point mutations that disrupt CBX7’s ability to bind to
either ANRIL or the H3K27me3 mark impair its ability
to repress INK4A and ARF and to prevent senescence
induction. However, this model conflicts with previous
studies, which reported a positive correlation between
expression levels of ANRIL, p16INK4A and p19ARF
(Pasmant et al., 2007; Liu et al., 2009). Recently, the
putative RNA helicase MOV10 has also been implicated
in the recruitment of the PRC1 complex (MessaoudiAubert et al., 2010). MOV10 knockdown displaces
polycombs from the INK4A-ARF locus, and results in
the upregulation of p16INK4a. Taken together, these data
imply that RNA-based mechanisms have an important
role in PRC1 recruitment.
Interestingly, transcription factors have also been
implicated in the regulation of the INK4A-ARF locus.
For example, the zinc-finger protein Zfp277 is reported
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to be involved in the transcriptional repression of the
INK4A and ARF genes through recruitment of PRC1,
by direct interaction with Bmi1 (Negishi et al., 2010).
Zfp277 associates with the INK4A-ARF locus in mouse
embryonic fibroblasts, an association that decreases
upon serial passaging. The loss of Zfp277 is correlated
with the progressive displacement of polycomb
association with the INK4A-ARF locus, and increased
mRNA expression of INK4A and ARF. The ectopic
expression of Bmi1 in Zfp277/ mouse embryonic
fibroblasts fails to bypass cellular senescence, showing
the importance of Zfp277 in polycomb-mediated regulation of the locus.
In contrast to what is described for PRC1, little is
known about the mechanisms of PRC2 recruitment to
the INK4A-ARF locus. However, it is also likely that
long ncRNAs and sequence-specific transcription factors are involved. Supporting this, recent work has
identified 49000 ncRNAs, which have been shown to
bind to the PRC2 complex by RNA immunoprecipitation
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(Khalil et al., 2009; Zhao et al., 2010). Alternatively, or
perhaps complementary to this, sequence-specific transcription factors may contribute to the recruitment and
maintenance of the PRC2 complex on the locus in
unstressed cells. Although numerous studies have shown
correlations between the expression of various transcription factors and INK4A and ARF levels, reliable
evidence of direct binding to DNA around the locus
by chromatin immunoprecipitation assay is required to
definitively show a direct link. Transcriptional regulators that have been reported to repress the INK4A and
ARF genes expression include the T-box transcription
factor TBX2, a potent repressor of mouse ARF (Jacobs
et al., 2000), and the transcriptional co-repressor CtBP,
which has been implicated as a direct repressor of
human INK4A expression (Mroz et al., 2008). Recent
work has also implicated an N-terminal fragment of the
GLI2 transcription factor in the repression of INK4A by
direct binding to its promoter (Bishop et al., 2010).
Although these factors are known repressors of the
INK4A-ARF locus, further work is required to evaluate
their potential interplay with polycomb group proteins.
Stress-induced derepression of the INK4A-ARF locus
A key feature of cellular senescence in response to
cellular stress is the displacement of polycomb repression complexes from the INK4A-ARF locus (Bracken
et al., 2007). In stressed cells, the key H3K27me3
methyltransferase, EZH2, an E2F target gene, is downregulated, leading to a loss of H3K27me3 on the locus,
and consequent displacement of the BMI1 containing
PRC1 complex (Bracken et al., 2003, 2007; Chen et al.,
2009). An alternative, or perhaps complementary,
mechanism of INK4A transcriptional activation has
been proposed to occur upon RAS activation (Agger
et al., 2009; Barradas et al., 2009). The histone
demethylase JMJD3 is upregulated, recruited to the
INK4A-ARF locus and actively removes the H3K27me3
mark, thereby leading to PRC1 displacement and
consequent gene activation. Chromatin remodelling
factors have also been shown to play a role in displacing
polycombs from the INK4A-ARF locus, including the
SWI/SNF complex (Kia et al., 2008), and the trithorax
protein MLL1, which functionally antagonises polycombs by catalysing the transcriptionally active
H3K4me3 mark (Kotake et al., 2009). Regulation of
the stability of the PRC1 complex also appears to be
another facet, as the de-ubiquitination of the PRC1
components BMI1 and MEL18 by the ubiquitin-specific
proteases 7 and 11 has been shown to increase their
abundance (Maertens et al., 2010), whereas the loss of
these ubiquitin-specific proteases promotes INK4A
transcriptional activation.
Transcriptional activators of the INK4A-ARF locus
In stressed cells, the loss of polycomb proteins from the
INK4A-ARF locus is thought to lead to an opening of
the chromatin state (Bracken et al., 2007), a configuration that allows for loading and induction by DNA
binding transcriptional activators. Candidates include
Ets1 and Ets2, which directly bind to the INK4A
promoter and activate INK4A expression in senescent
human diploid fibroblasts (Ohtani et al., 2001). Interestingly, the ability of Ets2 to activate INK4A can be
blocked by binding to the basic helix–loop–helix protein
ID1. In agreement with this, RasV12-induced cellular
senescence in the mouse mammary gland can be
bypassed by the additional overexpression of Id1
(Swarbrick et al., 2008). Furthermore, mouse embryonic
fibroblasts derived from Id1-deficient mice express high
levels of INK4A (Lyden et al., 1999; Alani et al., 2001),
suggesting that a balance between Ets2 and Id1
expression is maintained in unstressed cells, which can
be perturbed by increased Ets2 activity upon oncogenic
RasV12 activation, resulting in increased INK4A
expression.
Other potential transcriptional activators of the
INK4A-ARF locus include MEOX2, a homeobox
transcription factor (Irelan et al., 2009), MITF, a
transcriptional regulator expressed in melanocytes
(Loercher et al., 2005), and HMG-box containing
protein 1 (Li et al., 2010). Supporting a role for MITF
as an activator of INK4A expression, it was reported to
bind to the INK4A promoter in normal human
melanocytes. Furthermore, overexpression of MITF in
a mouse embryonic cell line resulted in the upregulation
of INK4A and inhibition of cellular growth, which could
be bypassed by INK4A inactivation (Loercher et al.,
2005). Recent work has reported that the transcription
factor HMG-box containing protein 1 binds to the
INK4A promoter in senescent late passage or RasV12
overexpressing human lung fibroblasts (Li et al., 2010).
Consistent with this, knockdown of HMG-box containing protein 1 impaired both the induction of senescence
and INK4A transcriptional activation, and was required
for RasV12-induced cellular transformation. Reduced
levels of HMG-box containing protein 1 mRNA
correlates with relapse of human breast tumours (Zhang
et al., 2006), suggesting a possible role as a tumour
suppressor. Additional regulators of the locus include
members of the E2F family of transcriptional regulators,
which can both activate (E2F1–3) and repress (E2F3b)
ARF but not INK4A (Aslanian et al., 2004; Komori
et al., 2005; del Arroyo et al., 2007). However, although
the transcriptional regulators discussed here are known
to bind to and regulate INK4A-ARF, the precise
mechanisms of how their activities relate to polycomb
protein function still remain to be elucidated.
Epigenetic regulation of the INK4A-ARF locus
The INK4A promoter is epigenetically silenced by DNA
hypermethylation in a range of human tumour types
(Merlo et al., 1995; Esteller et al., 2001). This loss can
also occur in histologically normal mammary tissues in
healthy women, supporting the notion that transcriptional and epigenetic alterations are among the earliest
events in cancer (Holst et al., 2003; Feinberg et al.,
2006). Studies on early-stage human cancers have shown
that INK4A expression is frequently lost upon
the transition from pre-neoplastic to invasive lesions
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F Lanigan et al
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(Collado et al., 2005; Bartkova et al., 2006; Fujita et al.,
2009; Bennecke et al., 2010).
One possible mechanism by which this might happen
is that polycomb group proteins are somehow misregulated in cancer, leading to the aberrant epigenetic
silencing of the INK4A-ARF locus. Supporting this
hypothesis, both EZH2 and CBX7 are reported to
recruit DNA methyltransferases to target gene promoters (Vire et al., 2006; Schlesinger et al., 2007;
Mohammad et al., 2009). Although this has not been
demonstrated for the INK4A promoter, it does suggest a
scenario whereby polycombs can directly contribute to
the altered DNA methylation profiles present in
tumours. In fact, polycomb target genes are as much
as 12 times more likely to become aberrantly silenced by
DNA methylation in cancer compared with nonpolycomb target genes (Ohm et al., 2007; Schlesinger
et al., 2007; Widschwendter et al., 2007). Furthermore,
poorly differentiated and aggressive human tumours
show preferential repression of polycomb target genes
(Ben-Porath et al., 2008). This is reflected in the fact that
polycombs and their recruiters are altered in a number
of tumour types. For example, BMI1 is amplified or
overexpressed in non-Hodgkin’s lymphoma (Bea et al.,
2001; Sauvageau and Sauvageau, 2010). Furthermore,
the levels of both CBX7 and ANRIL are elevated in
prostate cancer tissues, concurrent with a decrease in
INK4A expression levels (Yap et al., 2010), which would
seem to contradict the previously mentioned reports of a
positive correlation between ANRIL, INK4A and ARF
(Pasmant et al., 2007; Liu et al., 2009). Similarly, EZH2
is amplified or overexpressed and correlated with poor
prognosis in several cancer types, including prostate,
breast and melanoma (Bracken et al., 2003; Kleer et al.,
2003; Bachmann et al., 2006), and SUZ12 is overexpressed in lymphoma and melanoma, and translocated in some endometrial cancers (Koontz et al., 2001;
Martin-Perez et al., 2010). This frequent upregulation of
polycomb proteins and cofactors such as ncRNAs and
transcription factors could trigger the aberrant silencing
of the INK4A-ARF locus observed in multiple cancer
types. However, there are some contradictory reports,
which suggest that polycomb expression can be decreased
in certain tumour types, including the loss of CBX7 in
thyroid and pancreatic cancers (Pallante et al., 2008;
Karamitopoulou et al., 2010), the loss of BMI1 in
endometrial carcinoma and melanoma (Bachmann et al.,
2008; Engelsen et al., 2008) and the inactivation of EZH2
in myeloid disorders (Ernst et al., 2010). These studies
challenge the idea that tumourigenesis is exclusively
associated with an increase in polycomb function, and
imply that any imbalance in polycomb activity, resulting
in altered expression of polycomb target genes, may have
tumour-promoting effects.
Transcriptional regulation downstream of pRb
The remarkably stable state of cellular senescence
is thought to be maintained by compacted heterochromatic or ‘closed’ chromatin, called SAHF, which
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accumulates around genes encoding cell cycle and
proliferation proteins (Narita et al., 2003) (Figure 3).
Mechanistically, the pRb protein functions to recruit
repressive enzymatic activities to E2F-regulated target
gene promoters. These include histone deacetylases,
which remove permissive acetyl groups from histones
(Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin
et al., 1998); DNA methyltransferases (Robertson et al.,
2000), which methylate DNA and help to repress
transcription; and histone methyltransferases, including
SUV39H1, which tri-methylates lysine 9 on histone H3
(H3K9me3) (Rea et al., 2000; Nielsen et al., 2001;
Vandel et al., 2001). The H3K9me3 mark then acts as a
binding site for the chromodomain of the heterochromatin-associated protein HP1g (Bannister et al., 2001;
Lachner et al., 2001), which in turn promotes the
recruitment of chromatin remodellers such as HMGA2
(Narita et al., 2006), and variant histones such as
Figure 3 The pRb pathway and SAHF formation in senescent
cells. (a) In growing cells, the pocket proteins (pRB, p107 and
p130) are phosphorylated by cyclin D- and E-dependent kinases.
This blocks their ability to associate with E2F1–3 transcriptional
activators, which are then free to transactivate genes encoding
replication and cell cycle proteins. (b) Upon encountering cellular
stress upregulation of p16INK4a and/or p15INK4b leads to the
inhibition of the cyclin D/CDK complexes, resulting in the
hypophosphorylation of pRb, and sustained association with
E2F. To permanently silence E2F target genes, pRb recruits
histone deacetylases (HDACs), DNA methyltransferases
(DNMTs) and histone methyltransferases, such as SUV39H1.
SUV39H1 tri-methylates H3K9, creating a binding site for HP1g,
and resulting in the recruitment of molecules such as HMGA2 and
macroH2A histone. This results in the accumulation of heterochromatin around E2F target gene promoters in senescent cells,
stably silencing a subset of E2F-regulated genes and forming what
are known as SAHF (red, tumour suppressors; green, oncogenes).
Transcriptional regulation of cellular senescence
F Lanigan et al
2907
macroH2A histone (Zhang et al., 2005). All of this
results in the formation of heterochromatin around
E2F-responsive promoters, the so-called ‘SAHF’,
and the stable transcriptional silencing of a subset of
E2F-regulated, proliferation-associated genes. It is still
unclear as to whether SAHF formation requires
sustained p16–pRb activation, to allow the gradual
heterochromatinisation of E2F target genes. Significantly, in contrast to the transient, reversible repression
of E2F-regulated genes seen in quiescent cells, which is
mediated by p107 and p130, these genes are stably
repressed in senescent cells and do not respond to E2F1
expression (Narita et al., 2003).
The formation of SAHFs is relevant to cancer, and
defects in components of the SAHF machinery result in
an impaired cellular senescence response and increased
tumour formation. For example, the loss of the Suv39h1
histone methyltransferase in mice expressing activated
N-Ras in the haematopoietic compartment (Em-N-Ras)
leads to accelerated lymphomagenesis compared to
control mice (Braig et al., 2005). Significantly, the
cancer cells were apoptosis competent, but were incapable of undergoing cellular senescence. This suggests
that H3K9 methylation by Suv39h1 is essential for
chromatin compaction and formation of SAHF. A
recent study showed that pRb primarily targets E2F
target gene promoters in senescent human diploid
fibroblasts, supporting the notion that repression of
E2F target genes is the main function of pRb in
senescent cells (Chicas et al., 2010). However, pRb is
known to bind a variety of additional transcription
factors, including c-JUN, Pax3 and C/EBPb (Morris
and Dyson, 2001), and the importance of these
associations, and potentially others, in relation to
cellular senescence remains to be investigated.
Perspectives
Although our knowledge of the factors involved in
cellular senescence control has expanded in recent years,
there are a number of key questions that remain to be
answered. Firstly, what are the crucial p53 target genes
that mediate cellular senescence? How exactly is the
INK4A-ARF locus derepressed upon the induction of
cellular senescence? Does the contribution of polycombs, ncRNAs and transcription factors to the
regulation of INK4A vary depending on the stress
involved or cell lineage type? How is the INK4A-ARF
locus epigenetically silenced in cancer? Does pRb
regulation of SAHF formation involve additional
pRB-associated transcription factors?
Therapeutically, although tumour cell death may well
be a preferable end point, the induction of cellular
senescence could prove to be a viable alternative
approach (Shay and Roninson, 2004). Previously, the
study of apoptosis revealed several promising new
molecules, which can be targeted in cancer therapies
(Reed, 2006; Taylor et al., 2008). For example, treatments aimed at downregulating anti-apoptotic molecules such as BCL2 and survivin are currently in clinical
development, being tested both alone and in combination with existing chemotherapeutic treatments (Call
et al., 2008). It is likely that by studying the mechanisms
of cellular senescence, we will uncover novel therapeutic
targets that, when manipulated, may induce cellular
senescence in tumour cells. Cellular senescence has been
reported to be induced following chemotherapeutic
treatment of both human and mouse tumours, and,
along with apoptosis, may be a clinically beneficial end
point to chemotherapy (Schmitt et al., 2002; te Poele
et al., 2002; Haugstetter et al., 2010). In this regard,
pharmacological reactivation of p53 has been proposed
as an effective therapeutic intervention, as re-induction
of p53 expression in malignant tumours in mice leads to
rapid tumour regression owing to the induction of
cellular senescence or apoptosis, depending on the
tumour type (Ventura et al., 2007; Xue et al., 2007). In
humans, the reactivation of p53 could be achieved in
tumour cells using specific therapies such as the smallmolecule MDM2 inhibitor Nutlin-3a, which can induce
p53 expression in tumours with a still functional p53
(Vassilev et al., 2004; Kumamoto et al., 2008). This
would likely activate transcription of downstream genes,
resulting in the induction of apoptosis or cellular
senescence. However, recent work has cast doubt on the
therapeutic potential of p53 reactivation, suggesting that
this strategy may only be effective in late-stage tumours,
leaving early lesions that are likely to become cancerous
intact (Feldser et al., 2010; Junttila et al., 2010).
‘Epigenetic therapies’ such as DNA-demethylating
agents and histone deacetylase inhibitors have shown
some efficacy as cancer therapeutics (Schmitt, 2007),
mainly due to their capacity to induce a cellular
senescence response and derepress INK4A and other
tumour suppressors (Timmermann et al., 1998; Matheu
et al., 2005). However, the effects of these agents are
potentially very broad and they may have unintended
consequences. For example, in the Em-N-Ras transgenic
mice discussed previously (Braig et al., 2005), combined
treatment with the histone deacetylase inhibitor trichostatin A and the DNA-demethylating agent 2-deoxy-5azacytidine was found to promote the development of
T-cell lymphomas. The apparent tumour-promoting
activity of these treatments is at odds with their clinical
success in the treatment of cancer (Gore et al., 2006).
This study highlights the complex nature of epigenetic
modifications, and the need for caution with regard to
the use of ‘general’ epigenetic therapies, which could
potentially affect the expression of a multitude of genes
in both normal and cancer cells. The search for more
specific epigenetic-based therapies has identified the
histone methyltransferase inhibitor 3-deazaneplanocin
A as having efficacy in decreasing EZH2 levels. This
results in the inhibition of H3K27 methylation, the
reactivation of polycomb target genes, including
INK4A, and the induction of growth inhibition and
apoptosis in tumour cells in vitro (Tan et al., 2007;
Fiskus et al., 2009; Puppe et al., 2009; Hayden et al.,
2010; Yamaguchi et al., 2010). However, the fact that
EZH2 is known to be mutated and inactivated in
myeloid disorders (Ernst et al., 2010) suggests that
Oncogene
Transcriptional regulation of cellular senescence
F Lanigan et al
2908
EZH2-targeting drugs may only be effective in a subset
of tumour types.
Clearly, there is a need for the development of
additional, more specific epigenetic therapies. Established regulators of INK4A, such as polycombs,
ncRNAs and transcription factors, in theory provide
excellent targets. Practically, transcription factors,
although not generally ideal targets due to an abundance of protein–protein interaction sites and a lack of
hydrophobic pockets, can be specifically targeted by
drugs such as small-molecule inhibitors (Darnell, 2002;
Tan et al., 2007; Moellering et al., 2009). Long ncRNAs
on the other hand, as they do not code for protein, may
require alternative strategies, such as antisense oligonucleotides (Wahlestedt, 2006). With regard to the future
development of drugs targeting these factors, one issue
to consider is the lineage-specific expression of transcription factors and ncRNAs that regulate the INK4AARF locus (for example, MITF), meaning that drugs
targeting a specific transcription factor or ncRNA may
not be effective in inducing cellular senescence in all
tumour types.
Collectively, the work discussed in this review
confirms that transcription factors, chromatin remodellers and long ncRNAs are emerging as being centrally
involved in coordinating the activation and maintenance
of the senescent state. Future studies aimed at further
unravelling new players, delineating their functional
interplay with existing factors and examining their
function in different cell lineages will be crucial to fully
understand the mechanisms of cellular senescence.
Finally, this information must be harnessed for clinical
benefit, unlocking the potential of these mediators of
senescence as therapeutic targets in cancer.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Eros Lazzerini and Jean-Christophe Marine for
critical reading of the manuscript. Work in the Bracken Lab is
supported by Science Foundation Ireland under the Principal
Investigator Career Advancement Award (SFI PICA SFI/10/
IN.1/B3002), the Health Research Board under the Health
Research Awards 2010 (HRA_POR/2010/124), and the Irish
Research Council for Science, Engineering and Technology
(IRCSET).
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